Infectious Disease Reagents - HyTest Ltd.

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2 INFECTIOUS DISEASE REAGENTS

tury. According to the Centers for Desease Control(CDC) and the World Health Organization (WHO)data among 20 leading causes of mortality throughoutthe world in 2002 at least 9 were infectious, withthe first five positions kept by lower respiratory infections(6.8%), HIV/AIDS (4.9%), diarrheal diseases(3.2%), tuberculosis (2.7%) and malaria (2.2%).Thus it is still vitally important to modify and modernizetherapy methods, to develop new vaccines andto find fast, precise and simple methods for the diagnosticsof infectious diseases. This urgent need becameall too clear after September 11, 2001.Antibodies are used extensively as diagnostic toolsin a wide array of different analyses. Monoclonaland recombinant antibodies provide a never-endingsource of molecules and can produce endlesspossibilities for novel genetic constructs. Antibodiesare still very much in vogue and are now alsobeing used in microarray analysis of the proteomeusing protein chips. Although PCR – which is targetedto DNA or RNA infectious agent identification– has become a method of choice in the infectiousdiseases diagnostics in the latest decades, the antibody-basedmethods major trends, however, overthe past few decades have been increasing advancesin assay specificity, detection technologies andsensitivity and are increasingly used in research, investigationof pathogenesis mechanisms and in routineclinical tests. This is especially true for serologyanalysis and for microbial toxin detection.IntroductionIn spite of a considerable progress, which has beenachieved in the field of diagnostics, therapy and prophylaxisduring the recent decades, infectious diseasesstill keep the leading position among otherhuman illnesses at the beginning of the 21st cen-During the past 15 years Hytest has been involvedin the development and production of highly purifiedviral and microbial antigens as well as monoclonalantibodies against pathogens. Taking into considerationthe newly arisen global concern about bioterrorismand latest trends in most severe infectiousdiseases spreading, prompt supply of reagents forfast diagnosis is the major field of HyTest’s activitynow. We have widened the product spectrum for infectiousdisease diagnostics and encourage contactswith our clients.INFECTIOUS DISEASE REAGENTS3

Table of ContentsI INFLUENZA AND OTHER ACUTE RESPIRATORY DISEASES (ARDs) 71. Influenza A antigens 82. Influenza A monoclonal antibodies 102.1. Influenza A Haemagglutinin (HA) H1 and H3 monoclonal antibodies 102.1.1. Influenza A H1 and H3 immunodetection in ELISA 102.1.2. Influenza A quantitative sandwich immunoassay 112.1.3. Influenza A H1 and H3 immunodetection in Western blotting 112.2. Influenza A haemagglutinin H2 monoclonal antibodies NEW! 122.2.1. Influenza A immunodetection in ELISA 122.2.2. Influenza A H2 immunodetection in Western blotting 132.3. Influenza A haemagglutinin H5 monoclonal antibodies 142.4. Influenza A haemagglutinin H7 monoclonal antibodies New MAbs! 152.4.1. Influenza A immunodetection in ELISA 152.4.2. Influenza A H7 quantitative sandwich Immunoassay 152.5. Influenza A haemagglutinin H9 monoclonal antibodies NEW! 162.5.1. Influenza A immunodetection in ELISA 172.5.2. Influenza A H9 quantitative sandwich immunoassay 172.6. Influenza A matrix protein M2 monoclonal antibodies 182.7. Influenza A nonstructural (NS) protein monoclonal antibodies 182.8. Influenza A nucleoprotein (NP) monoclonal antibodies 192.8.1. Influenza A NP immunodetection in ELISA 192.8.2. Influenza A NP immunodetection in Western blotting 202.8.3. Influenza A NP quantitative sandwich immunoassay 203. Influenza B antigens 214. Influenza B monoclonal antibodies 234.1. Influenza B Nucleoprotein (NP) monoclonal antibodies 234.1.1. Influenza B immunodetection in ELISA 234.1.2. Influenza B quantitative sandwich immunoassay 244.1.3. Influenza B immunodetection in Western blotting 244.2. Influenza B haemagglutinin (HA) monoclonal antibodies 264.2.1. Influenza B HA immunodetection in ELISA 264.2.2. Influenza B HA immunodetection in Western blotting 264.3. Influenza B Matrix protein M1 monoclonal antibodies 274.3.1. Influenza B matrix protein M1 immunodetection in ELISA 274.3.2. Influenza B matrix protein M1 immunodetection in Western blotting 275. Respiratory Syncytial Virus (RSV) 285.1. Respiratory Syncytial virus (RSV) antigen 285.2. Respiratory Syncytial virus (RSV) monoclonal antibodies 296. Adenovirus 306.1. Adenovirus antigen 306.2. Adenovirus monoclonal antibodies 307. Parainfluenza 317.1. Parainfluenza antigens 318. Klebsiella pneumoniae 329. Newcastle disease virus (NDV) 33II HEPATITIS A AND B 341. Hepatitis A 342. Hepatitis B New MAb! 35III TORCH 3629 1. Toxoplasma gondii 362. Rubella virus 363. Cytomegalovirus (CMV) 374. Herpes simplex virus (HSV) 374 INFECTIOUS DISEASE REAGENTS

Table of ContentsIV EPSTEIN BARR VIRUS 38V SEXUALLY TRANSMITTED DISEASES (STD) 391. Chlamydia trachomatis 392. Herpes simplex virus 393. Treponema pallidum (Syphilis) 404. Candida albicans 405. Human papilloma virus (HPV) 415.1. HPV, type 6, oncoprotein E7 monoclonal antibodies 425.1.1. Applications 425.2. HPV, type 11, oncoprotein E7 monoclonal antibodies 435.2.1. E7 HPV type 11 immunodetection in ELISA 435.2.2. E7 HPV type 11 immunodetection in Western blotting 445.3. HPV, type 16, oncoprotein E7 monoclonal antibodies 445.3.1. E7 HPV type 16 immunodetection in ELISA 455.3.2. E7 HPV type 16 immunodetection in Western blotting 455.4. HPV, type 18, oncoprotein E7 monoclonal antibodies 465.4.1. E7 HPV type 18 immunodetection in ELISA 475.4.2. E7 HPV type 18 immunodetection in Western blotting 475.5. Human papilloma virus (HPV) antigens 48VI MALARIA 49VII TUBERCULOSIS 50VIII FOODBORNE PATHOGENS 521. Gastroenteritis viruses: rotavirus and adenovirus 522. Salmonella 543. Listeria monocytogenes 554. Legionella pneumophila 585. Campylobacter jejuni 596. Astrovirus 59IX MICROBIAL AND PLANT TOXINS ANTIBODIES 601. Antibodies for the detection of Staphylococcus aureus enterotoxins 601.1. Antibodies for the detection of Staphylococcus aureus enterotoxin 601.2. Antibodies for the detection of Staphylococcus aureus enterotoxin A 601.3. Antibodies for the detection of Staphylococcus aureus enterotoxin B 611.4. Antibodies for the detection of Staphylococcus aureus enterotoxin G 611.5. Antibodies for the detection of Staphylococcus aureus enterotoxin I 612. Antibodies for the detection of Cholera toxin (CT) 623. Antibodies for the detection of Escherichia coli heat-labile enterotoxin 624. Antibodies for the detection of Clostridium botulinum toxoids 635. Antibodies for the detection of Diphtheria 636. Antibodies for the detection of Ricin RCA 60from Ricinus communis 647. Antibodies for the detection of HT-2 toxin 658. Antibodies for the detection Microcystin-LR 659. Antibodies for the detection Nodularin 6510. Antibodies for the detection of Tetanus toxin 6611. Antibodies for the detection of Aflatoxin from Aspergillus flavus 66INFECTIOUS DISEASE REAGENTS5

Table of ContentsX BIODEFENSE ANTIBODIES 671. Antibodies for the detection of Bacillus anthracis 671.1. Antibodies for the detection of Bacillus anthracis Protective Antigen 681.2. Antibodies for the detection of Bacillus anthracis Lethal Factor 691.3. Antibodies for the detection of Bacillus anthracis Spore Antigen 702. Antibodies for the detection of Yersinia pestis 713. Antibodies for the detection of Francisella tularensis 744. Antibodies for the detection of Marburg and Ebola viruses 765. Antibodies for the detection of Vaccinia virus 766. Antibodies for the detection of Hemorrhagic fever with renal syndrome (HFRS) 78XI VETERINARY 791. Canine 791.1. Canine distemper virus (CDV) 791.2. Canine parvovirus (CPV) 791.3. Canine Adenovirus (CAV) 801.4. Rabies virus 801.5. Echinococcus granulosis 802. Bovine 812.1. Rotavirus 812.2. Bovine coronavirus 812.3. Brucella abortus (Brucellosis) 812.4. Alpha-1 – Acid Glycoprotein (AGP) 822.5. Foot-and-mouth disease (FMDV) 823. Equine 833.1. Burkholderia (Pseudomonas) mallei (Glanders) 834. Porcine 834.1. Transmissible Gastroenteritis (TGE) virus of Pigs 835. Piscine 835.1. Infectious Salmon Anemia virus 836. Avian 846.1. Newcastle disease virus (NDV) 846.2. Marek disease virus (MD) 846.3. Avian influenza 846.4. Infectious bursal disease virus (IBDV) 856.4.1. Immunodetection of VP2 and VP3 IBDV structure proteins in Western blotting 856.4.2. Direct ELISA 856.4.3. Histochemistry 866.4.4. Sandwich immunoassay for IBD virus detection 866.5. Infectious bronchitis virus (IBV) 876.5.1. Immunodetection of IBV nucleoprotein in Western blotting 876.5.2. Serological sandwich-type immunoassay 87XII MISCELLANEOUS 881. Borrelia burgdorferi (Borreliosis, Lyme Disease) 882. Tick-borne encephalitis virus (TBEV) 883. Cyclosporin A 894. Helicobacter pylori CagA 895. Hamster prion protein 896. FK 506 (Tacrolimus) 907. Allergen from Dermatotophagoides farinae 908. Streptavidin from Streptomyces avidinii 906 INFECTIOUS DISEASE REAGENTS

1. Influenza A antigensHyTest is offering following Influenza A antigens:Influenza A (H1N1) virus, strain A/Taiwan/1/86Influenza A (H1N1) virus, strain A/Beijing/262/95Influenza A (H1N1) virus, strain A/New Caledonia/20/99 (See Fig. 1)Influenza A (H1N1) virus, strain A/Solomon Islands/03/06Influenza A (H3N2) virus, strain A/Shangdong/9/93Influenza A (H3N2) virus, strain A/Panama/2007/99 (See Fig. 2)Influenza A (H3N2) virus, strain A/Kiev/301/94Influenza A (H3N2) virus, strain A/Wisconsin/67/05Influenza A (H3N2) virus, strain A/Brisbane/10/07The source is allantoic fluid of 10-12 days old embryonatedchicken eggs, inoculated with the appropriateinfluenza A strain. Purified viruses are inactivatedwith thimerosal and beta propiolactone treatment.Purity of all products is >90% and these antigenscan be used for detection of antibodies to influenzaA viruses in ELISA, HIT and Western blotting.Influenza A (H1N1) antigens do not have cross-reactivityin ELISA with panel of MAbs to HA of heter-ologicalsubtype of influenza A (H3N2) viruses,and Influenza A (H3N2) antigens do not have crossreactivityin ELISA with panel of MAbs to HA of heterologicalsubtype of influenza A (H1N1) viruses.Also these antigens are not cross-reacting withMAbs to HA of influenza B virus, MAbs to NP ofinfluenza B virus and in haemagglutination inhibitiontest with antisera to different subtypes of influenza Aand B viruses (See table 1).Table 1. Control investigation of influenza A antigens in haemagglutination inhibition test.Antibody titers in strain specific rabbit sera to:Influenza A virusInfluenza B virusVirus: SH1 Ssw1 SH3 SH2 SB1A/Panama/2007/99 (H3N2)

1,41,2+++MAb 3B3MAb 1C6MAb 4H721,81,6+++MAb 12/5MAb 1C611,4+MAb 4H7OD 4500,80,6++OD 4501,210,8MAb 3B30,40,6+0,40,20+++++++0,20+++++++5000 500 50 5 0,55000 500 50 5 0,5Concentration of MAbs (ng/ml)Concentration of MAbs (ng/ml)Figure 1. Control of specific activity and cross-reactivity of influenzaA/New Caledonia/20/99 virus in ELISA with monoclonal antibodies todifferent influenza viruses.MAb 3B3 to HA of influenza A/Beijing/262/95 (H1N1) virusMAb 1C6 to NP of influenza A/chick/Pennsylvania/1370/83 (H5N1) virusMAb 4H7 to HA of influenza B/Panama/45/90 virusFigure 2. Control of specific activity and cross-reactivity of influenzaA/Panama/2007/99 virus in ELISA with monoclonal antibodies to differentinfluenza viruses.MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virusMAb 1C6 to NP of influenza A/chick/Pennsylvania/1370/83 (H5N1) virusMAb 4H7 to HA of influenza B/Panama/45/90 virusMAb 3B3 to HA of influenza A/Beijing/262/95 (H1N1) virusOrdering information:Product Cat. # Strain RemarksInfluenza A (H1N1) virus 8IN73 A/Taiwan/1/86 EIA, HIT, WBInfluenza A (H1N1) virus-2 8IN73-2 A/Beijing/262/95 EIA, HIT, WBInfluenza A (H1N1) virus-3 8IN73-3 A/New Caledonia/20/99 EIA, HIT, WBInfluenza A (H1N1) virus-4 8IN73-4 A/Solomon Islands/03/06 EIA, HIT, WBInfluenza A (H3N2) virus 8IN74 A/Shangdong/9/93 EIA, HIT, WBInfluenza A (H3N2) virus-1 8IN74-1 A/Panama/2007/99 EIA, HIT, WBInfluenza A (H3N2) virus-2 8IN74-2 A/Kiev/301/94 EIA, HIT, WBInfluenza A (H3N2) virus-3 8IN74-3 A/Wisconsin/67/05 EIA, HIT, WBInfluenza A (H3N2) virus-4 8IN74-4 A/Brisbane/10/07 EIA, HIT, WBINFECTIOUS DISEASE REAGENTS9

2. Influenza A monoclonal antibodiesHyTest offers highly sensitive and specificmonoclonal antibodies for detection of Influenza Avirus. MAbs can be used in routine immunoassays(direct or indirect ELISA, sandwich immunodetectionsystems, Western blotting) and for specificdetection of the most important Influenza A anti-gens, such as Haemagglutnin (HA) and Nucleoprotein(NP) in different biological samples (nasalaspirates and swabs, cell lysates etc.). MAbs donot have cross-reactivity to Influenza B virus sothey can be used for differentiation between InfluenzaA and B.2.1. Influenza A haemagglutinin (HA) H1and H3 monoclonal antibodiesHosta Animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Influenza A H1N1 or H3N2 purified from allantoic fluidHaemagglutinin H1 OR h3Protein A or protein G affinity chromatographyPBS with 0.1% sodium azideHybridoma clones have been derived fromhybridization of Sp2/0 myeloma with the spleen cellsof Balb/c mice immunized with purified Influenza Aviruses: A/New Caledonia/20/99 (strain H1N1) andA/Shangdong/9/93 (strain H3N2). Haemagglutinin-specificantibodies selectively detect H1 or H3haemagglutinins of Influeza A in ELISA and Westernblotting.2.1.1. Influenza A H1 and H3 immunodetectionin ELISAAnti-haemagglutinin MAbs detect specific strain ofInfluenza A in direct and indirect ELISA. Titrationcurves of MAb InA4 (H1 specific) and MAb InA246(H3 specific) are shown of Fig. 3.0,8A0,4B0,6H1N1H3N20,3H1N1H3N2A, 490nm0,4A, 490nm0,20,20,100,1 1 10 100 1000 10000MAb concentration (ng/ml)00,1 1 10 100 1000 10000MAb concentration (ng/ml)Figure 3. Titration curves of MAbs specific to haemagglutinins H1 or H3 of Influenza A virus inindirect ELISA.A. MAb InA4 (H1 specific)B. MAb InA246 (H3 specific)Antigens:H1N1 – Influenza A/New Caledonia/20/99 - 0.1 µg/wellH3N2 – Influenza A/Shangdong/9/93 - 0.1 µg/well10 INFECTIOUS DISEASE REAGENTS

MAb C102 (Fig. 4) was obtained by use of avianinfluenza virus strain A (H1N1) as an immunogenand it is directed against relatively conservative H1epitope. MAb crossreactivity pattern shows that itdoes not react with H3 and other haemagglutininsbut interacts with H1 from human and avian influenzaviruses, having indirect ELISA titers not less than1:128 K. Thus MAb C102 may be used in EIA forsubtype differentiation of isolates. MAb C102 can alsobe used for immunocytochemistry, haemagglutinininhibition, ELISA and immunofluoresence.OD 4501,00,80,60,40,20,01E-3 0,01 0,1 1Concentration of MAb (µg/ml)MAb C1022.1.2. Influenza A quantitative sandwich immunoassayMAbs were tested in sandwich type fluoroimmunoassayas capture or detection MAbs. Pairs of MAbswere selected on their ability to detect specific strainof Influenza A with high specificity and sensitivity.Purified strains of Influenza A (H1N1 and H3N2)as well as recombinant H1 and H3 were used asantigens. For specific Influenza A H1 immunodetectionfollowing pairs are recommended (capture-detection):InA4 - InA88InA4 - InA134InA97 - InA134All pairs detect virus as well as recombinant haemagglutininH1 and can be used in Influenza AH1Nx-strain immunodetection systems. Calibrationcurve for one of the pairs is shown on Fig. 5.Figure 4. Specific activity of MAb C102 in ELISA with purified virus antigenA (H1N1).10000000CPS1000000100000100001000100H1N1H3N2Influenza B0,1 1 10 100 1000 10000 100000Virus concentration (ng/ml)Figure 5. Calibration curve for Influenza A sandwich fluoroimmunoassayusing anti haemagglutinin H1 antibodies.Capture: MAb InA97 – 1 µg/wellDetection (Eu-chelate labeled): MAb InA134 – 0.2 µg/wellIncubation time: 45 minAntigens:H1N1 – Influenza A/New Caledonia/20/99H3N2 – Influenza A/Shangdong/9/93Influenza B – mixture of Influenza B viruses (strains B/Qingdao/102/91,B/Tokio/53/99, B/Victoria/504/00)2.1.3. Influenza A H1 and H3 immunodetection inWestern blottingMw, kDa971 2 Mw, kDa 1 297MAbs detect haemagglutinin H1 or H3 in Westernblotting after SDS-PAGE in reducing conditions. MAbsbind to HA1 chain of processed or non-processedhaemagglutinin. Immunodetection of Influenza Ahaemagglutinins by specific antibodies is shown inFig. 6.664530664530Figure 6. Immunodetection of Influenza A viruses using anti-haemagglutininmonoclonal antibodies in Western blotting after PAGE in reducingconditions. Anti-mouse IgG conjugated with HRP was used for MAb haemagglutinincomplex visualization.Antigens (1 μg/well):H1N1 – Influenza A/New Caledonia/20/99H3N2 – Influenza A/Shangdong/9/93Antibodies (5 μg/ml):1: MAb InA4 – anti-Influenza A haemagglutinin H12: MAb InA246 – anti-Influenza A haemagglutinin H320H1N120H3N2INFECTIOUS DISEASE REAGENTS11

Ordering information:Product Cat. # MAb Isotype RemarksAnti-Influenza A haemagglutinin 3IH4 C102 IgG1 Haemagglutinin H1, EIA, IF, HIT, IHCAnti-Influenza A haemagglutinin H1 3AH1 InA4 IgG1 EIA (capture), WBAnti-Influenza A haemagglutinin H1 3AH1 InA16 IgG2a EIA, WBAnti-Influenza A haemagglutinin H1 3AH1 InA88 IgG2a EIA, WBAnti-Influenza A haemagglutinin H1 3AH1 InA97 IgG1 EIA (capture), WBAnti-Influenza A haemagglutinin H1 3AH1 InA134 IgG1 EIA (detection), WBAnti-Influenza A haemagglutinin H1 3AH1 InA139 IgG1 EIA, WBAnti-Influenza A haemagglutinin H3 3HG3 InA227 IgG1 EIA, WBAnti-Influenza A haemagglutinin H3 3HG3 InA246 IgG2a EIA, WB2.2. Influenza A haemagglutinin H2monoclonal antibodiesNEW!Host animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Influenza A H2N2 purified from allantoic fluidHaemagglutinin H2Protein A affinity chromatographyPBS with 0.1% sodium azideHybridoma clones have been derived fromhybridization of Sp2/0 myeloma with the spleen cellsof Balb/c mice immunized with purified InfluenzaA/Japan/305/57 H2N2 virus. Haemagglutinin-specificantibodies selectively detect H2 haemagglutinin ofInfluenza A virus in EIA and Western blotting.2.2.1. Influenza A immunodetection in ELISAAll MAbs detect haemagglutinin H2 of Influenza Avirus in direct and indirect ELISA. Representativetitration curves of some H2-specific MAbs (InA320and InA387) are shown on Fig. 7.3,5H1N1 virus3,0H1N1 virusA, 450nm3,02,52,01,5H3N2 virusH2N2 virusH7N7 virusParainfluenza virusrecombinant H2A, 450nm2,52,01,5H3N2 virusH2N2 virusH7N7 virusParainfluenza virusrecombinant H21,01,00,50,50,00 1 10 100 1 000 10 000InA320, ng/ml0,00 1 10 100 1 000 10 000InA387, ng/mlFigure 7. Titration curves of MAbs specific to haemagglutinins H2 in indirect ELISA.A. MAb InA320B. MAb InA387Antigens:Viruses – 200 ng/wellRecombinant H2 (A/Canada/720/2005)(aa 13-526) – 10 ng/well12 INFECTIOUS DISEASE REAGENTS

Cross-reactivity of all MAbs to some other InfluenzaA strains was also evaluated. MAbs were tested ontheir ability to interact with following viruses: InfluenzaA H1N1 (strain A/New Caledonia/20/99), InfluenzaA H2N2 (strain A/Japan/305/57), Influenza A H3N2(strain A /Panama/2007/99), Influenza A H7N7 (strainA/Netherlands/219/03) as well as Parainfluenzavirus (type 1 Sendai). MAbs InA317, InA318, InA320,InA382 do not cross-interact with tested InfluenzaA viruses (representative cross-reactivity for MAbsInA320 and InA387 is shown on Fig. 7). Only MAbsInA264 and InA382 reveal cross-reactivity to H1N1Influenza A virus (data not shown).2.2.2. Influenza A H2 immunodetection in WesternblottingAll MAbs detect haemagglutinin H2 in Westernblotting after SDS-PAGE in reducing conditions.Immunodetection of Influenza A haemagglutinin byspecific antibodies is shown on Fig. 8.Mw, kDa1 2 3 4 5 610075553527Figure 8. Immunodetection of Influenza A H2N2 virus by antihaemagglutininmonoclonal antibodies in Western blotting afterSDS-PAGE in reducing conditions. Anti-mouse IgG conjugatedwith HRP was used for MAb haemagglutinin complex visualization.Antigen: Influenza A /Japan/305/57 H2N2 – 0.5 μg/wellAntibodies: 10 μg/ml1 – InA264 4 – InA3202 – InA317 5 – InA3823 – InA318 6 – InA387Ordering information:New!New!New!New!New!New!Product Cat. # MAb Isotype RemarksAnti-Influenza A haemagglutinin H2 3HH2 InA264 IgG1 EIA, WBAnti-Influenza A haemagglutinin H2 3HH2 InA317 IgG2a EIA, WBAnti-Influenza A haemagglutinin H2 3HH2 InA318 IgG1 EIA, WBAnti-Influenza A haemagglutinin H2 3HH2 InA320 IgG2a EIA, WBAnti-Influenza A haemagglutinin H2 3HH2 InA382 IgG1 EIA, WBAnti-Influenza A haemagglutinin H2 3HH2 InA387 IgG1 EIA, WBINFECTIOUS DISEASE REAGENTS13

2.3. Influenza A haemagglutinin H5monoclonal antibodiesHost Animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Purified avian influenza virus type A (H5N1)Haemagglutinin H5Protein G affinity chromatographyPBS with 0.1% sodium azideAvian influenza viruses occurring naturally amongbirds cause avian influenza infection. Usually “avianinfluenza virus” refers to influenza A viruses foundmainly in birds, but infections with these virusescan occur also in humans. Avian influenza was firstidentified over 100 years ago during an outbreakin Italy. Since then, the disease has cropped up atirregular intervals in all world regions.There are many different subtypes of type A influenzaviruses and they differ because of changes incertain proteins on the surface of the influenza Avirus (haemagglutinin [HA] and neuraminidase [NA]proteins). Many different combinations of HA and NAproteins are possible and each combinationrepresents a different subtype. Of the 16 different haemagglutinintypes only strains within the H5 and H7subtypes cause highly pathogenic avian influenza,which is highly contagious and rapidly fatal in susceptibleavian species. When highly pathogenic influenzaH5 viruses cause outbreaks, the mortalityrate among poultry is usually between 90% - 100%.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Influenza A haemagglutinin H5 3H5N 11A9 IgG2a EIA, HIT, Dot blotAnti-Influenza A haemagglutinin H5 3H5N 15A6 IgG2a EIA, HIT, Dot blotAnti-Influenza A haemagglutinin H5 3H5N 18D5 IgG2a EIA, HIT, Dot blotAnti-Influenza A haemagglutinin H5 3H5N 19C11 IgG2a EIA, HIT, Dot blotAnti-Influenza A haemagglutinin H5 3H5N 8D2 IgG2a EIA, HIT, Dot blotAnti-Influenza A haemagglutinin H5 3H5N 6C8 IgG1 EIA, HITAnti-Influenza A haemagglutinin H5 3H5N 7E6 IgG2a EIA, HITAnti-Influenza A haemagglutinin H5 3H5N 1C7 IgG2a EIA, HITAnti-Influenza A haemagglutinin H5 3H5N 6B4 IgG2a EIA, HITAnti-Influenza A haemagglutinin H5 3H5N 9B3 IgG2a EIA, HITAnti-Influenza A haemagglutinin H5 3H5N 2D1 IgG1 EIAAnti-Influenza A haemagglutinin H5 3H5N 7D5 IgG2a EIAAnti-Influenza A haemagglutinin H5 3H5N 1B4 IgG2a EIA14 INFECTIOUS DISEASE REAGENTS

2.4. Influenza A haemagglutinin H7monoclonal antibodiesNew MAbs!Host animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Influenza A H7N7 or H7N1 purified from allantoic fluidHaemagglutinin H7Protein A or G affinity chromatographyPBS with 0.1% sodium azideHybridoma clones have been derived fromhybridization of Sp2/0 myeloma with the spleen cellsof Balb/c mice immunized with purified InfluenzaA/Netherlands/219/03 H7N7 virus (MAbs InA331,InA334 and InA414) or A/FPV H7N1 (other MAbs).Haemagglutinin-specific antibodies selectively detectH7 haemagglutinin of Influenza A virus in variousimmunoassays (direct and indirect ELISA, sandwichimmunoassay).2.4.1. Influenza A immunodetection in ELISAAll MAbs detect Influenza A H7 haemagglutinin indirect and indirect ELISA. Representative titrationcurve of anti-haemagglutinin H7 MAb InA334 isshown on Fig. 9.Cross-reactivity of all MAbs to other Influenza Astrains was evaluated. MAbs were tested on theirability to interact with following viruses: Influenza AH1N1 (strain A/New Caledonia/20/99), Influenza AH2N2 (strain A/Japan/305/57), Influenza A H3N2(strain A /Panama/2007/99), Influenza A H7N7 (strainA/Netherlands/219/03) as well as Parainfluenza virus(type 1 Sendai). All MAbs demonstrate high specificityto Influenza A H7 virus and do not bind to other testedInfluenza A viruses (representative cross-reactivity forMAb InA334 if shown on Fig. 9).A, 450nm4,03,53,02,52,01,51,00,50,0H7N7 virusrecombinant H7H1N1 virusH3N2 virusH2N2 virusParainfluenza virus0 1 10 100 1 000 10 000InA334, ng/mlFigure 9. Titration curve of MAb InA334 specific to haemagglutininH7 in indirect ELISAAntigens:Viruses – 200 ng/wellRecombinant H7 (A/Chicken/Netherlands/1/03)(aa 17-527) - 10 ng/well2.4.2. Influenza A H7 quantitative sandwichimmunoassayAll MAbs were tested in sandwich type immunoassayas capture or detection MAbs. Best pairs of MAbs wereselected on their ability to detect haemagglutinin H7of Influenza A virus with high sensitivity. Purified strainof Influenza A/Netherlands/219/03 H7N7 as well asrecombinant H7 were used as antigens in sandwichfluoroimmunoassay. For specific Influenza A H7immunodetection following pairs are recommended(capture-detection):InA334 – InA331InA334 – InA414Recommended pairs detect Influenza A H7N7 virusas well as recombinant haemagglutinin H7 and canbe used in Influenza A H7 immunodetection systems.Calibration curves for one of the recommended pairsare shown on Fig. 10.INFECTIOUS DISEASE REAGENTS15

100000,0A1000000,0B100000,010000,0CPS1000,0CPS10000,01000,0100,010 100 1 000 10 000 100 000virus H7N7, ng/ml100,00 1 10 100 1 000 10 000recombinant H7, ng/mlFigure 10. Calibration curve for Influenza A haemagglutinin H7 in sandwich fluoroimmunoassay utilizing pair of antibodies InA334-InA331.Capture MAb: InA334 – 1 µg/wellDetection MAb: (Eu-chelate labeled): InA331 – 0.1 µg/wellIncubation time: 45 minAntigens:A - Influenza A/Netherlands/219/03 H7N7 virusB - recombinant H7 (A/Chicken/Netherlands/1/03) (aa 17-527)Ordering information:New!New!New!Product Cat. # MAb Isotype RemarksAnti-Influenza A haemagglutinin H7 3HI7 InA331 IgG1 EIA (detection)Anti-Influenza A haemagglutinin H7 3HI7 InA334 IgG1 EIA (capture)Anti-Influenza A haemagglutinin H7 3HI7 InA414 IgG2b EIA (detection)Anti-Influenza A haemagglutinin H7 3HI7 1H11 IgG2a EIA, HITAnti-Influenza A haemagglutinin H7 3HI7 6B5 IgG2b EIA, HITAnti-Influenza A haemagglutinin H7 3HI7 9A9 IgG2a EIA, HITAnti-Influenza A haemagglutinin H7 3HI7 9F2 IgG1 EIA, HITAnti-Influenza A haemagglutinin H7 3HI7 10C6 IgG2a EIA, HITAnti-Influenza A haemagglutinin H7 3HI7 10H9 IgG1 EIA, HIT2.5. Influenza A haemagglutinin H9monoclonal antibodiesNEW!Host animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Recombinant H9Haemagglutinin H9Protein A affinity chromatographyPBS with 0.1% sodium azideHybridoma clones have been derived fromhybridization of Sp2/0 myeloma with the spleencells of Balb/c mice immunized with recombinantH9 haemagglutinins of Influenza A H9N2 virus:A/Chicken/Hong Kong/G9/97 and A/Hong Kong/1073/1999.16 INFECTIOUS DISEASE REAGENTS

2.5.1. Influenza A immunodetection in ELISAMAbs detect H9 haemagglutinin in direct and indirectELISA. Titration curve of MAb InA370 is shown onFig. 11.3,53rec (A/HongKong/1073/1999MAbs were tested on their ability to interact withother strains of Influenza A viruses: Influenza AH1N1 (strain A/New Caledonia/20/99), InfluenzaA H2N2 (strain A/Japan/305/57) Influenza A H3N2(strain A/Panama/2007/99), Influenza A H7N7 (strainA/Netherlands/219/03), Parainfluenza virus (type1 Sendai). MAbs InA370 and InA346 do not revealcross-reactivity with other Influenza A. MAb InA355demonstrates some cross-reactivity with H1N1 andH2N2 viruses (data not shown).MAbs InA355 and InA370 bind equally to strainsA/Chicken/Hong Kong/G9/97 and A/Hong Kong/1073/1999. MAb InA346 demonstrates lower binding tothe strain A/Chicken/Hong Kong/G9/97 in direct orindirect ELISA as compared to the strain A/HongKong/1073/1999. However, MAb InA346 works well insandwich immunoassay as the detection antibody forall tested H9 strains (see Fig. 12)A, 450nm2,521,510,50rec(A/Chicken/HongKong/G9/97virus InfluenzaA/HongKong/1073/991 10 100 1000 10000InA370, ng/mlFigure 11. Titration curve of MAb InA370 specific to haemagglutinin H9in indirect ELISA.Antigens: Virus – 200 ng/well, recombinant H9 – 10 ng/wellRecombinant H9: A/Chicken/ Hong Kong/G9/97 (a.a. 19-530) andA/Hong Kong/1073/1999 (a.a. 19-530) - 10 ng/wellVirus: Influenza A/Hong Kong/1073/99Antibody: InA370, titration from 10 µg/ml to 0.5 ng/ml2.5.2. Influenza A H9 quantitative sandwichimmunoassayMAbs detect H9 antigen in sandwich typefluoroimmunoassay. For specific Influenza A H9immunodetection following pairs are recommended(capture-detection):1000000,0100000,0InA355-InA370InA355-InA346Both pairs detect viral H9 haemagglutinin as well asrecombinant H9 haemagglutinin with high sensitivity(Fig. 12).CPS10000,01000,0virus A/HongKong/1073/99rec H9 A/HongKong/1073/1999rec H9A/Chicken/HK/G9/97Cross-reactivity of H9 specific pairs to some otherInfluenza strains was also evaluated. Pairs of MAbsdo not reveal cross-interaction with haemagglutininsof Influenza A H1N1 (strain A/New Caledonia/20/99),Influenza A H2N2 (strain A/Japan/305/57), InfluenzaA H3N2 (strain A/Panama/2007/99) and Influenza AH7N7 (strain A/Netherlands/219/03) (Fig. 13).100,00 1 10 100 1 000 10 000Antigen, ng/mlFigure 12. Haemagglutinin H9 immunodetection in sandwich fluoroimmunoassayutilizing pair of antibodies InA355-InA346 (capturedetection).Capture MAb: InA355 – 1 µg/wellDetection MAb (Eu-chelate labeled): InA346– 0.2 µg/wellIncubation time: 45 minINFECTIOUS DISEASE REAGENTS17

100000001000000CPS100000recombinant A/Hong Kong/1073/1999(H9N2)recombinant A/Chicken/Hong Kong/G9/97(H9N2)H1N1 virusH2N2 virusH3N2 virusH7N7 virusFigure 13. Haemagglutinin H9 immunodetection in sandwichfluoroimmunoassay utilizing pair of antibodies InA355-InA3470(capture-detection).Capture MAb: InA355 – 1 µg/wellDetection MAb (Eu-chelate labeled): InA370– 0.2 µg/wellIncubation time: 45 min1000010000 1 10 100 1 000 10 000Antigen, ng/mlNew!New!New!Ordering information:Product Cat. # MAb Isotype RemarksAnti-Influenza A haemagglutinin H9 3IA9 InA346 IgG1 EIA (detection)Anti-Influenza A haemagglutinin H9 3IA9 InA355 IgG1 EIA (capture)Anti-Influenza A haemagglutinin H9 3IA9 InA370 IgG1 EIA (detection)2.6. Influenza A matrix protein M2monoclonal antibodiesHost animal:Cell line used for fusion:Antigen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Purified Influenza A virusMatrix protein M2 of Influenza A virusProtein A affinity chromatographyPBS with 0.1% sodium azideOrdering information:Product Cat. # MAb Isotype RemarksAnti-Influenza A Matrix protein M2 3AM21 M2A10 IgG1 Indirect EIAAnti-Influenza A Matrix protein M2 3AM21 M2D2 IgG2a Indirect EIAAnti-Influenza A Matrix protein M2 3AM21 M2D4 IgG2b Indirect EIA2.7. Influenza A nonstructural (NS)protein monoclonal antibodiesMAbs were produced to non-structural antigens ofInfluenza A H5. MAbs can be used to detect antigenin ELISA or others methods, in antibody screening inthe competitive ELISA, etc.Host animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Nonstructural antigen of Influenza A H5Influenza A nonstructural antigen NS1Protein G affinity chromatographyPBS with 0.1% sodium azideOrdering information:Product Cat.# MAb Isotype RemarksAnti-Influenza A virus (NS protein) 3NS8 4A1 IgG2a EIAAnti-Influenza A virus (NS protein) 3NS8 9F10 IgG2b EIA18 INFECTIOUS DISEASE REAGENTS

2.8. Influenza A nucleoprotein (NP)monoclonal antibodiesHost Animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Purified influenza virus type A (H1N1)MAbs react with nucleoprotein (NP), which is common for Influenza A(H1N1), A(H3N2) and other Influenza A virusesProtein G affinity chromatography for MAb F8,Protein A affinity chromatography for othersPBS with 0.1% sodium azideFor the influenza virus type A determination we havea panel of MAbs against the nucleoprotein (NP).Influenza virus A (H1N1) was used as immunogen.All MAbs detect NP of Influenza A with high specificityand have no cross reactivity to NP of InfluenzaB virus.The investigation of F8 MAb specificity showed thatit recognizes the conservative epitope expressed onthe nucleoprotein, which is common for type A viruseswith different antigenic structure and speciesorigin. We investigated 25 strains of human and avianinfluenza virus A, isolated during different epidemicsin the period from 1934 till 1993 and in all the casesspecific reaction was observed. We investigated265 samples of nasal washings from patients duringinfluenza outbreaks in children’s communities bythe method of direct immunofluorescense. Sensitivityand specificity of the influenza virus A detectionreached 60% and 98.2% respectively.OD 4503,02.52.01,51,00,50,01E-3 0,01 0,1 1Concentration of MAb F8 (µg/ml)A (H1N1)A (H3N2)Figure 14. Specific activity of MAb F8 in ELISA with purified virus antigensA (H1N1) and A (H3N2).2.8.1. Influenza A NP immunodetection in ELISAAnti-NP MAbs equally detect different strains ofInfluenza A in ELISA. Titration curve of MAb InA108is shown of Fig. 15.0,8000,600A, 490nm0,4000,2000,00010 100 1 000 10 000MAb concentration (ng/m l)Figure 15. Titration curve of MAb InA108 specific to NP of Influenza Avirus in indirect ELISA.Antigen: Influenza A/New Caledonia/20/99 (H1N1) - 0.2 µg/wellINFECTIOUS DISEASE REAGENTS19

2.8.2. Influenza A NP immunodetection in WesternblottingMAbs InA108 and InA245 detect NP of Influenza Avirus in Western blotting after SDS-PAGE in reducingconditions. Immunodetection of Influenza A NPusing anti-NP monoclonal antibody InA108 is shownon Fig. 16.Mw, kDa9766451 22.8.3. Influenza A NP quantitative sandwich immunoassayMAbs were tested in sandwich type immunoassayas capture or detection MAbs. Pairs of MAbs wereselected on their ability do detect equally NP of H1N1and H3N2 strains of Influenza A virus. The best pairsof anti-Influenza A NP MAbs are as follows (capturedetection):InA108 – InA245InA180 – InA245All pairs detect NP of Influenza A virus of differentstrains. Calibration curve for one of the pairs isshown on Fig. 17.100000030H1N1H3N2100000Influenza B20CPS10000Figure 16. Immunodetection of Influenza A viruses using anti-NP monoclonalantibody 108 in Western blotting after PAGE in reducing conditions.Anti-mouse IgG conjugated with HRP was used for MAb NP complexvisualization.Antigens (1 μg/well):1: H1N1 – Influenza A/NewCaledonia/20/992: H3N2 – Influenza A/Shangdong/9/93Antibody (5 μg/ml):MAb InA108 – anti-Influenza A nucleoprotein (NP)100010 100 1 000 10 000 100 000Virus concentration (ng/ml)Figure 17. Calibration curve for Influenza A NP immunodetection insandwich fluoroimmunoassay.Capture: MAb InA108 – 1 µg/wellDetection (Eu-labeled): MAb InA245 – 0.2 µg/wellIncubation time: 45 minAntigens:H1N1 – Influenza A/New Caledonia/20/99H3N2 – Influenza A/Shangdong/9/93Influenza B – mixture of Influenza B viruses (strains B/Qingdao/102/91,B/Tokio/53/99, B/Victoria/504/00)Ordering information:Product Cat.# MAb Isotype RemarksAnti-Influenza A virus (nucleoprotein) 3IN5 F8 IgG2a EIA, IHCAnti-Influenza A virus (nucleoprotein) 3IN5 InA108 IgG1 EIA (capture), WBAnti-Influenza A virus (nucleoprotein) 3IN5 InA180 IgG3 EIAAnti-Influenza A virus (nucleoprotein) 3IN5 InA224 IgG1 EIA (capture)Anti-Influenza A virus (nucleoprotein) 3IN5 InA245 IgG2b EIA (detection), WB20 INFECTIOUS DISEASE REAGENTS

3. Influenza B antigensHyTest is offering following Influenza B antigens:Influenza B virus, strain B/Tokio/53/99 (See Fig. 19.)Influenza B virus, strain B/Victoria/504/00 (See Fig. 20.)Influenza B virus, strain B/Malaysia/2506/04Influenza B virus, strain B/Florida/07/04Influenza B virus, strain B/Florida/04/06The source is allantoic fluid of 10-12 days old embryonatedchicken eggs, inoculated with the appropriateinfluenza B strain. Purified viruses are inactivatedwith thimerosal and beta propiolactone treatment.Purity of all products is >90% and these antigenscan be used for detection of antibodies to influenzaB viruses in ELISA, HIT and Western blotting.Influenza B antigens do not have cross-reactivity inELISA with panel of MAbs to HA of heterological subtypeof influenza A (H3N2) viruses and MAbs to HAof influenza A (H1N1) viruses and in haemagglutinationinhibition test with antisera to influenza A (H3N2)and influenza A (H1N1) viruses (See table 2).Figure 18. Electron microscopic image of influenza B virus. (Influenza Bvirus particles 100-120 nm in diameter, magnification 1x 110 000).Table 2. Control investigation of influenza B antigens in haemagglutination inhibition test.Antibodies titers in strain specific immune rabbit and rat sera to:Influenza A virusInfluenza B virusVirus: SH1 Ssw1 SH3 SB1 SB2B/Tokio/53/99

10,8MAb 2/3MAb 12/5MAb 4H7Figure 19. Control of specific activity and cross-reactivity of influenzaB/Tokio/53/99 virus in ELISA with monoclonal antibodies to different influenzaviruses.MAb 2/3 to NP of influenza B/Beijing/184/93 virusMAb 4H7 to HA of influenza B/Panama/45/90 virus (Yamagata/16/88 lineage)MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virus+OD 4500,60,40,2++0++++5000 500 50 5 0,5 0,05Concentration of MAbs (ng/ml)1,41,21++MAb 2/3Figure 20. Control of specific activity and cross-reactivity of influenzaB/Victoria/504/00 virus in ELISA with monoclonal antibodies to differentinfluenza viruses.MAb 2/3 to NP of influenza B/Beijing/184/93 virusMAb 4H7 to HA of influenza B/Panama/45/90 virus (Yamagata/16/88 lineage)MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virusD 4500,80,6++MAb 12/5MAb 4H70,40,2+0++5000 500 50 5 0,5 0,05Concentration of MAbs (ng/ml)Ordering information:Product Cat. # Strain RemarksInfluenza B virus-2 8IN75-2 B/Tokio/53/99 EIA, HIT, WBInfluenza B virus-3 8IN75-3 B/Victoria/504/00 EIA, HIT, WBInfluenza B virus-4 8IN75-4 B/Malaysia/2506/04 EIA, HIT, WBInfluenza B virus-5 8IN75-5 B/Florida/07/04 EIA, HIT, WBInfluenza B virus-6 8IN75-6 B/Florida/04/06 EIA, HIT, WB22 INFECTIOUS DISEASE REAGENTS

4. Influenza B monoclonal antibodiesHyTest offers a panel of monoclonal antibodiesspecific to nucleoprotein (NP), haemagglutinin (HA)and matrix protein M1 of Influenza B virus. MAbswork with high affinity and specificity in differentimmunoassays: direct or indirect ELISA, Sandwichimmunodetection systems and in Western blotting.Anti-NP MAbs are highly specific to Influenza B nucleoproteinand do not bind to NP of Influenza A virus orany other viral proteins. Low detection limit of ourMAbs allows detection of Influenza B virus in differentsamples with low Influenza B titer. According tohigh specificity and affinity they are recommended tobe used in rapid Influenza B immunodetectionsystems.Anti-HA MAbs are specific to Influenza B haemagglutininHA 2and detect equally different strains of InfluenzaB virus.Anti-matrix protein MAbs are highly sensitive to M1matrix protein of Influenza B viruses and detect M1of different Influenza B strains in EIA and Westernblotting.4.1. Influenza B nucleoprotein (NP)monoclonal antibodiesHost Animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice SJL/J for MAb 2/3, mice Balb/c for other MAbsPx for MAb 2/3, Sp2/0 for other MAbsPurified influenza virus type BNucleoprotein of influenza virus type BProtein G or Protein A affinity chromatographyPBS with 0.1% sodium azide4.1.1. Influenza B immunodetection in ELISAAll anti-NP MAbs detect different strains of InfluenzaB in direct and indirect ELISA. Titration curves of selectedMAb are shown of Fig. 21.2,500A100000B2,000A, 490nm1,5001,000Influenza BInfluenza ACPS10000B/Tokyo/53/9910000,5000,0000,1 1 10 100 1000 10000MAb concentration (ng/ml)1000,01 0,1 1 10 100 1000InB114-Eu chelate concentration (ng/ml)Figure 21. Titration curves of MAb 114 specific to NP of Influenza B virus in indirect (A) anddirect (B) ELISA.A. MAb InB114 titration in indirect ELISAAntigens:Influenza B: Influenza B/Tokyo/53/99 - 0.5 µg/wellInfluenza A: mixture of two strains - A/Shangdong/9/93 and A/New Caledonia/20/99 - 0.5µg/wellB. MAb InB114 conjugated with Eu-chelate titration in direct ELISAAntigen:Influenza B/Tokyo/53/99 - 0.2 µg/wellINFECTIOUS DISEASE REAGENTS23

4.1.2. Influenza B quantitative sandwich immunoassayMAbs were tested in Sandwich type immunoassay asthe capture or detection MAbs. Pairs of MAbs wereselected on their ability to detect all tested strains ofInfluenza B with equal specificity and high sensitivity.Different strains of Influenza B (Influenza B/Leningrad/86/93,Influenza B/Tokyo/53/99, Influenza B/Victoria/504/00)as well as recombinant NP of InfluenzaB were used as antigens. For specific Influenza B NPimmunodetection following pairs are recommended(capture-detection):InB12 – InB27InB12 – InB64InB36 – InB64All pairs detect NP of influenza B and can be usedin Influenza B immunodetection systems. Calibrationcurve for one pair is shown on Fig. 22.100000MAbs are specific to different parts of NP molecule.For sensitive NP immunoassay MAbs that bind todiverse epitopes are recommended. Epitope specificityof some MAbs is shown in Table 3.Table 3. Epitope specificity of NP-specific MAbs.EpitopeMAbsFragment 1: (1-80 a.a.r.) InB12, InB36Fragment 2: (120-200 a.a.r.) InB27, InB64Fragment 3: (240-320 a.a.r.) InB204, InB210, 2/3Fragment 4: (480-560a.a.r) InB114, InB2134.1.3. Influenza B immunodetection in WesternblottingMAbs detect NP of Influenza B in Western blotting afterSDS-PAGE in reducing and non-reducing conditions.Immunodetection of Influenza B NP by selectedantibodies is shown on Fig. 23.10000CPSMw, kDa1 21000976610010 100 1 000 10 000 100 000Influenza B virus (ng/ml)45Figure 22. Calibration curve for Influenza B sandwich fluoroimmunoassayusing anti NP antibodies.Capture: MAb InB36 – 1 µg/well.Detection (Eu-chelate labeled): MAb InB64 – 0.2 µg/wellIncubation time: 45 min.Antigen: Influenza B/Tokio/53/99.3020Figure 23. NP of Influenza B virus immunodetection using anti-NPmonoclonal antibodies in Western blotting after PAGE in reducing conditions.Anti-mouse IgG conjugated with HRP was used for MAb NP complexvisualization.Antigen: Influenza B/Tokio/53/99 - 1 μg/well.Antibodies - 5μg/ml:1: MAb InB272: MAb InB6424 INFECTIOUS DISEASE REAGENTS

1,41,21MAb 2/3Figure 24. Specific activity of MAb 2/3 in ELISA with purified virusantigen B/Beijing/184/93.OD 4500,80,60,40,205000 500 50 5 0,5 0,05Concentration of MAb (ng/ml)Ordering information:Product Cat. # MAb Isotype RemarksAnti-Influenza Virus B (nucleoprotein) 3IF18 IB633 IgG1 WBAnti-Influenza Virus B (nucleoprotein) 3IF18 IB42 IgG2a EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB12 IgG2b EIA (capture), WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB27 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB36 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB64 IgG1 EIA (detection), WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB114 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB204 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB210 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 InB213 IgG1 EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 2/3 IgG2a EIA, WB, IFAnti-Influenza Virus B (nucleoprotein) 3IF18 8-5 IgG2a EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 13-9 IgG2a EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 14-12 IgG2a EIA, WBAnti-Influenza Virus B (nucleoprotein) 3IF18 15-12 IgG2a EIA, WBINFECTIOUS DISEASE REAGENTS25

4.2. Influenza B haemagglutinin (HA)monoclonal antibodiesHost animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Purified influenza B virusSpecific to haemagglutinin HA 2of Influenza BProtein A affinity chromatographyPBS with 0.1% sodium azide4.2.1. Influenza B HA immunodetection in ELISAAll of anti-haemagglutinin MAbs equally detect HAof different strains of Influenza B virus in direct andindirect ELISA. Titration curves of MAb InB190 isshown of Fig. 25.4.2.2. Influenza B HA immunodetection in WesternblottingAll MAbs detect Influenza B haemagglutinin HA 2chain of different strains in Western blotting afterSDS-PAGE in reducing conditions (Fig. 26).1,0000,800MW, kDa1 2 3A, 490nm0,6000,4000,200Influenza B/Tokyo/53/99Influenza B/Leningrad/86/93Influenza B/Victoria/504/00Influenza A mixture9766450,0000,1 1 10 100 1000 10000 100000MAb concentration (ng/ml)30Figure 25. Titration curves of MAb InB190 specific to HA of Influenza Bvirus in indirect ELISA.A. MAb InB190 titration in indirect ELISA.Antigens - 0.5 µg/well:Influenza B/Tokyo/53/99Influenza B/Leningrad/86/93Influenza B/Victoria/504/00Influenza A: mixture of two strains - A/Shangdong/9/93 and A/New Caledonia/20/9920Figure 26. Influenza B HA 2immunodetection after Western blotting.Anti-mouse IgG conjugated with HRP was used for MAb HA complexvisualization.Antigens - 1 μg/well:1 - Influenza B/Tokio/53/992 - Influenza B/Leningrad/86/933 - Influenza B/Victoria/504/00Antibody: MAb InB190 – 3 μg/mlOrdering information:Product Cat. # MAb Isotype RemarksAnti-Influenza Virus B (haemagglutinin) 3BH9 InB18 IgG2a HA2, EIA, WBAnti-Influenza Virus B (haemagglutinin) 3BH9 InB190 IgG2b HA2, EIA, WB26 INFECTIOUS DISEASE REAGENTS

4.3. Influenza B matrix protein M1monoclonal antibodiesHost animal:Cell line used for fusion:Immunogen:Specificity:Purification method:Presentation:Mice Balb/cSp2/0Purified Influenza B virusMatrix protein M1 of Influenza B virusProtein A affinity chromatographyPBS with 0.1% sodium azide4.3.1. Influenza B matrix protein M1 immunodetectionin ELISAMAbs InB4 and InB15 detect matrix M1 protein ofInfluenza B virus in direct and indirect ELISA. Titrationcurve of MAb InB4 is shown of Fig. 27.4.3.2. Influenza B matrix protein M1 immunodetectionin Western blottingAll MAbs detect Influenza B matrix protein M1 inWestern blotting after SDS PAGE in reducing conditions(Fig. 28). MAbs InB4 and InB15 equally detectM1 protein of different strains of Influenza B.A, 490nm2,5002,0001,5001,0000,500MW, kDa 1 29766450,0001 10 100 1000 10000 100000MAb concentration (ng/ml)30Figure 27. Titration curve of MAb InB4 specific to matrix protein M1 ofInfluenza B virus in indirect ELISA.MAb InB4 titration in indirect ELISAAntigen: Influenza B/Tokyo/53/99 - 0.5 µg/well20Figure 28. Influenza B matrix protein M1 immunodetection after Westernblotting. Anti-mouse IgG conjugated with HRP was used for MAb M1complex visualization.Antigen: Influenza B/Tokio/53/99 - 1 μg/wellMAbs: 3 μg/ml1 - InB42 - InB15Ordering information:Product Cat. # MAb Isotype RemarksAnti-Influenza Virus B (matrix protein M1) 3BM17 InB4 IgG1 EIA, WBAnti-Influenza Virus B (matrix protein M1) 3BM17 InB15 IgG1 EIA, WBINFECTIOUS DISEASE REAGENTS27

5. Respiratory Syncytial Virus (RSV)Respiratory syncytial virus is one of the most importantrespiratory pathogens in infants and childrenprovoking considerable morbidity, which oftenrequires bed care. Severe diseases caused by RespiratorySyncytial virus are most common among infantsduring the first six months of life and patientswith immunodeficiency. Serious lesions of the lowerrespiratory tract induced by Respiratory Syncytialvirus (bronchitis, bronchiolitis, pneumonia) are oneof the important causes of mortality in infants. 60-70% of infants less than six months of age fail to inducedetectable antibody response to natural infection.Repeated infections with Respiratory Syncytialvirus are common and result in neutralizing antibodyformation.5.1. Respiratory Syncytial virus (RSV) antigenSource: MA-104 cells, inoculated with Respiratory Syncytial virus, strain Long.Purity: >90%Inactivation: Viruses are inactivated with thimerosal and beta propiolactone treatment..Specificity: The identity of viral antigens, absence of contamination by other viruses (adenovirus, influenza A and B viruses andparainfluenza viruses) and immunoreactivity were checked in ELISA. See Fig. 30.Morphology: In investigation using electron microscopy: RS-virus particles 150 – 300 nm in diameter were observed. See Fig. 29.Applications: Detection of antibodies to Respiratory Syncytial virus in ELISA. Recommended concentration for ELISA is 2.5 – 5 μg/ml.1,21MAb 9C5MAb 7F10,8OD 4500,60,40,205000 500 50 5 0,5 0,05Concentration of MAbs (ng/ml)Figure 29. Electron microscopic image of Respiratory Syncytial virus(Virus particles 150-300 nm in diameter were observed, magnification1x110 000).Figure 30. Control of specific activity and cross-reactivity of RespiratorySyncytial virus in ELISA with monoclonal antibodies to different virusesMAb 9C5 to F-protein of Respiratory Syncytial virus.MAb 7F1 to hexon antigen of adenoviruses.Ordering information:Product Cat. # Strain RemarksRespiratory Syncytial virus 8RSV79 Long EIA28 INFECTIOUS DISEASE REAGENTS

5.2. Respiratory Syncytial virus (RSV)monoclonal antibodiesHost Animal:Cell line used for fusion:Immunogen:Purification method:Presentation:Mice SJL/JPxPurified Respiratory Syncytial virusProtein G affinity chromatographyPBS with 0.1 % sodium azideWe developed a panel of MAbs against RSV. In Westernblot MAb 8C5 reacts with protein having Mr 90 K,that corresponds in mobility to protein G. MAb 9C5specificity was determined by competitive ELISAwith MAbs 131-2A and 92-11C (CDC, Atlanta): theyare directed to the same F1a epitope, localized on F-protein. MAb 8B10 in directed against nucleoproteinN. MAb 9C5 is highly reactive with the surface domainsof both mature RSV virions and «empty» virionenvelopes without formed inner nucleocapsid structures.MAb 8B10 is reacting well only with maturevirions with completely assembled nucleocapsids.No cross-reactivity with influenza A and B viruses,adenovirus and parainfluenza type 1 and 2 viruses.MAbs 8C5 and 9C5 may be used in sandwich ELISAfor RSV detection both with themselves and in amixed combination, taking into account that 9C5 isespecially suitable for conjugation. MAbs 8C5 and9C5 have virus-neutralizing activity, 8C5 blocks RSVtargetcells binding, 9C5 hampers the virus penetrationinto the cell. MAb 8B10 is suitable for ELISA andcould be used to detect incomplete virus assembly.OD 4502,52,01,51,00,500,1E-30,01 0,1 18C5, µg/mlFigure 31. Specific activity of MAb 8C5 in ELISA with purified RSVantigen.OD 4502,01,51,00,50,01E-4 1E-3 0,01 0,1 19C5, µg/mlFigure 32. Specific activity of MAb 9C5 in ELISA with purified RSVantigen.0,80,70,6OD 4500,50,40,30,20,10,01E-3 0,01 0,1 18B10, µg/mlFigure 33. Specific activity of MAb 8B10 in ELISA with purified RSVantigen.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Respiratory Syncytial virus 3Res21 8C5 IgG2b G protein, EIAAnti-Respiratory Syncytial virus 3Res21 9C5 IgG2b F protein, EIAAnti-Respiratory Syncytial virus 3Res21 8B10 IgG1 Nucleoprotein, EIAINFECTIOUS DISEASE REAGENTS29

6. AdenovirusAdenoviruses are a large group (more than 80 types)of agents, which induce respiratory infections amonghuman beings, animals and birds. Clinical pattern ofadenoviral infection is characterized by pronouncedpharyngitis, conjunctivitis, general intoxication andpulmonary lesions with high fever in children. Adenovirustypes 3, 4, 7, 14 and 21 often spread in militaryunits and account for 72% of ARDs among recruits.A considerable part of these diseases resultsin hospitalization. Adenovirus types 3, 4, 7, 8 and 19are known as causative agents of epidemic keratoconjunctivitis.Some types of adenoviruses provokeoutbreaks of gastroenteritis with long (more than 2months) carriage of viruses.6.1. Adenovirus antigenSource: HeLa cells, inoculated with Adenovirus, type 6, strain Tonsil 99Purity: >90%Inactivation:Viruses are inactivated with thimerosal and beta propiolactone treatment..Specificity:The identity of viral antigens, absence of contamination by other viruses (RSV, influenza A and B viruses and parainfluenzaviruses) and immunoreactivity were checked in ELISA. See Fig. 35.Morphology: In investigation using electron microscopy - typical adenovirus particles 80 nm in diameter were observed. See Fig. 34.Applications:Detection of antibodies to Adenovirus in ELISA. Recommended concentration for ELISA is 2.5 - 5 μg/ml.1,61,49C57F11,2OD 45010,80,60,40,205000 500 50 5 0,5Figure 34. Electron microscopic image of Adenovirus, type 6 (Virus particles80 nm in diameter were observed, magnification 1x110 000).Figure 35. Control of specific activity and cross reactivity of adenovirusin ELISA with monoclonal antibodies to different viruses.MAb 9C5 to F-protein of RS-virusMAb 7F1 to hexon antigen of adenovirusesOrdering information:Product Cat. # Strain RemarksAdenovirus, type 6 8AV13 Tonsil 99 EIA6.2. Adenovirus monoclonal antibodiesHost Animal:Mice Balb/cCell line used for fusion: Sp2/0Immunogen: Purified human and canine adenoviruses (type 1)Purification method: Protein A affinity chromatographyPresentation:PBS with 0.1 % sodium azideAdenovirus antibodies react with Hexon antigen of atleast human, canine, bovine, monkey and rat adenoviruses.MAbs can be used in ELISA, immunodiffusionand immunohistochemistry. In sandwich ELISAwe recommend using MAb 8C4 for capture and MAb1E11 for detection.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Adenovirus hexon 3AV13 7C11 IgG2a + IgM EIA, ID, IHCAnti-Adenovirus hexon 3AV13 1E11 IgG2a + IgM EIA (detection), ID, IHCAnti-Adenovirus hexon 3AV13 8C4 IgG2a EIA (capture), ID, IHC30 INFECTIOUS DISEASE REAGENTS

7. ParainfluenzaParainfluenza virus types 1-3 are common agents ofacute respiratory infections predominating amongchildren less than 5 years old. They induce about15% of acute respiratory infections. They are mostlycausative agents of severe croup, bronchitis, bronchiolitisand pneumonia (Parainfluenza virus type 3)in infants. Children can be infected with Parainfluenzavirus several times during one year.7.1. Parainfluenza antigensWe produce extra purity grade parainfluenza virustype I (strain Sendai), parainfluenza type 2 (strainII-ALTB cc2056) and parainfluenza virus type 3(strain 3v29) viral antigens for serology tests suchas indirect EIA, HIT (haemagglutinin inhibition test),CFT (complement fixation test) and for the use asimmunogens in polyclonal antibody production.The purification technology is in general similar tothe one described earlier for influenza virus types Aand B, but parainfluenza virus types 2 and 3 weregrown in a monolayer of cells MA-104. Viruses are inactivatedwith thimerosal.The quality control was made by electron microscopy,SDS-PAGE and protein concentration measurementby BCA (Pierce) assay. The identity of viralantigens, absence of contamination by other virusesand immunoreactivity were checked in indirectELISA and HIT. Purity of the antigens is >90%.Parainfluenza virus type 1, strain SendaiParainfluenza virus type 2, strain II ALTB cc 2056 (See Table 4 and Fig. 36.)In investigation using electron microscopy parainfluenza virus type 2 particles 150-300 nmin diameter were observed.Low level of cross-reactivity with parainfluenza virus type 3 was observed in ELISA.Parainfluenza virus type 3, strain III v 2932 (See Table 4 and Fig. 37.)0,80,6+MAb PIV21MAb PIV23MAb 1B5OD 4500,40,20++++++5000 500 50 5 0,5 0,05Concentration of MAbs (ng/ml)Figure 36. Control of specific activity and cross-reactivity of parainfluenzavirus type 2 in ELISA with monoclonal antibodies to parainfluenzaviruses.PIV21: MAb to F-protein of parainfluenza virus type 2PIV23: MAb to F-protein of parainfluenza virus type 21B5: MAb to parainfluenza virus type 3Table 4. Results of the control investigation of parainfluenza virus types 2 and 3 inhaemagglutination inhibition test.Antibody titers in strain specific immune rabbit sera to:Parainfluenza viruses: S1 S2 S3PIV type 1 (Sendai strain) 320

+10,8++MAb PIV21MAb PIV23MAb 1B5+OD 4500,60,4+0,20++5000 500 50 5 0,5 0,05Concentration of MAbs (ng/ml)Figure 37. Control of specific activity and cross-reactivity of parainfluenzavirus type 3 in ELISA with monoclonal antibodies to parainfluenzaviruses.PIV21: MAb to F-protein of parainfluenza virus type 2PIV23: MAb to F-protein of parainfluenza virus type 21B5: MAb to parainfluenza virus type 3Ordering information:Product Cat. # Strain RemarksParainfluenza virus, type 1 8P76 Sendai EIA, HITParainfluenza virus, type 2 8P76-2 II ALTB cc 2056 EIA, HITParainfluenza virus, type 3 8P76-3 III v 2932 EIA, HIT8. Klebsiella pneumoniaeKlebsiella pneumoniae is a Gram-negative, non-motile,facultative anaerobic, rod shaped bacteriumfound in the normal flora of the mouth, skin, and intestines.It is clinically the most important member ofthe Klebsiella genus of Enterobacteriaceae. It naturallyoccurs in the soil and about 30% of strains canfix nitrogen in anaerobic condition.Members of the Klebsiella genus typically express2 types of antigens on their cell surface. The first, Oantigen, is a lipopolysaccharide of which 9 varietiesexist. The second is K antigen, a capsular polysaccharidewith more than 80 varieties. Both contributeto pathogenicity and form the basis for subtyping.K. pneumoniae can cause bacterial pneumonia, typicallydue to aspiration, though it is more commonlyimplicated in hospital-acquired urinary tract andwound infections, particularly in immunocompromisedindividuals (e.g. alcoholics). Klebsiella rankssecond to E. coli for urinary tract infections in olderpersons. It is also an opportunistic pathogen for patientswith chronic pulmonary disease, enteric pathogenicity,nasal mucosa atrophy, and rhinoscleroma.Monoclonal anti-Klebsiella pneumoniae antibodieswere produced from hybridoma clones derived fromhybridization of Sp2/0 myeloma cells with spleencells of Balb/c mice immunized with lyophilized Klebsiellapneumoniae, strain 204.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Klebsiella pneumoniae 204 3KP4 KpE7 IgG2a Indirect EIAAnti-Klebsiella pneumoniae 204 3KP4 KpE10 IgG2a Indirect EIAAnti-Klebsiella pneumoniae 204 3KP4 KpH11 IgG2a Indirect EIA32 INFECTIOUS DISEASE REAGENTS

9. Newcastle disease virus (NDV)Newcastle disease (ND) is a highly contagious andsometimes fatal illness affecting many domestic andwild bird species. The causal agent, Newcastle diseasevirus (NDV), is a negative-sense single-strandedRNA virus. NDV affects the respiratory, nervous,and digestive systems. Clinical signs are extremelyvariable depending on the strain of virus, speciesand age of bird, concurrent disease, and preexistingimmunity. NDV is so virulent that many birdsdie without showing any clinical signs.Transmission occurs by exposure to foecal andother excretions from infected birds, and throughcontact with contaminated food, water, equipmentand clothing. Virus-bearing material can be pickedup on shoes and clothing and carried from an infectedflock to a healthy one. Exposure of humansto infected birds (for example in poultry processingplants) can cause mild conjunctivitis and influenzalikesymptoms, but NDV otherwise poses no hazardto human health. MAbs are negative with parainfluenzatype 3 and avian influenza haemagglutinins.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Newcastle disease virus 3ND5 9F7 IgG1 EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 11F12 IgG2a EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 13H3 IgG2a EIA, WB, HITAnti-Newcastle disease virus 3ND5 9C6 IgG2a EIA, WB, HITAnti-Newcastle disease virus 3ND5 1C10 IgG2a EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 2H4 IgM EIA (capture), HITAnti-Newcastle disease virus 3ND5 8H2 IgG2a EIA (capture)New! Anti-Newcastle disease virus 3ND5 6H12 IgG2a EIA, IHC, IFINFECTIOUS DISEASE REAGENTS33

II Hepatitis A and BCurrently seven viruses, A, B, C, D, E, G and transfusiontransmitted virus (TTV) are recognized in thehepatitis virus alphabet. Hepatitis G virus and TTVprobably do not cause liver disease in humans. HepatitisA and E usually cause a self-limiting hepatitisfollowed by complete recovery but occasionallycause fulminant hepatic failure. Hepatitis B and Care major public health problems worldwide due totheir sequelae of chronic hepatitis, cirrhosis and primaryliver cancer. Chronic hepatitis C is a particularhealth issue for Western Europe already, accountingfor 40% of endstage cirrhosis and 30% of liver transplants.The contribution of hepatitis C to chronic liverdisease is predicted to rise in the future. Vaccinescan prevent hepatitis A and B. Interferon alpha is effectivetreatment in 25-30% of patients with chronichepatitis B or C. The prospects for treating chronichepatitis B have been improved by the introductionof reverse transcriptase inhibitors. Lamivudine is thefirst drug of this class to be licensed. The optimaluse of these new drugs is currently being studied.The success rate for treating chronic hepatitis C canbe raised to about 40% with combination therapy ofinterferon alpha and ribavirin. A large research effortto discover new antiviral agents against hepatitis C isalready giving the prospect of more effective therapiesin the next few years.Infections with the hepatitis B, C or D virus can alllead to chronic hepatitis. Serological and molecularmethods are essential for diagnosis and for differentiationbetween the different forms of chronic virushepatitis. In adults between 5% and 10% of all infectionswith the hepatitis B virus become chronic whilethe rate is as high as 80% with the hepatitis C virus.All forms of chronic hepatitis are frequently asymptomaticfor a long period of time. Complications areliver cirrhosis and hepatocellular carcinoma. Duringchronic hepatitis B infection in around 1% of the patientsper year the virus is eliminated spontaneouslywhile virus elimination occurs rarely in patients withchronic hepatitis C infection. In patients with chronichepatitis C infection over a period of 30 years around3% of the patients die due to chronic hepatitis C infection.Considerable evidence suggests that immunemechanisms are involved in the pathogenesis ofboth hepatitis B virus (HBV) and hepatitis C virus(HCV) infections. Both class I-restricted CD8+ T celland class II-restricted CD4+ T cell responses to viralantigens are important mechanisms that may be responsiblefor the hepatocyte damage in hepatitis Band C. CD4+ T cell proliferative responses to hepatitisB core antigen (HBcAg) in terms of stimulationindex are correlated with hepatitis activity. In termsof major histocompatibility complexes (MHC) classI-restricted, CD8+ cytotoxic T lymphocyte (CTL)response, antigenic peptides derived from HBcAg,hepatitis B surface antigen (HBsAg), and polymerasehave been demonstrated to be the targetsfor CTL recognition in hepatitis B patients. MultipleCTL epitopes within HBsAg, HBcAg and polymerasecan be detected by sensitizing target cells with syntheticpeptides. Likewise, multispecific, HCV-specificCTL responses can coexist with an extensive quasispeciesof viral variants. The mechanisms of viralpersistence in both hepatitis B and C remain to beclarified. The present situation of hepatitis infectionbeing widely spread in the world, the necessity forrapid diagnostics is self evident.1. Hepatitis AFor the detection of Hepatitis A virus in ELISA andWB we offer MAb MK01, which can also be used inantibody capture assay.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Hepatitis A Antigen 3HA18 MK01 IgG3 EIA, WB34 INFECTIOUS DISEASE REAGENTS

2. Hepatitis BFor the detection of Hepatitis B virus surface antigen(HBsAg) we offer a pair of MAbs, HB5 and HB6.These antibodies react equally well with subtypes adand ay and recognize “a” common epitope with 10 9M -1 binding constant. The detection limit in ELISA is0.5 ng/ml when HB5 is used for capture and HB6 forlabeling.MAbs HB11, HB12 and HB13 demonstrate a higheraffinity to HBsAg and may be used as capture forsandwich ELISA. We recommend to use non-infectiousrecombinant HBsAg (ayw subtype, Cat. #8HS7ay) and recombinant HBsAg (adw subtype,Cat. # 8HS7-2ad). These antigens are produced byyeast Saccharomyces cerevisiae, containing plasmidpCGA7. Both antigens could be used as a positivecontrol in immunoassay and as immunogen forantibody production. The purity of the antigens morethan 98% (SDS-PAGE). A supplementary product inthis line is a MAb match pair against Hepatitis B viruscore antigen (HBcAg). MAbs H3A4 and H6F5 recognize20 K and 40 K protein bands in WB and are suitedfor HBcAg detection in ELISA.Taking into consideration the growing requirementsfor the quantitative determination of HBsAg in EIAwe are focused on the production of new MAbs. Theexample of this could be the newest matched pairHs33-Hs41, which shows 50 pg/ml detection limit.(See Fig. 38)OD 4501,00,80,50,40,20,00,0 0,5 1,0 1,5 2,0 ng/mlFigure 38. Calibration curve for sandwich ELISA using the best recommendedpair Hs33 – Hs41.Coating: MAb Hs33, 5 μg/ml in 0.1 M carbonate buffer, detection: HRP-conjugatedMAb Hs41, 1/20000 in PBS-AT, substrate: TMB. Sensitivity 50 pg/ml.Ordering information:Product Cat. # Purity SourceHepatitis B Virus Surface Antigen, ayw subtype 8HS7ay >98% RecombinantHepatitis B Virus Surface Antigen, adw subtype 8HS7-2ad >98% RecombinantHepatitis B Virus Surface Antigen, adr subtype 8HS7-3 >98% RecombinantOrdering information:Product Cat. # MAb Isotype RemarksAnti-Hepatitis B Virus Surface Antigen 3HB12 Hs33 IgG2a EIA (capture)Anti-Hepatitis B Virus Surface Antigen 3HB12 Hs41 IgG1 EIA (detection)Anti-Hepatitis B Virus Surface Antigen 3HB12 HB5 IgG1 EIA (capture)Anti-Hepatitis B Virus Surface Antigen 3HB12 HB6 IgG1 EIA (detection)Anti-Hepatitis B Virus Surface Antigen 3HB12 HB11 IgG1 EIA (detection)Anti-Hepatitis B Virus Surface Antigen 3HB12 HB12 IgG1 EIA (capture)Anti-Hepatitis B Virus Surface Antigen 3HB12 HB13 IgG1 EIA (capture)Anti-Hepatitis B Virus Core Antigen 3HB17 H3A4 IgG2a EIA, WBAnti-Hepatitis B Virus Core Antigen 3HB17 H6F5 IgG2a EIA, WBAnti-Hepatitis B Virus “e” Antigen 3HBe24 AC5 IgG2b EIAAnti-Hepatitis B Virus “e” Antigen 3HBe24 HBe3 IgG2b EIA (detection)Anti-Hepatitis B Virus “e” Antigen 3HBe24 HBe5 IgG2b EIA (detection)Anti-Hepatitis B Virus “e” Antigen 3HBe24 HBe7 IgG1 EIA (capture)Anti-Hepatitis B Virus “e” Antigen 3HBe24 HBe8 IgG2b EIA (capture)New! Anti-Hepatitis B Virus Surface Antigen, subtype ay 3HBY3 3E7 IgG2a EIA, WBINFECTIOUS DISEASE REAGENTS35

III TORCHTORCH is an acronym for Toxoplasma, Rubella virus,Cytomegalovirus (CMV) and Herpes simplexvirus (HSV). Serologic tests for detection of antibodiesto these organisms are used for assessment ofcongenital infections. TORCH testing should reflect1. Toxoplasma gondiiPrenatal protozoal infection with Toxoplasma gondiiis associated with injury to the developing fetal nervoussystem. The severity of this condition is relatedto the stage of pregnancy during which the infectionoccurs; first trimester infections are associated witha greater degree of neurologic dysfunction. Clinicalfeatures include hydrocephalus, microcephaly, deafness,cerebral calcifications, seizures and psychomotorretardation. Signs of a systemic infection mayalso be present at birth, including fever, rash, andhepatosplenomegaly.We developed an optimal method of antigen preparationfrom T. gondii plasma membrane. We useddetection of IgM rather than IgG class antibodies.Infants with congenital infection due to the TORCHagents may have low birth weight, prema-turity, purpura,jaundice, anemia, microcephaly, cerebral calcification,choriorenitis, cataracts, microphtalmiaand pneumonia.tachyzoites from mice, strain RH, as the source forantigen and then performed extraction by a detergentmixture and high-speed centrifugation. On thefinal stage the main emphasis has been made on thedetergent removal from the preparation. It it possibleto use the antigen for the detection of specific antibodyin indirect ELISA and WB in the samples ofsera and plasma. The antigen may be used in captureformat, when labelled, to determine specificIgM. For the quality control of T. gondii antigen weuse MAb TP3, directed against p30. MAb TP3 reactswith tachyzoites and purified p30 in ELISA, Westernblotting and immunofluorescence.2. Rubella virusAn acute, usually benign, infectious disease causedby the Rubella virus is most often affecting childrenand nonimmune young adults, in which the virusenters the respiratory tract via droplet nuclei andspreads to the lymphatic system.MAb 1C11 recognizes Rubella virus core-protein inWestern blotting, whereas MAb 3D2 is evidently directedagainst E2 glycoprotein. MAb 3D2 is active inHIT. Recombinant Rubella virus antigen and MAb1C11 as a HRP-conjugate may be used for the determinationof Rubella-specific IgM in serum and plasmain capture assay.36 INFECTIOUS DISEASE REAGENTS

3. Cytomegalovirus (CMV)Infection with Cytomegalovirus (CMV) is characterizedby enlarged cells bearing intranuclear inclusions.Infection may be in almost any organ, but thesalivary glands are the most common site in children,as are the lungs in adults. Especially acutecharacter CMV acquires with immunocompromizedpatients and during transplantations.We offer monoclonal antibody against Cytomegalovirus,MAb B2, which reacts with 65K protein of cytomegalovirusin Western blotting. MAb B2 is suitablefor the CMV detection in ELISA, immunofluorescenceand Western blotting, as well as for EIA constructionfor specific IgM by capture method.4. Herpes simplex virus (HSV)MAb HSVA33 was obtained after mice immunizationby HSV 2, strain BH. According to Western blottingdata MAb HSVA33 reacts with 56-64 K protein bandin purified HSV preparation under reduced condi-tion and with a band 120-140 K in lyzates of the infectedVero cells under non-reduced condition, MAbHSVA33 is suited for HSV antigen detection in ELI-SA, Western blotting and immunofluorescence.New!New!New!Ordering information:Product Cat. # Purity SourceToxoplasma gondii Antigen 8T68 N/A RH Strain TachyzoitesRubella virus E1, mosaic 8RVE1 >80% RecombinantRubella virus E2 8RVE2 >80% RecombinantHerpes Simplex Virus, Type 2, gG2 8HVG2 >80% RecombinantOrdering information:Product Cat. # MAb Isotype RemarksAnti-Toxoplasma gondii 3Tx19 TP3 IgG2a EIA, WB, IF, P30 Antigen,Anti-Rubella Virus Structural Glycoprotein 3R23 Ru5 IgG2a EIA, WB, Structural E1 GlycoproteinAnti-Rubella Virus Structural Glycoprotein 3R23 Ru6 IgG2a EIA, WB, Structural E1 GlycoproteinAnti-Rubella Virus Structural Glycoprotein 3R23 3D2 IgG2a EIA, WB, Structural E2 GlycoproteinAnti-Rubella Virus Structural Glycoprotein 3R23 1C11 IgG1 EIA, WB, Structural Core proteinAnti-Cytomegalovirus 3CV14 B2 IgG2a EIA, WB, p65Anti-Herpes Simplex Virus, Type 2 3HS2 HSVA33 IgG1 EIA, WB, Glycoprotein DINFECTIOUS DISEASE REAGENTS37

IV Epstein Barr virusEpstein-Barr virus (EBV) is a ubiquitous virus associatedwith a variety of different diseases and disorders.There are manifestations of Epstein-Barrvirus-associated diseases or disorders within theliver, which involve a broad spectrum of histologicand clinical features, ranging from hepatitis throughlymphoproliferative disorders to lymphoma. An importantaspect of Epstein-Barr virus expression andinfection is the biology of the Epstein-Barr virus. Documentationof infection can be performed using serologyto detect the interaction of Epstein-Barr viruswith the immune system, and the detection of EBVproteins and use of molecular biologic techniquesto identify the presence of EBV RNA and DNA sequences.Of particular utility are in situ hybridization,Southern blot analysis, and polymerase chain reactionas diagnostic methods to identify specific RNAor DNA sequences. Epstein-Barr virus-associateddiseases and disorders including infectious mononucleosis,sporadic fatal infectious mononucleosis,X-linked proliferative disorder (Duncan’s disease),post-transplant lymphoproliferative disorders, lymphoma,and AIDS are described.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Epstein-Barr Virus 3EB20 1H1 IgG1 EIA, WB, IF, p120, p160 Capsid AntigenAnti-Epstein-Barr Virus 3EB20 4A8 IgG2b EIA, WB, IF, p120 Capsid Antigen38 INFECTIOUS DISEASE REAGENTS

V Sexually transmitted diseases (STD)More than 25 diseases are spread primarily throughsexual activity. The latest estimates indicate thatthere are 15 million new sexually transmitted diseasecases in the United States each year. Approximatelyone-fourth of these new infections are in teenagers.Nearly two-thirds of all STD cases occur in peopleyounger than 25 years.While some sexually transmitted diseases, such assyphilis, have been brought to all-time lows, others,like genital herpes, gonorrhea, and chlamydia, continueto resurge and spread through the population.Not including HIV, the most common sexually transmitteddiseases in the U.S. are chlamydia, gonorrhea,syphilis, genital herpes, human papillomavirus,hepatitis B, and trichomoniasis (Table 5.).Table 5. Most common sexually transmitted diseases in the U.S.Sexually transmitteddiseaseIncidence (estimated numberof new cases every year)Prevalence (estimated numberof people currently infected)Chlamydia 3 million 2 millionGonorrhea 650,000 n.a.Syphilis 70,000 n.a.Herpes 1 million 45 millionHuman papillomavirus (HPV) 5.5 million 20 millionHepatitis B 120,000 417,000Trichomoniasis 5 million n.a.1. Chlamydia trachomatisWe have available mouse monoclonal antibody,which reacts with major outer membrane protein(MOMP) of Chlamydia trachomatis.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Chlamydia trachomatis MOMP 3CT1 HT10 IgG2a EIA, WB2. Herpes simplex virusMAb HSVA33 was obtained after mice immunizationby HSV 2, strain BH. According to Western blottingdata MAb HSVA33 reacts with 56-64 K protein bandin purified HSV preparation under reduced conditionand with a band 120-140 K in lyzates of the infectedVero cells under non-reduced condition, MAbHSVA33 is suited for HSV antigen detection in ELI-SA, Western blotting and immunofluorescence.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Herpes Simplex Virus, Type 2 3HS2 HSVA33 IgG1 EIA, WBOrdering information:Product Cat. # Purity SourceNew! Herpes simplex virus type 2, gG2 8HVG2 >80% RecombinantINFECTIOUS DISEASE REAGENTS39

3. Treponema pallidum (Syphilis)Recent studies indicate that the STDs that causeulcerative diseases (herpes simplex virus type 2,Treponema pallidum and Haemophilus ducreyi) increasethe risk of HIV transmission by at least two- tofive-fold. Treponema pallidum is the causative agentof syphilis, which is the second most common causeof sexually transmitted ulcers in the United States.Unlike other STDs, syphilis has several defined clinicalstages. Syphilis is characterized by multiple clinicalstages and long periods of latent, asymptomaticinfection. The primary infection is localized, but organismsrapidly disseminate and cause manifestationsthroughout the body. Although effective therapieshave been available since the introductionof penicillin, syphilis remains an important globalhealth problem.We have developed a MAb which reacts with ultrasonicatedlysates of Treponema pallidum and Treponemareiter.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Treponema pallidum 3T11 Tr33 IgG1 EIA, WB4. Candida albicansCandidiasis, commonly called as yeast infection orthrush, is a fungal infection of any of the Candidaspecies, of which Candida albicans is probably themost common. C. albicans is normally found in themouth, gastrointestinal tract, vagina and the skin.In healthy individuals the presence of C. albicansis considered a normal part of the bowel flora. Usually,C. albicans is kept under control by the nativebacteria and by the body’s immune defenses. If the“healthy” bacterial environment is compromised theC. albicans cells get an opportunity to proliferate andtransform into a harmful fungus. Especially immunocompromisedpatients ranging from premature infantsto AIDS patients are in risk.Ordering information:Product Cat. # MAb Isotype RemarksAnti- Candida albicans 3CA4 CDA IgG1 EIA, WB40 INFECTIOUS DISEASE REAGENTS

5. Human papilloma virusHuman papillomavirus (HPV) belongs to Papillomaviruses,a diverse group of DNA-based viruses thatinfect the skin and mucous membranes of humansand a variety of animals. Over 100 different humanpapillomavirus (HPV) types have been identi-fied onthe basis of difference in the virus genome nucleotidesequences (e.g. type 1, 2, 3 etc.). Today genitalHPV infection is one of the most widespread sexuallytransmitted diseases. Approximately 20 millionpeople around the world are currently infected withHPV. At least 50 percent of sexually active men andwomen acquire genital HPV infection at some pointin their lives. By age 50, at least 80 percent of womenwill have acquired genital HPV infection. In accordancewith WHO information, genital HPV infectionwas a reason of over 99% of cervical cancer cases,i.e. about 1.4 million women were affected worldwideand 239 000 of them died each year.All HPVs are transmitted by skin-to-skin contact. Agroup of about 30-40 HPVs is typically transmittedthrough sexual contact and infect the anogenital region.Some sexually transmitted HPVs, types 6 and11, may cause genital warts. However, other HPVtypes which may infect the genitals do not cause anynoticeable signs of infection.Persistent infection with a subset of about 13 socalled“high-risk” sexually transmitted HPVs, includingtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59and 68 — different from the ones that cause warts —may lead to the development of cervical intraepithelialneoplasia (CIN), vulvar intraepithelial neoplasia(VIN), penile intraepithelial neoplasia (PIN), and/oranal intraepithelial neoplasia (AIN). These are precancerouslesions and can progress to invasive cancer.HPV infection is a necessary factor in the developmentof nearly all cases of cervical cancer.The HPV lifecycle begins from infection of epithelialtissues through micro-abrasions. At this point,the viral genome is transported to the nucleus andestablishes itself at a copy number between 10-200viral genomes per cell. A sophisticated transcriptionalcascade then occurs as the host keratinocyte beginsto divide and become increasingly differentiatedin the upper layers of the epithelium. The viraloncogenes, E6 and E7, are thought to modify the cellcycle so as to make them amiable to the amplificationof viral genome replication and consequent lategene expression. In the upper layers of the host epithelium,the late genes L1 and L2 are transcribed/translated and serve as structural proteins which encapsidatethe amplified viral genomes.HyTest offers a wide spectrum of monoclonal antibodiesspecific to oncoprotein E7 of “high-risk” HPVtypes 16 and 18 as well as to less oncogenic HPVtype 6 and 11. MAbs can be used in routine immunoassays(direct or indirect ELISA, sandwich immunodetectionsystems, Western blotting). Some MAbsdisplay high specificity to definite type of HPV whileothers can be used for determination of E7 proteinsfor all four types of viruses.INFECTIOUS DISEASE REAGENTS41

5.1. HPV, type 6, oncoprotein E7monoclonal antibodiesHost animal:Mice Balb/cCell line used for fusion: Sp2/0Immunogen:Recombinant oncoprotein E7, type 6, conjugated with hsp70Specificity: Human papilloma virus, type 6 (for cross reactivity information see Fig. 39)Purification method: Protein G affinity chromatographyPresentation:PBS with 0.1 % sodium azide5.1.1. ApplicationsMAb 706-C5 against E7 HPV type 6 can be used inroutine immunoassays like HPV enzyme immunoassayand HPV immunodetection in Western blotting.It should be noted that it is cross-reacting withHPV types 11, 16 and 18 (Fig. 39).OD4502,521,51,8252,2082,3001,46910,500,057706-HSP70 711-HSP70 E7, type16 E7, type18 HSP70Figure 39. Testing of MAb 706-C5 cross-reactivity in indirect ELISA.Coating: 1 mg/ml of each antigen; MAb 706-C5, 3 mg/mlFigure 40. Results of the MAb mapping.42 INFECTIOUS DISEASE REAGENTS

5.2. HPV, type 11, oncoprotein E7monoclonal antibodiesHost animal:Mice Balb/cCell line used for fusion: Sp2/0Immunogen:Recombinant oncoprotein E7, type 11, conjugated with hsp70Specificity: Human papilloma virus, type 11 (for cross reactivity information see table 7)Purification method: Protein G affinity chromatographyPresentation:PBS with 0.1 % sodium azideTable 6. MAbs main characteristics.MAbMAbisotypeHPV typeImmunogen (hsp70conjugated)E7 oncoproteinfragment711-13 IgG1 11 Whole molecule711-45 IgG2a 11 Whole molecule711-66 IgG1 11 Whole moleculeTable 7. MAbs specificity.Study of cross-reactivity with HPV types 11, 16 and 18Indirect ELISA, Coating: 5 mg/ml of each antigen;MAbs: 3 mg/mlMAbsCross reactivity withE7, type 11 E7, type 16 E7, type 18711-13 100% 100% 89%711-45 100% 38% 27%711-66 100% 12% 12%Figure 41. Results of the MAbs mapping.5.2.1. E7 HPV type 11 immunodetection in ELISAThe best combination of monoclonal antibodies forE7 HPV type 11 sandwich ELISA were selected fromseveral MAb combinations. The pairs were selectedon the basis of MAb mapping data (maximal spatialdeterminant separation), sensitivity, specificity andkinetics characteristics.OD4501,61,41,210,80,60,4Recommended pairs for sandwich ELISA are(capture - detection):711-45 – 711-13 (see Fig. 42.)711-66 – 711-130,200 2 4 6 8 10 12[E7-11], ng/mlFigure 42. Calibration curves for E7 HPV type 11 sandwich immunoassays:711-45 – 711-13.Coating: MAb 711-45, 5 µg/ml in 0.1 M, Carbonate buffer, pH 9.2Detection: HRP-conjugted MAb 711-13, 1/20 000Substrate: TMBINFECTIOUS DISEASE REAGENTS43

5.2.2. E7 HPV type 11 immunodetection inWestern blottingThe results of MAb E7 HPV type 11 immunodetectionin Western blotting after antigen SDS-gel electrophoresisand its transfer onto nitrocellulose membraneare presented on Fig. 43. Most of the testedMAbs recognize both monomer and dimer forms ofHPV type 11.Dimer formMonomer formFigure 43. Detection of E7 HPV type 11 (conjugated with hsp70) in Westernblotting by different monoclonal antibodies after 12% SDS-PAAGelectrophoresis.Strip 1: MAb 711-13, Strip 2: MAb 711-45, Strip 3: MAb 711-66,E7 HPV type 11 quantity: 15.0 μg/strip.5.3. HPV, type 16, oncoprotein E7monoclonal antibodiesHost animal:Mice Balb/cCell line used for fusion: Sp2/0Immunogen:Recombinant oncoprotein E7, type 16 (whole or fragments), conjugated with hsp70Specificity: Human papilloma virus, type 16 (for cross reactivity information see table 9)Purification method: Protein G affinity chromatographyPresentation:PBS with 0.1 % sodium azideTable 8. MAbs main characteristics.MAbMAbisotypeHPVtypeImmunogen (hsp70conjugated)E7 oncoproteinfragment716-281 IgG2b 16 Whole molecule716-325 IgG2a 16 Whole molecule716-332 IgG1 16 Whole molecule716-A6** IgG2a 16 Whole molecule716-B2** IgG1 16 Whole molecule716-C4** IgG1 16 Whole molecule716-D1 IgG2a 16 Whole molecule716-E11 IgG1 16 Whole molecule716-F10 IgG1 16 Whole moleculeST1-A8** IgG1 16 36-54 a.a.r. fragmentST1-A9** IgG1 16 36-54 a.a.r. fragmentST1-B7** IgG1 16 36-54 a.a.r. fragmentST1-B11** IgG1 16 36-54 a.a.r. fragmentCom1-D9** IgG1 16 CR1 fragment(common for all E7)Com2-D9** IgG1 16 CR2 fragment(common for all E7)Com2-C11** IgG1 16 CR2 fragment(common for all E7)Com2-D4** IgG1 16 CR2 fragment(common for all E7)Table 9. MAbs specificity.Study of cross-reactivity with HPV types 11, 16 and 18Indirect ELISA, Coating: 5 mg/ml of each antigen;MAbs: 3 mg/mlCross reactivity withMAbs E7, type 11 E7, type 16 E7, type18716-281 0% 100% 37%716-325 0% 100% 0%716-332 0% 100% 43%716-A6** 0% 100% 9%716-B2** 0% 100% 83%716-C4** 0% 100% 42%716-D1 0% 100% 114%716-E11 0% 100% 97%716-F10 0% 100% 100%Com1-D9** *) 0% 13% 0%Com2-D9** *) 0% 35% 100%Com2-C11** *) 0% 0% 21%** MAb available only on special request.*) 100% reaction with corresponding peptide CR1 and CR2** MAb available only on special request.44 INFECTIOUS DISEASE REAGENTS

5.3.1. E7 HPV type 16 immunodetection in ELISAThe best combination of monoclonal antibodies forE7 HPV type 16 sandwich ELISA were selected fromseveral MAb combinations. The pairs were selectedon the basis of MAb mapping data (maximal spatialdeterminant separation), sensitivity, specificity andkinetics characteristics.Figure 44. Results of the MAbs mapping.Recommended pairs for sandwich ELISA are (capture- detection):716-D1 – 716-332 (see Fig. 45)716-D1 – 716-E11716-D1 – 716-F10MAbs 716-D1, 716-281, 716-325 are equally suitablefor capture of both HPV type 16 and 18.5.3.2. E7 HPV type 16 immunodetection inWestern blottingThe results of MAb E7 HPV type 16 immunodetectionin Western blotting after antigen SDS-gel electrophoresisand its transfer onto nitrocellulose membraneare presented on Fig. 46. Most of tested MAbsrecognize both monomer and dimer (most commonin physiological media) forms of HPV type 16.OD4501,81,61,41,210,80,60,40,200 1 2 3 4 5[E7-16], ng/mlDimer form(46 kDa)Monomer form(23 kDa)Figure 45. Calibration curves for E7 HPV type 16 sandwich immunoassays:716-D1 - 716-332.Coating: MAb 716-D1, 5 μg/ml, 0.1 M Carbonate buffer, pH 9.2Detection: HRP-conjugated MAb 716-332, 1/50 000Substrate: TMBFigure 46. Detection of E7 HPV type 16 in Western blotting by differentmonoclonal antibodies after 15% SDS-PAAG electrophoresis.Strip 1: MAb 716-281, Strip 2: MAb 716-332, Strip 3: MAb 716-A6,Strip 4: MAb 716-B2, Strip 5: MAb 716-C4, Strip 6: MAb 716-D1,Strip 7: MAb 716-E11, Strip 8: MAb 716-F10;E7 HPV type 16 quantity: 15.0 μg/strip.INFECTIOUS DISEASE REAGENTS45

5.4. HPV, type 18, oncoprotein E7monoclonal antibodiesHost animal:Mice Balb/cCell line used for fusion: Sp2/0Immunogen:Recombinant oncoprotein E7, types 18 (whole or fragments), conjugated with hsp70Specificity: Human papilloma virus, type 18 (for cross reactivity information see table 11)Purification method: Protein G affinity chromatographyPresentation:PBS with 0.1 % sodium azideTable 10. MAbs main characteristics.MAbImmunogen (hsp70 conjugated)MAbisotypeE7 oncoproteinHPV typefragment718-15 IgG1 18 Whole molecule718-67 IgG2a 18 Whole molecule718-85** IgG2b 18 Whole molecule718-238 IgG2b 18 Whole molecule718-A7** IgG2a 18 Whole molecule718-B5** IgG1 18 Whole molecule718-B6** IgG1 18 Whole molecule718-F9** IgG2b 18 Whole molecule718-G9** IgG2a 18 Whole moleculeTable 11. MAbs specificity.Study of cross-reactivity with HPV types 11, 16 and 18Indirect ELISA, Coating: 5 mg/ml of each antigen;MAbs: 3 mg/mlMAbCross reactivity withE7, type 11 E7, type 16 E7, type 18718-15 7% 43% 100%718-67 0% 9% 100%718-85** 0% 32% 100%718-238 0% 8% 100%718-B6** 5% 43% 100%718-F9** 0% 21% 100%718-G9** 0% 13% 100%** MAb available only on special request.Figure 47. Results of the MAbs mapping.46 INFECTIOUS DISEASE REAGENTS

5.4.1. E7 HPV type 18 immunodetection in ELISAThe best combination of monoclonal antibodies forE7 HPV type 18 sandwich ELISA were selected fromseveral MAb combinations. The pairs were selectedon the basis of both MAb mapping data (maximalspatial determinant separation), sensitivity, specificityand kinetics characteristics.OD45032,521,510,5Recommended pairs for sandwich ELISA are (capture- detection):00 1 2 3 4 5[E7-18], ng/ml716-D1 – 718-238 (see Fig. 48)718-15 – 718-85718-67 – 718-238Figure 48. Calibration curves for E7 HPV type 18 sandwich immunoassays:716-D1 – 718-238Coating: MAb 716-D1 5 ug/ml, 0.1 M Carbonate buffer, pH 9.2Detection: HRP-conjugated MAb 716-238, 1/20 000Substrate: TMB5.4.2. E7 HPV type 18 immunodetection in WesternblottingThe results of MAb E7 HPV type 18 immunodetectionin Western blotting after antigen SDS-gel electrophoresisand its transfer onto nitrocellulose membraneare presented on Fig. 42. As can be seen, twotested MAbs, 716-281 and 716-D1, have ability torecognize E7 oncoprotein of both HPV type 16 and18 that makes them suitable as a capture antibodyfor determination of both types of HPV. MAbs 718-67,718-85 and 718-238 were found to be able to recognizeboth monomer and dimer forms of HPV type 18and can be recommended for an ELISA application.Dimer form(36-40 kDa)Monomer form(18-20 kDa)Figure 49. Detection of E7 HPV type 18 in Western blotting by different monoclonal antibodies after 15% SDS-PAAG electrophoresis:Strip 1: MAb 716-281, Strip 2: MAb 716-D1, Strip 3: MAb 718-15, Strip 4: MAb 718-67, Strip 5: MAb 718-85, Strip 6: MAb 718-238, Strip 7: MAb 718-A7;Strip 8: MAb 718-B5; Strip 9: MAb 718-B6; Strip 10: MAb 718-F9; Strip 11: MAb 718-G9; E7. HPV type 18 quantity: 15.0 μg/strip.INFECTIOUS DISEASE REAGENTS47

Ordering information:Product Cat. # MAb Isotype RemarksAnti-E7 HPV type 6 3HP6 706-C5 IgG3 EIA, WB, C/r with types 11, 16 and 18Anti-E7 HPV type 11 3HP11 711-13 IgG1 EIA, WBAnti-E7 HPV type 11 3HP11 711-45 IgG2a EIA, WBAnti-E7 HPV type 11 3HP11 711-66 IgG3 EIA, WBAnti-E7 HPV type 16 3HP16 716-281 IgG2b EIA (capture), WBAnti-E7 HPV type 16 3HP16 716-325 IgG2a EIA (detection), WBAnti-E7 HPV type 16 3HP16 716-332 IgG1 EIA (detection), WB, dimer and monomerAnti-E7 HPV type 16 3HP16 716-A6** IgG2a WB, dimer and monomerAnti-E7 HPV type 16 3HP16 716-B2** IgG1 WBAnti-E7 HPV type 16 3HP16 716-C4** IgG1 WB, dimer and monomerAnti-E7 HPV type 16 3HP16 716-D1 IgG2a EIA (capture), WB, dimer and monomerAnti-E7 HPV type 16 3HP16 716-F10 IgG1 EIA (detection), WB, dimer and monomerAnti-E7 HPV type 16 3HP16 716-E11 IgG1 EIA (detection), WBAnti-E7 HPV type 16 3HP16 ST1-A8** IgG1 WBAnti-E7 HPV type 16 3HP16 ST1-A9** IgG1 WBAnti-E7 HPV type 16 3HP16 ST1-B7** IgG1 WBAnti-E7 HPV type 16 3HP16 ST1-B11** IgG1 WBAnti-E7 HPV type 16 3HP16 Com1-D9** IgG1 WBAnti-E7 HPV type 16 3HP16 Com2-D9** IgG1 WBAnti-E7 HPV type 16 3HP16 Com2-C11** IgG1 WBAnti-E7 HPV type 16 3HP16 Com2-D4** IgG1 WBAnti-E7 HPV type 18 3HP18 718-15 IgG1 EIA (capture), WBAnti-E7 HPV type 18 3HP18 718-67 IgG2a EIA (capture), WBAnti-E7 HPV type 18 3HP18 718-85** IgG2b EIA (detection), WBAnti-E7 HPV type 18 3HP18 718-238 IgG2b EIA (detection), WBAnti-E7 HPV type 18 3HP18 718-A7** IgG2a WBAnti-E7 HPV type 18 3HP18 718-B5** IgG1 WBAnti-E7 HPV type 18 3HP18 718-B6** IgG1 WBAnti-E7 HPV type 18 3HP18 718-F9** IgG2b WBAnti-E7 HPV type 18 3HP18 718-G9** IgG2a WB** MAb available only on special request.5.5. Human papilloma virus (HPV) antigensOrdering information:Product Cat. # Purity SourceHuman Papillomavirus L1 protein (HPVL1), type 16 8HPV16 >90% RecombinantHuman Papillomavirus L1 protein (HPVL1), type 18 8HPV18 >90% Recombinant48 INFECTIOUS DISEASE REAGENTS

VI MalariaMalaria is a serious and sometimes fatal diseasecaused by a parasite. Forty-one percent of theworld’s population live in areas where malaria istransmitted (e.g., parts of Africa, Asia, the MiddleEast, Central and South America, Hispaniola, andOceania). It is a leading cause of death and diseaseworldwide, especially in developing countries. Mostdeaths occur in young children. The World HealthOrganization estimates that each year 300-500 millioncases of malaria occur and more than 1 millionpeople die of malaria.Patients with malaria typically are very sick with highfevers, shaking chills, and flu-like illness. Four kindsof malaria parasites can infect humans: Plasmodiumfalciparum, P. vivax, P. ovale, and P. malariae. Amongthese four malaria species, P. vivax and P. ovale candevelop dormant liver stages that can reactivate aftersymptomless intervals of up to 2 (P. vivax) to 4years (P. ovale). Infection with any of the malaria speciescan make a person feel very ill; infection with P.falciparum, if not promptly treated, may be fatal.The Plasmodium genus of protozoal parasites have alife cycle which is split between a vertebrate host andan insect vector. The Plasmodium species, with theexception of P. malariae (which may affect the higherprimates) are exclusively parasites of man. The mos-quito is always the vector and only female mosquitosare involved as the males do not feed on blood. Thebasic life cycle of the parasite is following:The gametocyte is the form that infects the mosquitoand reproduces itself, as if it were both sexes. Insidea mosquito gametocytes pass into the salivaryglands where they develop into a new form, the sporozoite.The sporozoite can be passed on to man when themosquito bites. Sporozoites are more antigenic, andbear circumsporozoite polypeptide on their plasmalemma.The sporozoite travels with the blood tothe liver where it enters the liver cells and some ofthe sporozoites divide (tachysporozoites) and becomethousands of merozoites.The merozoites are released to the blood where theyare taken up by the red blood corpuscles. Some ofthese turn into ring-formed trophozoites, which splitagain to form schizonts.Schizonts burst the red blood corpuscles at a certainmoment, releasing the merozoites. This releasecoincides with the violent rises in temperature duringthe attacks seen in malaria.Ordering information:Product Cat. # Mab Isotype RemarksAnti-Plasmodium vivax merozoite surface protein 1 (MSP1) 3PLV5 PVM-1 IgG1 EIA, WBAnti-Plasmodium vivax merozoite surface protein 1 (MSP1) 3PLV5 PVM-2 IgG2b EIA, WBAnti-Plasmodium vivax merozoite surface protein 1 (MSP1) 3PLV5 PVM-3 IgG2b EIA, WBAnti-Plasmodium vivax merozoite surface protein 1 (MSP1) 3PLV5 PVM-4 IgG2b EIA, WBAnti-Plasmodium vivax merozoite surface protein 1 (MSP1) 3PLV5 PVM-5 IgG1 EIA, WBAnti-Plasmodium vivax circumsporozoite protein (CSP) 3PLV2 PVC-1 IgG1 EIA, WBAnti-Plasmodium vivax circumsporozoite protein (CSP) 3PLV2 PVC-2 IgG1 EIA, WBAnti-Plasmodium falciparum S-antigen (Sag) 3PLF3 PFS-1 IgG1 EIA, WBAnti-Plasmodium falciparum S-antigen (Sag) 3PLF3 PFS-2 IgG1 EIA, WBAnti-Plasmodium falciparum S-antigen (Sag) 3PLF3 PFS-3 IgG1 EIA, WBAnti-Plasmodium falciparum merozoite surface protein 1 (MSP1) 3PLF1 PEM-1 IgG2a EIA, WBAnti-Plasmodium falciparum merozoite surface protein 1 (MSP1) 3PLF1 PEM-2 IgG1 EIA, WBAnti-Plasmodium falciparum merozoite surface protein 1 (MSP1) 3PLF1 PEM-3 IgG1 EIA, WBINFECTIOUS DISEASE REAGENTS49

VII TuberculosisTuberculosis (TB) is a disease of global concern.About one third of the world population is infectedwith Mycobacterium tuberculosis. Every year, approximately8 million people get the disease and2 million die of TB. The currently available vaccineagainst TB is the attenuated strain of Mycobacteriumbovis, Bacillus Calmette Guerin (BCG), which hasfailed to provide consistent protection in differentparts of the world. The commonly used diagnosticreagent for TB is the purified protein derivative (PPD)of M. tuberculosis, which is nonspecific because ofthe presence of antigens cross-reactive with BCGand environmental mycobacteria. Thus there is aneed to identify M. tuberculosis antigens as candidatesfor new protective vaccines and specific diagnosticreagents against TB.Unfortunately, due to the great heterogeneity of theantibody response in tuberculosis patients no commerciallyavailable serological test has so far shownuseful levels of sensitivity and specificity. It is thereforegenerally accepted that it will be necessary toinclude several antigens in a future serodiagnosticassay and the necessary improvements in sensitivitycan be achieved by combining the best antigensavailable.By using the techniques of recombinant DNA, syntheticpeptides, antigen-specific antibodies and Tcells etc., several major antigens of M. tuberculosishave been identified, e.g. heat shock protein (hsp)60, hsp70, Ag85, ESAT-6 and CFP10 etc. These antigenshave shown promise as new candidate vaccinesand/or diagnostic reagents against TB. In addition,recent comparisons of the genome sequenceof M. tuberculosis with BCG and other mycobacteriahave unraveled M. tuberculosis specific regionsand genes. Expression and immunological evaluationof these regions and genes can potentially identifymost of the antigens of M. tuberculosis importantfor developing new vaccines and specific diagnosticreagents against TB. Moreover, advances in identificationof proper adjuvant and delivery systems canpotentially overcome the problem of poor immunogenicity/short-livedimmunity associated with proteinand peptide based vaccines. HyTest has startedwork over the development of a new line of TB diagnosticpreparations. Our first products in this seriesare recombinant hsp70 and hsp65 and relatedmonoclonal antibodies. We are offering also otherM. tuberculosis related monoclonal antibodies, includingthe one specific to a number of antigens correspondingto dormancy regulon of M. tuberculosis.Ordering information:Product Cat. # Purity SourceM. tuberculosis 65 kDa Heat Shock Protein (HSP65) 8HSP65 >95% RecombinantM. tuberculosis 70 kDa Heat Shock Protein (HSP70) 8HSP70 >95% Recombinant50 INFECTIOUS DISEASE REAGENTS

Ordering information:Product Cat. # MAb Isotype RemarksAnti-M. tuberculosis CFP10 3CFP1 KFB16 IgG1 EIA (capture)Anti-M. tuberculosis CFP10 3CFP1 KFB34 IgG1 EIA (capture)Anti-M. tuberculosis CFP10 3CFP1 KFB42 IgG2b EIA (detection)Anti-M. tuberculosis ESAT6 3ES6 RSB14 IgG1 EIAAnti-M. tuberculosis ESAT6 3ES6 RSB34 IgG2a EIAAnti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) 3HSP70 TS489 IgG2b EIA (detection), WBAnti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) 3HSP70 TS29 IgG1 EIA (capture), WBAnti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) 3HSP70 TS31 IgG2a EIA (capture), WBAnti-M. tuberculosis, Rv1734 dormant protein from H37Rv strain 3RV17 17A9 IgG2b EIA, WBAnti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain 3RV20 31C11 IgG1 Indirect EIA, WBAnti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain 3RV20 31D7 IgG1 Indirect EIA, WBAnti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain 3RV20 31F11 IgG2a Indirect EIA, WBAnti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain 3RV20 31F12 IgG2b Indirect EIAAnti-M. tuberculosis, RV2623 Rec. Protein of Dormancy 3RV26 A10 IgG1 EIA, WBAnti-M. tuberculosis, RV2623 Rec. Protein of Dormancy 3RV26 E1 IgG3 EIA, WBAnti-M. tuberculosis, RV2623 Rec. Protein of Dormancy 3RV26 E3 IgG2a EIA, WBAnti-M. tuberculosis, RV2623 Rec. Protein of Dormancy 3RV26 E5 IgG2b EIA, WBAnti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain 3RV66 26A8 IgG2a Indirect EIA, WBAnti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain 3RV66 26A11 IgG2a Indirect EIA, WBAnti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain 3RV66 26C5 IgG2a Indirect EIA, WBAnti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain 3RV66 26F6 IgG2a Indirect EIA, WBAnti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain 3RV66 26H6 IgG2b Indirect EIA, WBAnti-M. tuberculosis, RV3134 Rec. Protein of Dormancy 3RV31 B10 IgG3 EIA, WBAnti-M. tuberculosis, RV3134 Rec. Protein of Dormancy 3RV31 D5 IgG1 EIA, WBAnti-M. tuberculosis, RV3134 Rec. Protein of Dormancy 3RV31 G1 IgG1 EIA, WBAnti-M. tuberculosis recombinant 16 kDa Ag 3MT16 HTM61 IgG1 EIA, WBAnti-M. tuberculosis recombinant 16 kDa Ag 3MT16 HTM62 IgG2a EIA, WBAnti-M. tuberculosis recombinant 16 kDa Ag 3MT16 HTM63 IgG2a EIA, WBAnti-M. tuberculosis recombinant 38 kDa Ag 3MT38 HTM81 IgG1 EIA, WBAnti-M. tuberculosis recombinant 38 kDa Ag 3MT38 HTM82 IgG2b EIA, WBAnti-M. tuberculosis recombinant 38 kDa Ag 3MT38 HTM83 IgG1 EIA, WBINFECTIOUS DISEASE REAGENTS51

VIII Foodborne pathogens1. Gastroenteritis viruses:rotavirus and adenovirusAcute gastroenteritis is among the most common illnessesof humankind, and its associated morbidityand mortality are greatest among those at the extremesof age, children and the elderly. In developingcountries, gastroenteritis is a common cause ofdeath in children < 5 years that can be linked to awide variety of pathogens. In developed countries,while deaths from diarrhoea are less common, muchillness leads to hospitalization or doctor visits. Muchof the gastroenteritis in children is caused by virusesbelonging to four distinct families rotaviruses, caliciviruses,astroviruses and adenoviruses. Other viruses,such as the toroviruses, picobirnaviruses, picornavirus(the Aichi virus), and enterovirus 22, mayplay a role as well. Viral gastroenteritis occurs withtwo epidemiologic patterns, diarrhoea that is endemicin children and outbreaks that affect peopleof all ages. Viral diarrhoea in children is caused bygroup A rotaviruses, enteric adenoviruses, astrovirusesand the caliciviruses; the illness affects all childrenworldwide in the first few years of life regardlessof their level of hygiene, quality of water, food or sanitation,or type of behaviour. For all but perhaps thecaliciviruses, these infections provide immunity fromsevere disease upon reinfection. Epidemic viral diarrhoeais caused primarily by the Norwalk-like virusgenus of the caliciviruses. These viruses affect peopleof all ages, are often transmitted by food or watercontaminated with foeces, and are therefore subjectto control by public health measures. The tremendousantigenic diversity of caliciviruses and shortlivedimmunity to infection permit repeated episodesthroughout life. In the past decade, the molecularcharacterization of many of these gastroenteritis viruseshas led to advances both in our understandingof the pathogens themselves and in developmentof a new generation of diagnostics. Application ofthese more sensitive methods to detect and characterizeindividual agents is just beginning, but has alreadyopened up new avenues to reassess their diseaseburden, examine their molecular epidemiology,and consider new directions for their prevention andcontrol through vaccination, improvements in foodand water quality and sanitary practices.Rotavirus (Reoviridae family) has 11 segments ofdouble-stranded RNA. The virion has four majorstructural proteins, which form a capsid with threelayers.The adenovirus (family Adenoviridae, genus Mastadenovirus)virion contains double-stranded DNAsurrounded by a capsid 70-90 nm in diameter, whichis comprised of 252 capsomers. The hexon capsomersshare a cross-reacting group common antigenon their surface.Rotavirus and adenovirus antigens can be detectedin stool specimens by EIA or electron microscopy.We developed a representative panel of murineMAbs against rotavirus. Purified bovine rotavirus,cell culture adapted strain MR was used as an immunogen.Further on we chose one MAb 3C10, being of thegreatest diagnostic potential. Western blot showedthat MAb 3C10 recognized p42-major inner-capsidantigen. MAb 3C10 demonstrates broad cross-reactivityand interact equally well with monkey rotavirus(SA-11), porcine rotavirus (PP) and with numeroushuman rotaviruses (field strains).Specific antibody titer is 10 -6 in ELISA. In combinationwith polyclonal bovine antibody to rotavirus ascapture and labeled MAb 3C10 form a perfect sandwichfor the quantitative virus determination in clinicalspecimens (see Fig. 50).We used a purified human adenovirus type 2 as animmunogen for the MAb panel generation. After selection,2 MAbs were chosen: 1E11 and 7C11, directedagainst genus-specific hexon antigen, which ispresent in all human serotypes, and being of greateraffinity to adenovirus. Traditional sandwich ELISAwith the use of a homologous pair of MAb 1E11/1E11,as well as a combination of MAb 7C11 (capture) and1E11 (detection) provides for the detection limit of viralantigens 1ng/ml. (See Fig. 51).52 INFECTIOUS DISEASE REAGENTS

The new MAb 8C4 having the same specificityshowed even better performance as capture antibodyin sandwich ELISA with MAb 1E11-HRP conjugate.We tested 100 faecal specimes and 100 nasal washingsusing in-house ELISA with MAb 7C11 and 1E11,compared to adenovirus Antigen EIA (Biotrin International)and a complete coinsidence of results wasobserved. FITC-labelled MAb 7C11 provided for theadenovirus detection with the sensitivity and specificity71.4 and 100% respectively at testing of 20 clinicalspecimens of nasal washings (see Fig. 52).The fermentation conditions for bulk production ofrota- and adenovirus antigens as positive controlshave been optimised for ELISA kits production.0,1 1 10 100 1000 10000Ab (poly) 5 µg/ml, 3C10 0.25 µg/ml 7C11 1E11Figure 50. ELISA calibration curve for Rotavirus antigen determination.Figure 51. ELISA calibration curve for adenovirus antigen determination.ABNegative Positive Negative PositiveFigure 52. Determination of Adenovirus antigen in biologic materials. Comparison in-house ELISA versus BIOTRIN ELISA.A: Nasal washing, B: Stool specimens.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Rotavirus Group Specific Antigen 3R10 3C10 IgG2a EIA, IHC, WB, P42 AntigenAnti-Adenovirus hexon 3AV13 7C11 IgG2a + IgM EIA, ID, IHCAnti-Adenovirus hexon 3AV13 1E11 IgG2a + IgM EIA (detection), ID, IHCAnti-Adenovirus hexon 3AV13 8C4 IgG2a EIA (capture), ID, IHCOrdering information:Product Cat. # Source RemarksAdenovirus, type 6 8AV13 Tonsil 99 EIAINFECTIOUS DISEASE REAGENTS53

2. SalmonellaGenus Salmonella (Enterobacteriaceae family) is animportant enteric pathogen that is responsible fora variety of diseases, including gastroenteritis andenteric fever. Human infection is primarily acquiredby ingestion of contaminated poultry or meat. Waterthat has been contaminated by faeces or urinefrom humans or animals may also serve as a vehicleof transmission. Ninety-five percent of the salmonellaserotypes isolated from humans belong to Subgroup1 containing serogroup A, B, C 1 , C 2 , D andE. For immunoserologic identification one shouldbe able to detect Vi capsular antigen of S. typhi andgroup-specific O-antigens. For production of monoclonalantibodies anti-Salmonella O-antigens the followingheat-inactivated (100 °C, 60 min) microor-ganisms were used for immunization: S. paratyphi A(group A), S. typhimurium (group B) and S. enteriditis(group D). Finally we produced a panel of MAbs, beingof a different cross-reactivity spectrum in respectto Salmonella spp. (see Table 12).Thus for serogroup typing of Salmonella spp. we offerthe following MAbs:5D12A: Group A, B, C 1, C 2, D, E 1, E 2(pan-Salmonella)10D9H: Group A, B, D, E 1, E 210B10G: Group A1E6: Group BTable 12. O-Antigen specificity of the anti-Salmonella MAbs.ImmunogenMAbImmunogenserogroupMouse IgGsubisotypeS. paratyphi AS. typhimuriumS. choleraesuisS. newportS. enteriditisS. anatumS. selandiaE. coli 055:B5E. coli K12Klebsiellapneum.Tentative LPSantigenicdeterminant10B10G5D12A1E610D9HAABDIgG1IgG1IgG1IgG1A B C1 C2 D E1 E2+ - - - - - -+ + + + + + +- + - - - - -+ + - - + ± ±------------0-2core0-40-12To prove cross-reactivity we determined bindingconstants (Ka, M -1 ) for MAbs with lipopolysaccharides(LPS) of A, B, D and E groups (Table 13).Table 13. Binding constants (Ka, M 1 ).MAb LPS A LPS B LPS D LPS E10B10G 2.0 x 10 710D9H 2.1 x 10 9 1.1 x 10 9 6.5 x 10 8 1.0 x 10 65D12A 1.0 x 10 9 1.0 x 10 7 1.0 x 10 7 1.0 x 10 10MAbs may be used for Salmonella spp. detection indot-blot, ELISA and immunofluorescence. For productionof monoclonal antibody anti-S. typhimuriumlipopolysaccharides of S. typhimurium have beenused as immunogen. Obtained MAb 1E6 is specificfor LPS of S. typhimurium. Later on it was shownthat Eu-labelled MAb 1E6 has a very broad crossreactivityrange recognizing E. coli 1243 and Listeriamonocytogenes (ATCC 7644) species as well, beinga potential positive control antibody for a variety ofassays.For production of anti-Salmonella virchow MAbs hybridomaclones deriving from hybridization of Sp2/0myeloma cells with spleen cells of Balb/c mice immunizedwith lyophilized Salmonella virchow, serotypeC has been used. MAbs SvB3 and SvE2 crossreactwith S. typhimurium. MAbs SvA1 and SvB3 bindLPS. MAb SvE2 binds a protein of approximately 38-40 kDa in Western blotting.54 INFECTIOUS DISEASE REAGENTS

Ordering information:Product Cat. # MAb Isotype RemarksAnti-Salmonella O-Antigens 3SO22 10B10G IgG1 A-group, C/r Data AvailableAnti-Salmonella O-Antigens 3SO22 5D12A IgG1 Pan Salmonella, C/r Data AvailableAnti-Salmonella O-Antigens 3SO22 10D9H IgG1 A, B and D-group, C/r Data AvailableNew! Anti-Salmonella O-Antigens 3SO22 4G7C IgG1 D-group, C/r Data AvailableAnti-Salmonella typhimurium 3S9 1E6 IgG1 LPS of Salmonella typhimuriumAnti-Salmonella virchow 3SV4 SvA1 IgG2b LPSAnti-Salmonella virchow 3SV4 SvB3 IgG2a LPS, C/r with S. typhimuriumAnti-Salmonella virchow 3SV4 SvE2 IgG2a 38-40 kDa in WB, C/r with S. typhimurium3. Listeria monocytogenesThe genus Listeria comprises six species: L. monocytogenes,L. innocua, L. welshimeri, L. seeligeri, L.ivanovii and L. grayi. Listeria monocytogenes, themost commonly isolated pathogenic member, is associatedwith a wide spectrum of human and animaldiseases. In the smear from the original tissue, L.monocytogenes may appear as gram-positive coccobacillithat may be confused with Streptococcusagalactiae (group B), enterococci, or Corynebacteriumspp. Listeria is differentiated from streptococciby a positive catalase test.L. monocytogenes is the only species of the genusListeria that has been clearly documented as apathogen for humans. The forms of disease causedby this organism are myriad and age-related. Themost common clinical manifestations are meningitisand septicemia. An electrophoresis picture of L.monocytogenes can be seen in Fig. 53.946743Figure 53. 12% SDS-PAGE (Laemmli, silver stain)Lane 1: Positive control (KPL) without β-mercaptoethanolLane 2: L. monocytogenes cell suspension without β-mercatoethanolLane 3: L. monocytogenes OM fraction without β-mercaptoethanolLane 4: L. monocytogenes OM fraction with β-mercaptoethanolLane 5: L. monocytogenes cell suspension with β-mercaptoethanolLane 6: Positive control (KPL) with β-mercaptoethanolLane 7: Molecular mass standards from top to bottom:94K, 67K, 43K, 30K, 20K, 14K3020141 2 3 4 5 6 7The antibodies are working in indirect ELISA andthey are specific and show high immunoreactivity(end-point dilution is about 1 – 3 ng/ml) against outermembrane (OM) fraction of L. monocytogenes. Thebest calibration curves were obtained with antibodiesLZG7 and LZF7 (Fig. 54).The best pair for sandwich ELISA was selectedamong the antibodies by testing all possible pairs.The best pair according to the results was LZH1 –LZF7 (capture – detection) (Fig. 55, 56 and 57).INFECTIOUS DISEASE REAGENTS55

0,80,25++++++0,70,60,50,40,30,2OD 4500,200,150,10LZH1 - LZF7-POD 1/10 000LZH1 - LZF7-POD 1/20 000LZH1 - LZF7-POD 1/50 000LZH1 - LZF7-POD 1/100 000+++++++++++010 1 0,1 0,01 0,001 0,0001MAbs, µg/ml+LZF7 LZG5 LZG7 LZH1 LZA20,10,050,000,1 1 10OM fraction L. monocytogenes, ng/mlFigure 54. Calibration curves of anti-L. monocytogenes antibodies inindirect ELISA.Figure 55. Calibration curves for L. monocytogenes detection at differentconjugate dilutions using pair LZH1 – LZF7.0,61,61,41,22,5 µg/ml5,0 µg/ml10,0 µg/ml0,50,42,5 µg/ml5,0 µg/ml10,0 µg/mlOD 4501,00,80,60,40,2OD 4500,30,20,10,00,1 1 10 100OM fraction L. monocytogenes, ng/ml0,01 10 100 1000 10 000Cell/mlFigure 56. Calibration curves for L. monocytogenes detection with different MAb coating concentrations using pair LZH1 – LZF7.1,4OD 4501,61,41,21,00,80,60,40,21/20001/50001/10000OD 4501,21,00,80,60,40,21/20001/50001/100000,00,00,1 1 10 100OM fraction L. monocytogenes, ng/ml1 10 100 1000 10000Cell/mlFigure 57. Calibration curves for L. monocytogenes detection at different conjugate dilutions and 2.5 µg/ml coating concentration using pair LZH1– LZF7.Using pair LZH1 – LZF7 the detection limit 0.3 ng/ml for L. monocytogenes OM fraction and 100 cellsper well for L. monocytogenes cell suspension havebeen achieved.All antibodies recognize L. monocytogenes wholecell lysate and OM fraction in Western blotting. Theantibodies detect 23 kDa protein band (Fig. 58).56 INFECTIOUS DISEASE REAGENTS

L. monocytogenes cell suspension L. monocytogenes OM fraction946743302014Lanes 1 and 16: Molecular weight markersLanes 2 and 9: MAb LZF7Lanes 3 and 10: MAb LZG5Lanes 4 and 11: MAb LZG7Lanes 5 and 12: MAb LZH1Lanes 6 and 13: MAb LZA2Lanes 7 and 14: Anti-Legionella pneumophilaantibodiesLanes 8 and 15: Control (anti-mouse IgG,hrp-conjugated)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Figure 58. Western blotting of L. monocytogenes with different antibodies.All antibodies recognize specifically L. monocytogenesOM fraction, L. monocytogenes cell suspensionand Listeria-genus specific positive control indot-blot EIA (Fig. 59).LZF7, hrp-conjugate, 1/10000123Line 1: L. monocytogenes OM fraction in concentrations50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.3 μg/ml, 3.1μg/ml 2 μl/point, i.e. 100 ng, 50 ng, 25 ng, 12.5 ng,6.3 ng per dot.LZG7, hrp-conjugate, 1/1000123Line 2: L. monocytogenes cell suspension from initialstock with step 1/10 2 μl/point, i.e. 2 x 10 6 cells, 2x 10 5 cells, 2 x 10 4 cells etc. per dot.123LZH1, hrp-conjugate, 1/3000Figure 59. Dot-blot EIA with conjugated anti-L. monocytogenes antibodies.Line 3: Listeria-genus specific positive control wasreconstituted according to manufacture’s instructionand spotted from initial stock with 10-fold serial dilutions2 μl/point. (KPL, Cat # 50-90-90).INFECTIOUS DISEASE REAGENTS57

The detection limits for L. monocytogenes OM fractionswere 6 - 12 ng for conjugated LZF7 (diluted1/10000), 25 ng for conjugated LZG7 (diluted 1/1000)and 6 ng for conjugated LZH1 (diluted 1/3000). UsingL. monocytogenes cells the detection limit for allconjugates was 2000 cells/dot (the fourth dilutiondot). Using Listeria-genus specific positive controlfrom KPL only the initial concentration was detectedwith conjugated LZF7. With conjugated LZG7 thesecond dilution (1/10) was detected and conjugatedLZH1 detected also the third dilution (1/1000).Cross-reactivity:Anti-L. monocytogenes antibodies have been testednegative for cross-reactivity (ELISA) with the followingspecies: E. coli, Salmonella, Y. pestis, F. tularensis,L. pneumophila and B. abortus.MAbs LZH1, LZF7 and LZG5 have been tested forcross-reactivity with L. innocua and L. ivanovii. Theantibodies are cross-reacting with these species.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Listeria monocytogenes 3L1 LZF7 IgG2a EIA (detection), WBAnti-Listeria monocytogenes 3L1 LZG5 IgG2a EIA, WBAnti-Listeria monocytogenes 3L1 LZG7 IgG2a EIA, WBAnti-Listeria monocytogenes 3L1 LZH1 IgG1 EIA (capture), WBAnti-Listeria monocytogenes 3L1 LZA2 IgM EIA, WB4. Legionella pneumophilaLegionella pneumophila is the bacterium associatedwith Legionnaires’ disease and Pontiac fever. Respiratorytransmission of this organism can lead toinfection, which is usually characterized by a gradualonset of flu-like symptoms. Patients may experiencefever, chills, and a dry cough as part of theearly symptoms. Patients can develop severe pneumonia,which is not responsive to penicillins or aminoglycosides.Legionnaires’ disease also has thepotential to spread into other organ-systems of thebody such as the gastrointestinal tract and the centralnervous system. Accordingly, patients with advancedinfections may experience diarrhea, nausea,disorientation, and confusion. Pontiac fever is alsocaused by L. pneumophila but does not produce theseverity of the symptoms found in Legionnaires’ disease.The flu-like symptoms are still seen in Pontiacfever patients but pneumonia does not develop andinfection does not spread beyond the lungs. Most L.pneumophila infections are easily treated with erythromycin.MAbs react with LPS of Legionella pneumophila serogroup1 and could be used in sandwich ELISA(2F10 for capture, 5F4 for detection), WB and dotblotas well as in lateral flow devices with MAb 5F4-colloidal gold conjugate. MAbs do not cross-reactwith Pseudomonas fluorescens, Pseudomonascepacia, Pseudomonas aerugenose, Bordetella pertusis,Leptospira interrogens (Australia), Leptospirainterrognes (Pomona), Leptospira interrognes (Icterogemorrhagia),Toxoplasma gondii, Hemophilusinfluenza (type B), Brucella abortus, Bacillus anthracis,Yersinia pseudotuberculosis, Salmonella typhi,Escherichia coli K88, Francisella tularensis.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Legionella pneumophila LPS 3L15 2F10 IgG3 EIA (capture)Anti-Legionella pneumophila LPS 3L15 5F4 IgG3 EIA (detection)58 INFECTIOUS DISEASE REAGENTS

5. Campylobacter jejuniCampylobacters are bacteria that are a major causeof diarrheal illness in humans and are generally regardedas the most common bacterial cause of gastroenteritisworldwide. In developed and developingcountries, they cause more cases of diarrheathan, for example, foodborne Salmonella bacteria.In developing countries, Campylobacter infectionsin children under the age of two years are especiallyfrequent, sometimes resulting in death. In almostall developed countries, the incidence of humancampylobacter infections has been steadily increasingfor several years. The reasons for this are unknown.We used Campylobacter jejuni intact cell suspensionas immunogen and succeeded in developing3 monoclonal antibodies E10, H3 and H2, whichrecognize C. jejuni in indirect ELISA and show nocross-reactivity against Salmonella spp and E. coli.MAbs H3, H2 and E10 recognize 38 kDa C. jejuni antigenin Western blotting. MAbs H2 and H3 could beused to capture C. jejuni in double antibody sandwichELISA using MAb E10 as a conjugate in thepresence of nonidet NP40.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Campylobacter jejuni 3CJ2 E10 IgG2b EIA (detection), WBAnti-Campylobacter jejuni 3CJ2 H3 IgG2b EIA (capture), WBAnti-Campylobacter jejuni 3CJ2 H2 IgG1 EIA (capture), WB6. AstrovirusAstroviruses are small, nonenveloped and singlestrandedRNA viruses. In humans they are transmittedprimarily through the foecal-oral route (includingfood- and waterborne transmission) and occasionallyby aerosols. Numerous studies indicate that humanastrovirus serotype 1 is the most predominantserotype worldwide. It has been postulated that theincidence of astrovirus-associated gastroenteritishas been underestimated and that astrovirus infectionsmay be one of the common infections of childhood.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Astrovirus 3AS6 ASV IgG3 EIA, WBINFECTIOUS DISEASE REAGENTS59

IX Microbial and plant toxins antibodies1. Antibodies for the detection ofStaphylococcus aureusenterotoxinsStaphylococcal enterotoxins (S.enterotoxins) representa group of proteins, which are secreted byStaphylococcus aureus and cause food poisoning.Their molecular masses range between 27-30 kDa.At present, seven S. enterotoxins are known, namelyA, B, C1, C2, C3, D and E. Their amino acid sequenceshave been determined and it was shown that allare single-chain polypeptides containing one disulfidebond formed by two half-cystines located in themiddle of the polypeptide chain, which form the socalledcysteine loop. S. enterotoxins are known to bemost potent T cell mitogens. T cell activation accompaniedby induction of interleukin 2 and interferon isconditioned by high–affinity interaction of S. enterotoxinswith class II major histocompatibility complex(MHC) molecules and subsequent presentation ofthe complex formed to a variable region of the subunitof the T cell receptor.1.1. Antibodies for the detection ofStaphylococcus aureus enterotoxinSpecificity of MAbs was established in indirectELISA using standard staphylococcal enterotoxinsA, B, C1, C2, D, E from Serva (Germany) at appropriatedilution of ascitic fluids.Percentage of cross-reactivity is shown in Table 14.At 0.1 nM MAb S5 inhibits enterotoxins inducedstimulation of T-lymphocytes in vitro.Table 14. Percentage of cross-reactivity with enterotoxins of S. aureusMAb A B C1 C2 D ES1 21 0 0 0 100 16S2 8 0 0 0 100 0S5 2 100 7 76 29 01.2. Antibodies for the detection ofStaphylococcus aureus enterotoxin AWe used S. aureus enterotoxin A (SEA) as animmunogen to produce the MAbs C4, E8, E11, F12,G10, H5 and H10. All MAbs are working in Westernblotting and EIA applications. In Table 15 are the recommendedpairs for sandwich immunoassay andthe detection limits with these pairs.Table 15. Recommended pairs for sandwich immunoassayand the detection limits with these pairs.Capture Detection MAb Detection limit ng/mlMAbH5 C4 < 0.1H10 C4 0.1E8 C4 0.1G10 C4 0.1F12 H5 0.1-0.2C4 H5 0.1-0.2E11 C4 0.1-0.2C4 E8 0.1-0.260 INFECTIOUS DISEASE REAGENTS

1.3. Antibodies for the detection ofStaphylococcus aureus enterotoxin B3,5MAbs S222 and S643 recognize two differentepitopes of SEB molecule. There is no cross-reactivitywith S. aureus enterotoxins A, C1, C2, C3, Dand E.OD 4503,02,52,01,51,0meanFig. 53 shows the calibration curve for SEB antigenquantification using double antibody sandwichELISA with MAbs S222 and S643. The detection limitfor the immunoassay is 50 pg/ml.0,50,00 2 4 6 8 10 12 14ng/mlFigure 60. Calibration curve for double antibody sandwich with MAbS222 used for capture and MAb S643 as an HRP conjugate for SEBantigen detection.1.4. Antibodies for the detection ofStaphylococcus aureus enterotoxin GWe used S. aureus enterotoxin G (SEG) as animmunogen to produce the MAbs SEG-59 and SEG-16. Both MAbs are working in Western blotting andEIA applications. Recommended pair for sandwichimmunoassay is (capture - detection):SEG-59 - SEG-16The detection limit for the immunoassay is 0.1 ng/ml.1.5. Antibodies for the detection ofStaphylococcus aureus enterotoxin IWe used S. aureus enterotoxin I (SEI) as an immunogento produce the MAbs SEI-17A and SEI-68. BothMAbs are working in Western blotting and EIA applications.Recommended pair for sandwich immunoassayis (capture - detection):SEI-17A - SEI-68The detection limit for the immunoassay is 0.1 ng/ml.Ordering information:Product Cat.# MAb Isotype ApplicationAnti-S. aureus enterotoxin 2S3 S1 Ig2a A, D and E enterotoxin, EIAAnti-S. aureus enterotoxin 2S3 S2 Ig2a A and D enterotoxin, EIAAnti-S. aureus enterotoxin 2S3 S5 Ig2a A, B, C1, C2 and D enterotocin, EIAAnti-S. aureus enterotoxin A 2S7 C4 IgG1 EIA (capture, detection), WBAnti-S. aureus enterotoxin A 2S7 E8 IgG1 EIA (capture, detection), WBAnti-S. aureus enterotoxin A 2S7 E11 IgG2a EIA (capture), WBAnti-S. aureus enterotoxin A 2S7 F12 IgG1 EIA (capture), WBAnti-S. aureus enterotoxin A 2S7 G10 IgG1 EIA (capture), WBAnti-S. aureus enterotoxin A 2S7 H5 IgG1 EIA (capture, detection), WBAnti-S. aureus enterotoxin A 2S7 H10 IgG2b EIA (capture), WBAnti-S. aureus enterotoxin B 2S4 S222 IgG1 EIA (capture), N/cr with A, C, D and E enterotoxinAnti-S. aureus enterotoxin B 2S4 S643 IgG1 EIA (detection), N/cr with A, C, D and E enterotoxinAnti-S. aureus enterotoxin G 2S6 SEG-59 IgG1 EIA (capture), WBAnti-S. aureus enterotoxin G 2S6 SEG-16 IgG2a EIA (detection), WBAnti-S. aureus enterotoxin I 2S5 SEI-17A IgG2a EIA (capture), WBAnti-S. aureus enterotoxin I 2S5 SEI-68 IgG1 EIA (detection), WBINFECTIOUS DISEASE REAGENTS61

2. Antibodies for the detection ofCholera toxin (CT)Cholera is an acute intestinal infection caused by ingestionof food or water contaminated with the bacteriumVibrio cholerae. The bacterium may also livein the environment in brackish rivers and coastalwaters. In an epidemic, the source of the contaminationis usually the foeces of an infected person.The disease can spread rapidly in areas with inadequatetreatment of sewage and drinking water.The infection is often mild or without symptoms, butsometimes it can be severe (approximately one in20 cases). It has a short incubation period, from lessthan one day to five days, and produces an enterotoxinthat in severe cases causes painless and waterydiarrhoea that can quickly lead to severe dehydrationand death even within hours if treatmentis not promptly given. Vomiting and leg cramps canalso occur.Cholera toxin and the closely related E. coli heatlabileenterotoxin are 85 kDa hexameric assembliesconsisting of a single 240 residue A subunit and fiveidentical 103 residue B subunits. The B subunits assembleinto a regular pentamer containing a centralpore, through which the C-terminal tail of the A subunitextends to hold the AB 5holotoxin together.All MAbs react with B subunit of cholera toxin. MAb3D11 reacts also with E.coli heat-labile enterotoxin.Specific antibody titer in indirect ELISA is 10 -6 .Ordering information:Product Cat. # MAb Isotype RemarksAnti-Cholera toxin 2C4 3D11 IgG1 EIA, B-subunit of CTAnti-Cholera toxin 2C4 B8 IgG1 EIA (capture), B-subunit of CTAnti-Cholera toxin 2C4 E6 IgG2a EIA (capture), B-subunit of CTAnti-Cholera toxin 2C4 E10 IgG2a EIA (detection), WB, B-subunit of CTAnti-Cholera toxin 2C4 G9 IgG1 EIA (detection), B-subunit of CT3. Antibodies for the detection ofEscherichia coli heat-labileenterotoxinWe have used recombinant A- and B-chains of E.coli heat-labile enterotoxin as immunogens for MAbdevelopment. 19 clones were produced and testedfor reactivity in ELISA and WB against LTA, LTB andCholera toxin.Ordering information:Product Cat. # MAb Isotype RemarksAnti-E. coli heat-labile enterotoxin A-chain 2LTA2 AB3 IgG2b EIA (capture), C/r with Cholera toxinAnti-E. coli heat-labile enterotoxin A-chain 2LTA2 AE4 IgG2b EIA (detection), No C/r with Cholera toxinAnti-E. coli heat-labile enterotoxin A-chain 2LTA2 AE7 IgG2b WB, C/r with Cholera toxinAnti-E. coli heat-labile enterotoxin B-chain 2LTB2 BB2 IgG2b EIA, WBAnti-E. coli heat-labile enterotoxin B-chain 2LTB2 BB12 IgG2b EIA, WBAnti-E. coli heat-labile enterotoxin B-chain 2LTB2 BG12 IgG1 EIA, WB62 INFECTIOUS DISEASE REAGENTS

4. Antibodies for the detection ofClostridium botulinum toxoidsClostridium botulinum is an anaerobic, Gram-positive,spore-forming rod that produces a potent neurotoxin.The spores are heat-resistant and can survivein foods that are incorrectly or minimally processed.Foodborne disease caused by C. botulinum is referredto as botulism. Seven types (A, B, C, D, E,F and G) of botulism are recognized, based on theantigenic specificity of the toxin produced by eachstrain. Only types A, B, E and F cause illness inhumans. Types C and D cause most cases of botulismin animals. Toxin is called a neurotoxin becauseit affects the nervous system. Symptoms appear 12to 48 hours after eating a food, which contains thetoxin. The symptoms include double vision, droopyeyelids, trouble with speaking and swallowing anddifficulty with breathing. Without treatment deathmay result from suffocation because the nerves canno longer stimulate breathing. We have used formaldehydeinactivated C. botulinum toxins A, B, D andE to generate monoclonal antibodies. New MAbs2A33 and 24A29 were produced against two syntheticpeptides, a.a.r. 869-887 and a.a.r. 1177-1195,of Clostridium botulinum toxin A heavy chain. Thesenew MAbs recognize natural and non-inactivatedtoxin A.Ordering information:Product Cat. # MAb Isotype RemarksAnti-C. botulinum toxin A 3Cb19 2A33 IgGM a.a.r. 869-887, C. botulinum toxin A heavy chainAnti-C. botulinum toxin A 3Cb19 24A29 IgGM a.a.r. 1177-1195, C. botulinum toxin A heavy chainAnti-C. botulinum A Toxoid 3Cb20 KBA211 IgG1 EIA (capture), C/r data AvailableAnti-C. botulinum A Toxoid 3Cb20 KBA468 IgG2a EIA (detection), C/r data AvailableAnti-C. botulinum B Toxoid 3Cb21 KBB18 IgG1 EIA (detection), C/r data AvailableAnti-C. botulinum B Toxoid 3Cb21 KBB27 IgG1 EIA (capture), C/r data AvailableAnti-C. botulinum B Toxoid 3Cb21 KBB36 IgG1 EIA (capture), C/r data AvailableAnti-C. botulinum D Toxoid 3Cb23 KB21 IgG1 EIA, IF, C/r data availableAnti-C. botulinum E Toxoid 3Cb24 KBE169 IgG1 EIA (capture), WB, C/r data AvailableAnti-C. botulinum E Toxoid 3Cb24 KBE42 IgG1 EIA (detection), WB, C/r data AvailableOrdering information:Product Cat. # Host animal RemarksPolyclonal Anti-C. botulinum D Toxoid 3Cb23/2 goat EIA5. Antibodies for the detection ofDiphtheriaDiphtheria is an acute disease caused by a bacteriumCorynebacterium diphtheriae, which produces atoxin that is carried in the bloodstream. Diphtheria ispassed from person to person by droplet transmission,usually by breathing in diphtheria bacteria afteran infected person has coughed, sneezed or evenlaughed. Diphtheria is a very contagious and potentiallylife-threatening infection. It usually attacksthe throat and nose causing breathing problems butin more serious cases it can attack the nerves andalso cause heart failure, paralysis and even death.Because of widespread immunization, diphtheria isvery rare. However, some people are not adequatelyvaccinated, and cases still occur. People carryingdiphtheria germs are contagious for up to 4 weekswithout antibiotic therapy, even if they themselves donot develop symptoms.MAbs 1H2 and 3B6 react with different determinantsof Diphtheria toxin and anatoxin. They do not reactwith free A or B subunits of Diphtheria toxin. Antibodiescan be used for detection of Diphtheria toxinby different immunochemical technique. We recommendto use MAb 3B6 for capture and MAb 1H2 forlabeling.INFECTIOUS DISEASE REAGENTS63

MAb 8A4 reacts with free A subunit of Diphtheria toxin.It does not react with the whole Diphtheria toxinand free B subunit of Diphtheria toxin. MAb 7F2 reactwith the epitope exposed on free A subunit andon whole Diphtheria toxin molecule. It does not reactwith free B subunit of Diphtheria toxin. Antibodiescan be used for detection of Diphtheria toxin Asubunit by different immunochemical technique. Werecommend to use MAb 8A4 for capture and MAb7F2 for labeling. MAb DiB4 reacts with free A subunitand with whole Diphtheria toxin and does not reactwith free B subunit (WB results). On the contrary,MAb DiD1 reacts in WB with the epitope of free Bsubunit and on whole Diphtheria toxin molecule anddo not react with free A subunit. Antibodies can beused for detection of Diphtheria toxin by different immunochemicaltechnique. We recommend using thefollowing pairs (capture- detection):DiH1 - DiB4DiH1 - DiD1Ordering information:Product Cat. # MAb Isotype RemarksAnti-Diphtheria Toxin 2DT13 1H2 IgG1 EIA (detection), N/cr with Free A- and B-subunitsAnti-Diphtheria Toxin 2DT13 3B6 IgG1 EIA (capture), N/cr with Free A- and B-subunitsAnti-Diphtheria Toxin 2DT13 DiD1 IgG2b EIA, WBAnti-Diphtheria Toxin 2DT13 DiB4 IgG2b EIA, WBAnti-Diphtheria Toxin 2DT13 DiH1 IgG1 EIAAnti-Diphtheria Toxin, A-subunit 2DT14 8A4 IgG2a N/cr with Whole Toxin and Free B-subunit, EIA (capture)Anti-Diphtheria Toxin, A-subunit 2DT14 7F2 IgG1 N/cr with Free B-subunit, EIA (detection), Whole Toxin6. Antibodies for the detection ofRicin RCA 60 from Ricinus communisRicin toxin is a protein derived from the beans of thecastor plant (Ricinus communis). The naturally occurringtoxin is fairly easily removed from the beanpulp waste, which remains after castor oil extraction.The toxin is stable in powder and aerosolizedform, can be disseminated as an aerosol, an injection,or as a food or water contaminant, and has noknown treatment or vaccine. Ricin poisoning cannotbe spread from person to person through casualcontact. Symptoms of ricin poisoning depend on thedose received and route of exposure and they usuallybegin within six hours of ingestion exposure andwithin eight hours of inhalation exposure.MAbs CP23, CP37, CP75 and RA999 recognize theA-chain of RCA 60 . MAb RB999 recognizes the B-chain of RCA 60 and also the RCA 120 . The antibodiesare working in ELISA. All MAbs (except CP23) areworking also in Western blotting. The matched pairRA999 (capture) and RB999 (detection) has a detectionlimit of 100 pg/ml for RCA 60 and shows no crossreactivityfor RCA 120 .Ordering information:Product Cat. # MAb Isotype RemarksAnti-Ricin, RCA 60 from Ricinus communis 2R1 CP23 IgM A-chain, EIAAnti-Ricin, RCA 60 from Ricinus communis 2R1 CP37 IgG2a A-chain, EIA, WBAnti-Ricin, RCA 60 from Ricinus communis 2R1 CP75 IgG1 A-chain, EIA, WBAnti-Ricin, RCA 60 from Ricinus communis 2R1 RA999 IgG1 A-chain, EIA (capture), WB, C/r with RCA 120Anti-Ricin, RCA 60 from Ricinus communis 2R1 RB999 IgG1 B-chain, EIA (detection), WB64 INFECTIOUS DISEASE REAGENTS

7. Antibodies for the detection ofHT-2 toxinHT-2 toxin is mycotoxin of the group trichothecenestype A produced by fungi of the Fusarium genus, i.e.Fusarium sporotrichioides, F.poae, F.equiseti, andF.acuminatum which are commonly found in variouscereal crops (wheat, maize, barley, oats, and rye)and processed grains (malt, beer and bread). HT-2toxin (together with related T-2-toxin) often occurs ininfected cereals. The fungi producing trichothcenesare soil fungi and are important plant pathogenswhich grow on the crop in the field. The trichothecenesare in general very stable compounds, bothduring storage/milling and processing/cooking offood, and they do not degrade at high temperatures.Both HT-2 and T-2 toxins have a multiple toxic actions,including inhibition of DNA and RNA synthesisand of initiation phase of protein synthesis, affectingthe permeability of cell membranes, causing apoptosisin living tissues (e.g. thymic and splenic lymphocytes).Monoclonal mouse anti-HT-2 toxin antibody wereproduced from ascitic fluid of tumour bearing BALB/cmice, inoculated with corresponding hybridomaclones using HT-2 toxin conjugated with HAS asan immunogen. All MAbs recognize HT-2 toxin andC6B4 and C6D4 also react with T2-toxin.Ordering information:Product Cat. # MAb Isotype RemarksAnti-HT-2 Toxin 2HT2 C6B4 IgG1 C/r with T2-toxinAnti-HT-2 Toxin 2HT2 C6D4 IgG1 C/r with T2-toxinAnti-HT-2 Toxin 2HT2 C6E6 IgG1Anti-HT-2 Toxin 2HT2 C6F1 IgG18. Antibodies for the detection ofMicrocystin-LRMicrocystin-LR is a cyanobacterial toxin and a potentinhibitor of protein phosphatase types 1 and 2A (hasno effect on protein kinase). It is a tumor promoterrather than a carcinogen. Among more than 80 microcystinsidentified to date, only few occur frequentlyand in high concentrations but Microcystin-LR isamong the most frequent and most toxic microcystincongeners. Frequently occurring cyanobacterial generathat contain these toxins are Microcystis, Planktothrixand Anabaena. Microcystins usually occur withinthe cells; substantial amounts are released to thesurrounding water only in situations of cell rupture.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Microcystin-LR 2MC2 C64A1 IgG1 Indirect EIAAnti-Microcystin-LR 2MC2 C64C12 IgG1 Indirect EIA9. Antibodies for the detection ofNodularinNodularin is cyanobacterial toxins and it inhibits proteinphosphatases 1 and 2A with the same potencyas does microcystin-LR. Nodularin might be a tumorpromoter in rat liver. Nodularin itself is a new liver carcinogen.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Nodularin 2ND3 C66H3 IgG1 Indirect EIAAnti-Nodularin 2ND3 C66B6 IgG1 Indirect EIAAnti-Nodularin 2ND3 C66A2 IgG1 Indirect EIAINFECTIOUS DISEASE REAGENTS65

10. Antibodies for the detectionof Tetanus toxinTetanus toxin (tetanospasmin, TeNT) is the neurotoxinproduced by the vegetative spore of Clostridium tetaniin anaerobic conditions, causing tetanus – a medicalcondition that is characterized by a prolongedcontraction of skeletal muscle fibers, which caneventually cause respiratory failure.Tetanospasmin is one of the most potent toxinsknown: the estimated minimum human lethal dose is2.5 nanograms per kilogram of body weight, or 175nanograms in a 70 kg human.The toxin acts at several sites within the centralnervous system, including peripheral nerve terminals,the spinal cord, and brain, and within the sympatheticnervous system. The clinical manifestations oftetanus are caused when tetanus toxin blocksinhibitory impulses by interfering with the release ofneurotransmitters. This leads to unopposed musclecontraction and spasm. Seizures may occur, and theautonomic nervous system may also be affected.The peptide tetanospasmin has a molecular weight of150 kDa. It is made up of two parts: a 100 kDa heavy orB-chain and a 50 kDa light or A-chain. The chains areconnected by a disulfide bond. The B-chain binds todisialogangliosides (GD2 and GD1b) on the neuronemembrane. The A-chain, a zinc endopeptidase,attacks the vesicle-associated membrane protein.The chains are non-toxic after separation.Using supernatant from C. tetani we managed toproduce three monoclonal antibodies specific totetanus toxin having toxin neutralization activity (invivo assay) and suitable for EIA and WB applications.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Tetanus Toxin 2TE8 TetE3 IgG1 EIA, WBAnti-Tetanus Toxin 2TE8 TetG2 IgG2a EIA, WBAnti-Tetanus Toxin 2TE8 TetH6 IgG1 EIA, WB11. Antibodies for the detection ofAflatoxin from Aspergillus flavusAspergillus flavus is a plant, animal, and humanpathogen that produces the carcinogen, aflatoxin.Aflatoxins are potent toxic, carcinogenic, mutagenicand immunosuppressive agents, produced as secondarymetabolites by the fungus Aspergillus flavusand A. parasiticus on variety of food products.Among 18 different types of aflatoxins identified, majormembers are aflatoxin B1, B2, G1 and G2. AflatoxinsM1 and M2 are major metabolites of aflatoxinB1 and B2 respectively, found in milk of animals thathave consumed feed contaminated with aflatoxins.We used aflatoxin from Aspergillus flavus conjugatedto BSA as immunogen to produce monoclonal antibodyagainst aflatoxin. MAb ATB recognizes free aflatoxinsB1 and B2. No cross-reactivity with G1, G2and M1 was observed.Ordering information:Product Cat.# MAb Isotype RemarksAnti-Aflatoxin 3Af27 ATB IgG1 EIA66 INFECTIOUS DISEASE REAGENTS

X Biodefense antibodies1. Antibodies for the detection ofBacillus anthracisAccording to JAMA Consensus Statement, of the numerousbiological agents that may be used as weapons,the Working Group on Civilian Biodefense hasidentified a limited number of organisms that couldcause disease and deaths in sufficient numbers tocripple a city or region. Anthrax is one of the mostserious of these diseases. For centuries, anthraxhas caused disease in animals and, uncommonly,serious illness in humans throughout the world. Researchon anthrax as a biological weapon beganmore than 80 years ago. Today, at least 17 nationsare believed to have offensive biological weaponsprograms; it is uncertain how many are working withanthrax. In humans, 3 types of anthrax infection occur:inhalational, cutaneous, and gastrointestinal.Naturally occurring inhalational anthrax is now a rarecause of human disease. Historically, wool sorters atindustrial mills were at highest risk. Cutaneous anthraxis the most common naturally occurring form,with an estimated 2000 cases reported annually, diseasetypically follows exposure to anthrax-infectedanimals, whereas gastrointestinal anthrax is uncommonlyreported. However, gastrointestinal outbreakshave been reported in Africa and Asia.B. anthracis derives from the Greek word for coal,anthrakis, because the disease causes black, coallikeskin lesions. B. anthracis is an aerobic, grampositive,spore-forming, nonmotile Bacillus species.The nonflagellated vegetative cell is large (1-8 μm inlength, 1-1.5 μm in breadth). Spore size is approximately1 μm. Spores grow readily on all ordinarylaboratory media at 37 °C, with a “jointed bamboorod” cellular appearance and a unique “curledhair”colonial appearance, and display no hemolysis onsheep agar. This cellular and colonial morphologytheoretically should make its identification by an experiencedmicrobiologist straightforward, althoughfew practicing microbiologists outside the veterinarycommunity have seen anthrax colonies otherthan in textbooks. Anthrax spores germinate whenthey enter an environment rich in amino acids, nucleosides,and glucose, such as that are found in theblood or tissues of an animal or human host. TheFigure 61. Gram Stain of Bacillus anthracis.rapidly multiplying vegetative anthrax bacilli, on thecontrary, will only form spores after local nutrientsare exhausted, such as when anthrax-infected bodyfluids are exposed to ambient air. Full virulence requiresthe presence of both an antiphagocytic capsuleand 3 toxin components (ie, protective antigen,lethal factor, and edema factor). Vegetative bacteriahave poor survival outside of an animal or humanhost; colony counts decline to undetectable within24 hours following inoculation into water. This contrastswith the environmentally hardy properties ofthe B. anthracis spore, which can survive for decades.For diagnostics, identification and investigation ofpathogenesis mechanisms of B. anthracis HyTest offersa wide spectrum of immunochemical reagents.We have produced 13 monoclonal antibodies to ProtectiveAntigen (PA), which according to WB recognizeboth 83K and truncated 63K forms of PA. Traditionalsandwich ELISA with the use of the matchpair BAP0105 (capture) – BAP0106 (HRP conjugate)easily provides for the detection limit 100 pg/ml. Forthe detection of the lethal factor we offer match pairBAL0105 and BAL0106. HyTest has started the processof production of immunogenic preparationsfrom B. anthracis spores. As a result of the rabbitsimmunization and a special exhaustion procedurewe now have at our disposal IgG fraction of rabbitserum to spore antigen of B. anthracis which interactonly with anthrax spores and do not cross-react withspores of other bacilli: B. cereus, B. turingiensis, B.subtilis, B. megaterium and others. According to WBthey recognize S-layer proteins (92-94K) and workin dot-blot ELISA, immunofluorescence as well as insandwich-ELISA with MAb when used as capture.INFECTIOUS DISEASE REAGENTS67

1.1. Antibodies for the detection ofBacillus anthracis Protective AntigenBacillus anthracis produces a three-component toxin,which contributes to the symptoms and lethalityof anthrax. The anthrax toxin consists of three proteins,termed Protective Antigen (PA), Edema Factor(EF) and Lethal Factor (LF). The components arenon-toxic individually until they combine to each otherand enter cells. Full size PA molecule (83K) triggersthis process via binding to the cell surface ATRreceptor. After binding PA is cleaved enzymatically,yielding 63K fragment, which combine to form aring-shaped heptamer. The heptamer captures EFand LF and is transported to endosome. Monoclonalantibodies against PA are useful for anthrax diagnostics,toxin neutralization and analyses of howit enters cells.Immunogen: Purified B. anthracis Protective Antigen, 83K.Immunoreactivity: MAbs C3, BAP0101, BAP0102, BAP0103, BAP0104, BAP0105, BAP0106 are directed against different PA epitopes.They recognize both 83K and truncated 63K forms of PA. There is no cross-reactivity with B. anthracis LF, spore andvegetative microbe surface antigens.Applications:Western blotting, ELISA. Fig. 62 shows the detection of crude B. anthracis Protective Antigen preparation in Westernblotting using BAP0101, BAP0103, BAP0105 and BAP0106. Fig. 63 shows the calibration curve for PA quantificationusing double antibody sandwich ELISA with MAbs BAP0105 and BAP0106. The detection limit for the immunoassayis 50 pg/ml.kDa2,094674330OD 4501,51,00,5PA1 2 3 45200,00 10 20 30 40 50 60ng/mlFigure 62. Detection of B. anthracis Protective Antigen in Western blottingusing MAbs BAP0101 (1); BAP0103 (2); BAP0105 (3); BAP0106 (4);molecular mass standards (5). 10% SDS-PAGE was run under non-reducingconditions.Figure 63. Calibration curve in ELISA for B. anthracis Protective Antigendetermination using MAb BAP0105 for capture and MAb BAP0106 as aHRP-conjugate.Ordering information:Product Cat. # MAb Isotype RemarksAnti-B. anthracis Protective Antigen 3BA16 C3 IgG1 EIA (capture), WBAnti-B. anthracis Protective Antigen 3BA16 BAP0101 IgG2b EIA, WBAnti-B. anthracis Protective Antigen 3BA16 BAP0102 IgG2b EIA (detection), WBAnti-B. anthracis Protective Antigen 3BA16 BAP0103 IgG2b EIA (detection), WBAnti-B. anthracis Protective Antigen 3BA16 BAP0104 IgG2b EIA, WBAnti-B. anthracis Protective Antigen 3BA16 BAP0105 IgG1 EIA (capture), WBAnti-B. anthracis Protective Antigen 3BA16 BAP0106 IgG1 EIA (detection), WB68 INFECTIOUS DISEASE REAGENTS

1.2. Antibodies for the detection ofBacillus anthracis Lethal FactorB. anthracis full virulence requires the presence ofboth an anti-phagocyte capsule and 3 toxin collaboratingcomponents: Protective Antigen (PA), LethalFactor (LF) and Edema Factor (EF). Up to three copiesof EF or LF or a combination of the two bind to theProtective Antigen heptamer on the outer cell surfaceand are delivered to an endosome. Mild acidityin the endosome compartment causes EF and LFtransport into the cytosol. The exact mechanism ofthe LF action is not known. It was shown that LF is aprotease and cleaves enzymes belonging to MAPKKfamily. Anti-LF monoclonals could be useful for anthraxtoxin neutralization and LF intracellular localization.Immunogen:Immunoreactivity:Applications:Purified B. anthracis Lethal Factor.MAbs BAL0105 and BAL0106 recognize two different epitopes of LF molecule with high affinity.There is no cross-reactivity with B.anthracis PA, spore and vegetative microbe surface antigens.Western blotting, ELISA. Fig. 64 shows the detection of crude B. anthracis Lethal Factor preparationin Western blotting using MAbs BAL0105 and BAL0106. Fig. 65 shows the calibration curve forLF quantification using double antibody sandwich ELISA with MAbs BAL0105 and BAL0106.The detection limit for the immunoassay is 5 ng/ml.kDa94674330OD 450 nm2,52,01,51,00,5stand. curve1 2200,00100 200 300 400 500[c] Ag ng/mlFigure 64. Detection of B. anthracis Lethal Factor in Western blottingusing MAbs BAL0105 (1); BAL0106 (2). 10% SDS-PAGE was run undernon-reducing conditions.Figure 65. Calibration curve in ELISA for Lethal Factor determinationusing MAb BAL0106 for capture and MAb BAL0105 as an HRP-conjugateOrdering information:Product Cat. # MAb Isotype RemarksAnti-B. anthracis Lethal Factor 3BA17 BAL0105 IgG1 EIA (detection), WBAnti-B. anthracis Lethal Factor 3BA17 BAL0106 IgG1 EIA (capture), WBAnti-B. anthracis Lethal Factor 3BA17 LFA58 IgG1 EIA (capture), WBAnti-B. anthracis Lethal Factor 3BA17 LFA27 IgG2a EIA (detection), WBINFECTIOUS DISEASE REAGENTS69

1.3. Antibodies for the detection ofBacillus anthracis Spore AntigenB. anthracis is an aerobic, gram-positive, sporeforming,nonmotile Bacillus species. Spore sizeis approximately 1 µm. Anthrax spores germinatewhen they enter an environment rich in amino acids,nucleosides and glucose. The rapidly multiplyingvegetative anthrax bacilli will only form sporesafter local nutrients are exhausted, such as whenanthrax-infected body fluids are exposed to ambientair. Spores can survive for decades in an environment.Current methods for anthrax spore identificationare based on microscopy, germination andspore destruction PCR/DNA-probes. The use of immunochemicalassays was limited in connectionwith poor antigens expositions and cross-reactivitywith thousands of similar but harmless spore-formingmicroorganisms that colonize air, water and soil.Immunogen:Immunoreactivity:Applications:B. anthracis spore extract made by chaotropic agents and detergent treatment.MAbs SA26 and SA27 recognize S-layer protein (92K). There is no cross-reactivity with PA, LF andB. anthracis vegetative form. They do not bind to spores and vegetative forms of B. thuringiensis, B. subtilis,B. cereus, B. brevis, B. megaterium.Western blotting, ELISA. Fig. 66 shows the detection of crude B.anthracis spore preparation in Westernblotting using MAbs SA26 and SA27. Fig. 67 shows the calibration curve for B. anthracis spore quantificationusing double antibody sandwich ELISA with MAbs SA26 and SA27.The detection limit for the immunoassay is 3 x 10 4 spores/ml.1 2 33,02,52,0BCDEFOD 4501,51,00,50,010 100 1000thousand spore/mlFigure 66. Detection of B. anthracis spore in Western blotting usingMAbs SA26 (1); SA27 (2); Molecular mass standards are 94, 67, 43,30, 20 K from top to bottom (3). 10% SDS-PAGE was run under reducingconditions.Figure 67. Calibration curves for B. Anthracis spore detection usingMAb SA26 for capture and SA27 as an HRP conjugate. The spores weresuspended in PBST (B), 0.05% SDS (C), 0.5M Urea (D), 0.4% Formaldehyde(E), 1% Formaldehyde (F).Ordering information:Product Cat. # MAb Isotype RemarksAnti-B. anthracis Spore Antigen 3BA19 SA26 IgG2a EIA (capture), WBAnti-B. anthracis Spore Antigen 3BA19 SA27 IgG2a EIA (detection), WBOrdering information:Product Cat. # Host animal RemarksPolyclonal B. anthracis Spore Antigen 3BA18 rabbit EIA, WB70 INFECTIOUS DISEASE REAGENTS

2. Antibodies for the detection ofYersinia pestisIn AD 541, the first recorded plague pandemic beganin Egypt and swept across Europe with attributablepopulation losses of between 50% and 60% in NorthAfrica, Europe, and central and southern Asia. Thesecond plague pandemic, also known as the ”blackdeath or great pestilence”, began in 1346 and eventuallykilled 20 to 30 million people in Europe, onethird of the European population. The pandemic lastedmore than 130 years and had major political, cultural,and religious ramifications. The third pandemicbegan in China in 1855, spread to all inhabitedcontinents, and ultimately killed more than 12 millionpeople in India and China alone. Small outbreaks ofplague continue to occur throughout the world. Advancesin living conditions, public health, and antibiotictherapy make future pandemics improbable.However, plague outbreaks following use of a biologicalweapon are a plausible threat. In World WarII, a secret branch of the Japanese army, Unit 731,is reported to have dropped plague-infected fleasover populated areas of China, thereby causing outbreaksof plague.In the ensuing years, the biological weapons programsof the United States and the Soviet Uniondeveloped techniques to aerosolize plague directly,eliminating dependence on the unpredictableflea vector. In 1970, the World Health Organization(WHO) reported that, in a worst-case scenario, if 50kg of Y. pestis were released as an aerosol over acity of 5 million, pneumonic plague could occur inas many as 150,000 persons, 36,000 of whom wouldbe expected to die. Human plague most commonlyoccurs when plague-infected fleas bite humans whothen develop bubonic plague. As a prelude to humanepidemics, rats frequently die in large numbers,precipitating the movement of the flea populationfrom its natural rat reservoir to humans. Althoughmost persons infected by this route develop bubonicplague, a small minority will develop sepsis withno bubo, a form of plague termed ”primary septicemicplague”. Neither bubonic nor septicemic plaguespreads directly from person to person. A smallpercentage of patients with bubonic or septicemicplague develop secondary pneumonic plague andcan then spread the disease by respiratory droplet.Persons contracting the disease by this route developprimary pneumonic plague. Plague remains anenzootic infection of rats, ground squirrels, prairiedogs, and other rodents on every populated continentexcept Australia. Worldwide, on average in thelast 50 years, 1700 cases have been reported annually.In the United States, 390 cases of plague werereported from 1947 to 1996, 84% of which were bubonic,13% septicemic, and 2% pneumonic. Concomitantcase fatality rates were 14%, 22%, and 57%,respectively. Y. pestis is a nonmotile, gram-negativebacillus, sometimes coccobacillus, that shows bipolar(also termed safety pin) staining with Wright, Giemsa,or Wayson stain. (Fig. 66)Figure 68. PeripheralBlood Smear From PatientWith SepticemicPlague.INFECTIOUS DISEASE REAGENTS71

Y. pestis is a lactose nonfermenter, urease and indolenegative, and a member of the Enterobacteriaceaefamily. It grows optimally at 28 °C on blood agaror MacConkey agar, typically requiring 48 hours forobservable growth, but colonies are initially muchsmaller than other Enterobacteriaceae and may beoverlooked. Y. pestis has a number of virulence factorsthat enable it to survive in humans by facilitatinguse of host nutrients, causing damage to host cells,and subverting phagocytosis and other host defensemechanisms. Capsular F1 antigen is a main immunochemicalcomponent of Y. pestis surface. Its synthesisis determined by pFra plasmid and induced at37 °C. F1 is aggregated on the outer membrane ofthe Y. pestis microbe as an olygomeric protein, forminga granular layer and gradually diffusing into theenvironment. Capsular polymer consists of multiplesimilar hydrophobic protein subunits, aggregating atphysiological conditions. F1 subunit of the antigen isoriginally synthesized from 170 aminoacids and hasa molecular mass 17.6 kD, then, after the eliminationof the signal peptide during the secretion onto thesurface, a protein with molecular mass 15.6 kD andisoelectric point 4.1 is formed. We used highly purifiedF1 antigen for hybridoma development.To confirm capsular antigen identity Ouchterlonydouble immunodiffusion in agarose gel and Westernblotting analysis were performed with F1-specificrabbit polyclonal antibodies. (Fig. 70 and 71)32 14Figure 70. Ouchterlony immunodiffusion plate.Well 1: Commercial F1, 1 mg/ml positive control;Wells 2, 3, 4: Samples of F1: batch 7 non diluted; batches 7 and 8diluted 4-fold;Well 5: LPS EV 76 negative control.51 2 3 4 5 694Figure 69. Electrophoretic analysis of Y. pestis F1 antigensLane 1: Cytochrome C 12.5 kDLane 2 F1 referenceLane 3: F1 batch 8Lane 4: F1 batch 7Lane 5: F1 referenceLane 6: Molecular mass standards67433020141 2 3Figure 71 Immunoblotting of capsular F1 antigen with rabbit antisera.Lane 1: F1 batch 7Lane 2: F1 batch 8Lane 3: F1 commercial preparationElectrophoresis and immunoblotting data testify tothe contents of F1 in the immunogen preparationsat not less than 95%. For the detection of Y. pestiswe produced MAb YPF19, which is characterized byhigh affinity towards F1 capsular antigen. This antibodywas checked in different formats: sandwichELISA, hem-and-latex agglutination, lateral flow deviceas a colloidal gold conjugate, Western blottingand immunofluorescence. Data confirm high specificityand sensitivity of the assays on the base ofthese MAb. There is no cross-reactivity with Y. pseudotuberculosisand Y. enterocolitica.72 INFECTIOUS DISEASE REAGENTS

V antigen of Yersinia pestis is a multifunctional proteinthat has been implicated as a protective antigen,a virulence factor, and a regulatory protein. Somestudies suggest that V antigen is also a virulencefactor that reduces local expression of the host cytokinestumor necrosis factor alpha and gamma interferonin response to Yersinia infection, thus allowingthe bacterium to become established. V antigenalso has a role in the regulation of the low-calciumresponse.To produce MAbs Va13, Va22, Va48, Va52 and Va68we used Y. pestis, V antigen as an immunogen. Allthese MAbs are working in ELISA and Western blotting.Recommended pair for sandwich immunoassayis (capture - detection): Va13 - Va48.Anti-Yersinia pestis V antigenImmunogen:Y. pestis full-size recombinant V antigen.Immunoreactivity: MAbs Va13 and Va22 are not cross-reacting with B. anthracis protective antigen and spore,E. coli or Y. pestis F1 antigen.Applications:Western blotting, ELISAAnti-Yersinia pestis capsular F1 antigenImmunogen:Purified Y. pestis strain EV76 F1 capsular antigen.Immunoreactivity: MAb YPF19 recognize Y. pestis F1 capsular antigen. There is no cross-reactivity with Y. pseudotuberculosisand Y. enterocolitica.Applications:Western blotting, ELISA. Fig. 72 shows the detection of purified Y. pestis F1 antigen preparation in Westernblotting using MAb YPF19. Fig. 73 shows the calibration curve for Y.pestis F1 antigen quantification usingdouble antibody sandwich ELISA with MAb YPF19. The detection limit for the immunoassay is 0.5 ng/ml.2,01,5OD 4921,00,5con. 20 tcon. 40 t0,01 2 3 40 2 4 6 8 10 12 14ng/mlFigure 72. Detection of Y. pestis F1 capsular antigen in Western blottingusing MAb YPF19 (2). Molecular mass standards are 67, 43, 30, 20, 14 Kcontrol and track 3 shows F1 antigen staining with Amido black. 10% SDS-PAGE was run under reducing conditions.Figure 73. Calibration curve for double antibody sandwich with MAbYPF19 used for capture and as an HRP conjugate for Y. pestis F1 antigendetection. The conjugate working dilutions were 1/20000 (black squares)and 1/40000 (red squares).Ordering information:Product Cat. # MAb Isotype RemarksAnti-Yersinia pestis V Antigen 3YPV8 Va13 IgG1 EIA (capture), WBAnti-Yersinia pestis V Antigen 3YPV8 Va22 IgG1 EIA, WBAnti-Yersinia pestis V Antigen 3YPV8 Va48 IgG1 EIA (detection), WBAnti-Yersinia pestis V Antigen 3YPV8 Va52 IgG1 EIA, WBAnti-Yersinia pestis V Antigen 3YPV8 Va68 IgG1 EIA, WBAnti-Yersinia pestis capsular F1 Antigen 3YP8 YPF19 IgG1 EIA (capture, detection), WB, IHCINFECTIOUS DISEASE REAGENTS73

3. Antibodies for the detection ofFrancisella tularensisTularemia was first described as a plague-like diseaseof rodents in 1911 and, shortly thereafter, wasrecognized as a potentially severe and fatal illnessin humans. Tularemia’s epidemic potential becameapparent in the 1930s and 1940s, when large waterborneoutbreaks occurred in Europe and the SovietUnion and epizootic-associated cases occurredin the United States. As well, F. tularensis quicklygained notoriety as a virulent laboratory hazard.Public health concerns impelled substantial early investigationsinto tularemia’s ecology, microbiology,pathogenicity, and prevention.Tularemia occurs throughout much of North Americaand Eurasia. In the United States, human caseshave been reported from every state except Hawaii;however, most cases occur in south-centraland western states (especially Missouri, Arkansas,Oklahoma, South Dakota, and Montana). In Eurasia,the disease is also widely endemic, although thegreatest numbers of human cases are reported fromnorthern and central Europe, especially Scandinaviancountries and those of the former Soviet Union.Tularemia is almost entirely a rural disease, althoughurban and suburban exposures occasionally do occur.Throughout its range, F. tularensis is found inwidely diverse animal hosts and habitats and can berecovered from contaminated water, soil, and vegetation.A variety of small mammals, including voles,mice, water rats, squirrels, rabbits, and hares, arenatural reservoirs of infection. They acquire infectionthrough bites by ticks, flies, and mosquitoes, and bycontact with contaminated environments. Althoughenzootic cycles of F. tularensis typically occur withoutnotice, epizootics with sometimes extensive dieoffsof animal hosts may herald outbreaks of tularemiain humans.Humans become infected with F. tularensis by variousmodes, including bites by infective arthropodshandling infectious animal tissues or fluids, directcontact with or ingestion of contaminated water,food or soil, and inhalation of infective aerosols. Personsof all ages and both sexes appear to be equallysusceptible to tularemia. Certain activities, such ashunting, trapping, butchering, and farming, are mostlikely to expose adult men. Laboratory workers areespecially vulnerable to infection, either by accidentallyinoculating themselves or by inhaling aerosolizedorganisms. Ordinary exposures during examinationof an open culture plate can cause infection.Although F. tularensis is highly infectious and pathogenic,its transmission from person to person hasnot been documented. Francisella tularensis is asmall, nonmotile, aerobic, gram-negative coccobacillus.It has a thin lipopolysaccharide-containing envelopeand is a hardy nonspore-forming organismthat survives for weeks at low temperatures in water,moist soil, hay, straw, and decaying animal carcasses.Francisella tularensis has been divided into 2 majorsubspecies (biovars) by virulence testing, biochemicalreactions, and epidemiological features.Francisella tularensis biovar tularensis (type A) maybe highly virulent in humans and animals, producesacid from glycerol, demonstrates citrulline ureidaseactivity, and is the most common biovar isolatedin North America. Francisella tularensis biovarpalaearctica (type B) is relatively avirulent, does notproduce acid from glycerol, and does not demonstratecitrulline ureidase activity. In Europe and Asia,all human tularemia is thought to be caused by themilder type B strains, although recent studies therehave identified naturally occurring F. tularensis relatedto F. tularensis biovar tularensis. A few rapidlygrowing strains of F. tularensis have been recoveredfrom the blood of immunocompromised patients notshowing seroreactivity to F. tularensis.74 INFECTIOUS DISEASE REAGENTS

Lipopolysaccaride (LPS) is a main species-specificantigen of Francisella tularensis. According to thedata available LPS of tularemia microbe differs fromLPS of other gram-negative bacteria. LPS of tularemiamicrobe haven’t yet revealed the properties ofclassical endotoxin, which may be connected withnon-typical structure of lipid A – a toxic componentof LPS. We used LPS purified from f vaccine strain 15of Francisella tularensis for hybridoma development.Fig. 67 shows the results of electrophoresis in 15%SDS-PAGE and silver stained by the method of Tsaiand Frasch .Balb/c mice were repeatedly immunized with ultrasonicdisintegrate of F. tularensis strain 15 cells. PrimaryMAbs screening was done using highly purifiedLPS as capture antigen in indirect ELISA. TwoMAbs T14 and FB11 were established, both MAbsare suitable for F. tularensis detection in sandwichELISA (detection limit 100 pg/ml)(Fig. 68), immunofluorescenceand lateral flow devices. Antibodies arenot cross-reacting with the following microorganisms:F. novicida, B. abortus, B. suis, B. melitensis, B.ovis, B. neotomae, Y. pestis, Y. pseudotuberculosis,Y. enterocolitica, S. typhimurium, V. cholera, E. coli.1 20,300,25MeanMaxMin0,20OD 4920,150,100,051 0,5 0,25 0,125 0ng/mlFigure 74. Electrophoresis of F. tularensis strain 15.Lane 1: Ra-lipopolysaccaride Salmonella Minnesota SF 1112Lane 2: LPS15-lipopolysaccaride F. tularensis 15. LPS F. tularensis is presentedon the gel as a brown spot in the region 5-10 kDa.Figure 75. Determination of the detection limit for F. tularensis LPS insandwich ELISA using MAb T14 as capture and HRP conjugate.Ordering information:Product Cat. # MAb Isotype RemarksAnti-F. tularensis LPS 3FT6 T14 IgG3 EIA (capture, detection) IFAnti-F. tularensis LPS 3FT6 FB11 IgG2a EIA, IFINFECTIOUS DISEASE REAGENTS75

4. Antibodies for the detection ofMarburg and Ebola virusesMarburg virus (MBG) is a representative of Filoviridaefamily of RNA containing viruses. MBG is an exceptionallydangerous pathogen, which induces a severecontagious and highly lethal (53-88%) febrile diseasewith hemorrhagic syndrome. The name “thread viruses”is based on their morphology. MBG made itsappearance in 1967 in the form of frightening nosocomialoutbreak, initially among polio vaccine productionworkers in Germany in contact with Ugandangreen monkeys and their kidney tissues. The pathoandimmuno-genesis of MBG fever have so far beenlittle studied, no specific agents or treatment methodshave been developed. We used purified formaldehydeinactivated Marburg virus as an immunogen to produceMAbs FM213, FM11, FM32 and FM44. All MAbsreact with MBG in Western blotting and indirect ELISA.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Marburg virus 3M1 FM213 IgG1 EIA, WBAnti-Marburg virus 3M1 FM11 IgG1 EIA, WBAnti-Marburg virus 3M1 FM32 IgG1 EIA, WBAnti-Marburg virus 3M1 FM44 IgG1 EIA, WBEbola haemorrhagic fever (EHF) is one of the mostvirulent viral diseases known to humankind, causingdeath in 50-90% of all clinically ill cases. Severaldifferent species of Ebola virus have beenidentified. The Ebola virus is transmitted by directcontact with the blood, body fluids and tissues ofinfected persons. Transmission of the Ebola virushas also occurred by handling ill or dead infectedchimpanzees.Ebola virus is one of at least 18 known viruses capableof causing the viral hemorrhagic fever syndrome.Although agents of the viral hemorrhagic feversyndrome constitute a geographically diversegroup of viruses, to date, all are RNA viruses, all areconsidered zoonoses, all damage the microvasculatureresulting in increased vascular permeability,and all are members of 1 of 4 families: Arenaviridae,Bunyaviridae, Flaviviridae, and Filoviridae.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Ebola virus, Zaire strain 3E1 FE18 IgG2a EIA, WBAnti-Ebola virus, Zaire strain 3E1 FE25 IgG2a EIAAnti-Ebola virus, Zaire strain 3E1 FE37 IgG2a EIA, WB5. Antibodies for the detection ofVaccinia virusVaccinia virus is a double-stranded DNA orthopoxvirus.Orthopoxviruses are the largest viruses found.The viruses are about 200 nm in diameter, 250-300nm long and 250 nm high. Smallpox (variola) is a viraldisease characterized by a skin rash and a highdeath rate. Smallpox is highly contagious from oneperson to another. It may spread extremely rapidlye.g. from bed sheets and clothing and from salivadroplets. In the past smallpox was found all overthe world causing illness and death where ever itoccurred. It was primarily a disease of children andyoung adults. Vaccinia virus can be used as an effectiveimmunizing agent against human smallpox.In 1980 WHO declared that the world is free of smallpoxand the vaccinations were stopped. The vaccineis, however, still used for the people working in laboratoriesdirectly involved with smallpox, monkeypox,vaccinia and other closely related orthopoxviruses.Even it has been successfully eradicated; smallpoxmay still pose a threat to humanity.We used Vaccinum variola vivim siccum (live drysmallpox vaccine) as an immunogen to producemonoclonal antibodies against vaccinia. Pep-76 INFECTIOUS DISEASE REAGENTS

tone was used as cryoprotector and stabilizer. Forscreening purposes vaccinia virus preparation usedas immunogen was dialyzed against PBS to removepeptone. 10% SDS-PAGE of vaccinia virus preparationbefore and after the dialysis under non-reducingand reducing conditions are presented on Fig. 76.As a reference on Fig. 75 there are 10% SDS-PAGEimages of different highly purified orthopoxviruses.According to Ichihashi et al: 1988, Virology, Vol.163,133-144, major polypeptides of vaccinia virus havethe following molecular masses: 27, 34, 57 and 61kDa, as is shown on the picture. The same proteinsare found in the immunogen preparation. The antibodiesare working in ELISA and stain 27 kDa proteinband at the concentration 10 μl/ml in Westernblotting (see Fig. 78). Cross-reaction against cowpoxvirus, ectromelia virus, avipox virus and smallpoxvirus in ELISA and Western blotting are currentlybeing determined. Neutralization was not observedagainst live vaccinia virus in cell culture.kDa94kDa1 2 3 M 4216132676157784330342745,732,52018,41 2 3 4 5Figure 76. SDS-PAGE of vaccinia antigen used as immunogen and forscreening.Lane 1: Vaccinia antigen before dialysis, non-reducing conditionsLane 2: Vaccinia antigen before dialysis, reducing conditionsLane 3: Molecular mass standards(from top to bottom: 94, 67, 43, 30, 20 kDa)Lane 4: Vaccinia antigen after dialysis, non-reducing conditionsLane 5: Vaccinia antigen after dialysis, reducing conditionsFigure 77. SDS-PAGE analysis of orthopoxviruses.Lane 1: Vaccinia virus, ElstreeLane 2: Ectromelia virus K-1, CAMLane 3: Ectromelia virus K-1, cell cultureLane M: Molecular mass standardsLane 4: Cowpox virus, GrishakkDa94674330201427Figure 78. 10% SDS-PAGE (lanes 1-3 non-reducing conditions, lanes 4-6reducing conditions), Western blotting.Lane 1: Molecular mass standards(from top to bottom: 94, 67, 43, 30, 20, 14 kDa)Lane 2: Anti-Vaccinia, clone TV43, ascites, dilution 1:1000Lane 3: Anti-Vaccinia, clone TV46, ascites, dilution 1:1000Lane 4: Anti-Vaccinia, clone TV43, ascites, dilution 1:1000Lane 5: Anti-Vaccinia, clone TV46, ascites, dilution 1:1000Lane 6: Molecular mass standards(from top to bottom: 94, 67, 43, 30, 20, 14 kDa)Ordering information:Product Cat. # MAb Isotype RemarksAnti-Vaccinia virus 3V1 TV43 IgG2a EIA, WBAnti-Vaccinia virus 3V1 TV46 IgG2a EIA, WBINFECTIOUS DISEASE REAGENTS77

6. Antibodies for the detection ofHemorrhagic fever with renal syndrome(HFRS)Hemorrhagic fever with renal syndrome (HFRS) is agroup of clinically similar illnesses caused by hantavirusesfrom the family Bunyaviridae. HFRS includesdiseases such as Korean hemorrhagic fever, epidemichemorrhagic fever and nephropathis epidemica.The viruses that cause HFRS include Hantaan,Dobrava-Belgrade, Seoul, and Puumala strains.Symptoms of HFRS usually develop within 1 to 2weeks after exposure to infectious material, but inrare cases, they may take up to 8 weeks to develop.Initial symptoms begin suddenly and include intenseheadaches, back and abdominal pain, fever, chills,nausea, and blurred vision. The severity of the diseasevaries depending upon the virus causing theinfection: Hantaan and Dobrava virus infections usuallycause severe symptoms, while Seoul and Puumalavirus infections are usually more moderate. Completerecovery can takes weeks or months.Hantaviruses are carried and transmitted by rodentsand the occurrence of the disease in endemic areasis at its highest when rodent densities are highest(May-June and October-November). The disease isworldwide but is most prevalent in Korea and neighboringprovinces of China. People can become infectedwith these viruses and develop HFRS after exposureto aerosolized urine, droppings, or saliva ofinfected rodents or after exposure to dust from theirnests. In addition, individuals who work with live rodentscan be exposed to hantaviruses through rodentbites from infected animals. Transmission fromone human to another may occur, but is extremelyrare.Like other members of the bunyavirus family, hantaviruseshave spherical virions with diameters of 90-100 nm enveloped with glycoproteins G1 (aka Gn)and G2 (Gc) and contain no matrix proteins.For production of HFRS virus (Puumala strain) specificmonoclonal antibodies a lysate of virus-infectedcells (MAb PG10) and synthetic peptide conjugatedwith hsp70 (MAb G2D11) have been used as immunogens.All MAbs are specific to HFRS virus, Puumalastrain without cross reactivity with Dobrava, Hantaand Seoul strains. MAb G2D11 was proved to bespecific to glycoprotein G2 of the viral envelope.Ordering information:Product Cat.# MAb Isotype RemarksAnti-HFRS 3CCH5 PG10 IgG2a Reacts with Puumala strain, N/cr with Dobrava, Hanta andSeoul strains, virus neutralizing activityAnti-HFRS 3CCH5 G2D11 IgG1 Reacts with Puumala strain, G2 protein, WBAnti-HFRS 3CCH5 Puu-H6 IgG1 EIA, fluorescent immunoassay, reacts with Puumala,Dobrava, Hanta and Seoul strainsAnti-HFRS 3CCH5 Puu-D21 IgG1 EIA, fluorescent immunoassay, reacts with Puumala,Dobrava, Hanta and Seoul strainsAnti-HFRS 3CCH5 Dob-A4 IgG2a EIA, fluorescent immunoassay, reacts with Puumala,Dobrava, Hanta and Seoul strainsAnti-HFRS 3CCH5 Dob-F1 IgG2b EIA, fluorescent immunoassay, reacts with Puumala,Dobrava, Hanta and Seoul strainsAnti-HFRS 3CCH5 HANT-E9 IgG1 EIA, fluorescent immunoassay, reacts with Hanta strainAnti-HFRS 3CCH5 HANT-B5 IgG1 EIA, fluorescent immunoassay, reacts with Hanta strain78 INFECTIOUS DISEASE REAGENTS

XI Veterinary1. Canine1.1. Canine distemper virus (CDV)Canine distemper is a contagious, incurable, oftenfatal, multisystemic viral disease that affects the respiratory,gastrointestinal, and central nervous systems.Distemper is caused by the canine distempervirus (CDV). It occurs among domestic dogs andmany other carnivores, including raccoons, skunks,and foxes. CDV is fairly common in wildlife. Infecteddogs shed the virus through bodily secretions andexcretions, especially respiratory secretions. Theprimary mode of transmission is airborne viral particlesthat dogs breathe in. The development of a vaccinein the early 1960s led to a dramatic reductionin the number of infected domestic dogs. It tends tooccur now only as sporadic outbreaks.For the production of MAb to CDV, splenocytes ofthe Balb/c mice recovered after acute infection byCDV envelope protein (Onderstepoort) were used.MAbs 8-1 and 5-4 against CDV could be used in ELI-SA with the native CDV and purified CDV, in immunofluorescencewith infected Vero cells and in immunohistochemistry.These MAbs may be used for thedetection of CDV in clinical samples (blood, saliva,tears, feces) of animals.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Canine distemper virus 3CD10 8-1 IgG2a EIA (detection), PLAAnti-Canine distemper virus 3CD10 5-4 IgG2a EIA (capture), PLA1.2. Canine parvovirus (CPV)Canine parvovirus (CPV) is a highly contagious andserious disease caused by a virus that attacks thegastrointestinal tract of puppies, dogs, and wild canids.Parvoviral infection must be considered as apossible diagnosis in any young dog with vomitingand/or diarrhea. Puppies and dogs usually becomeinfected when they ingest virus that is passed in thefeces of an infected dog. There is a 3-7 day incubationperiod before the puppy seems obviously ill.Canine parvovirus causes lethargy; loss of appetite;fever; vomiting; and severe, often bloody, diarrhea.Vomiting and diarrhea can cause rapid dehydration,and most deaths from parvovirus occur within 48 to72 hours following onset of clinical signs. Virus isshed for the first two weeks or less after infection inthe stool of an infected dog.MAbs 5G7 and 8H7 are directed against CPV. TheseMAb are working in ELISA with the native CPV andpurified CPV, in immunodiffusion, haemagglutinin inhibition,immunohistochemistry and immunofluorescence.These MAb may be used for the detectionof CPV in clinical samples (foeces). MAbs 5G7, 8H7are cross-reacting with Mink enteritis virus and FPLV(Feline panleucoperia).Ordering information:Product Cat. # MAb Isotype RemarksAnti-Parvovirus, canine 3PV16 5G7 IgG2a EIA (capture), WB, ID, HITAnti-Parvovirus, canine 3PV16 8H7 IgG2a EIA (detection), WB, ID, HITINFECTIOUS DISEASE REAGENTS79

1.3. Canine Adenovirus (CAV)MAbs 8C4, 1E11 and 7C11 were produced by immunizationwith human adenovirus type 1 and boostingwith canine hepatitis virus. These MAbs are workingin ELISA with the native CAV and purified CAV in immunodiffusion,immunohistochemistry and immunofluorescence.These MAbs may be used for the detectionof Canine adenovirus infections in clinicalsamples (blood, nasal swabs) of animals.New!New!New!New!Ordering information:Product Cat. # MAb Isotype RemarksAnti-Adenovirus hexon 3AV13 8C4 IgG2a EIA (capture), ID, IHCAnti-Adenovirus hexon 3AV13 7C11 IgG2a+IgM EIA, ID, IHCAnti-Adenovirus hexon 3AV13 1E11 IgG2a+IgM EIA (detection), ID, IHC1.4. Rabies virusRabies is a disease caused by a virus found in thesaliva of infected animals and is transmitted to petsand humans by bites, or possibly by contaminationof an open cut. It infects the central nervoussystem, causing encephalopathy and ultimatelydeath. The time between exposure to the virusand the onset of symptoms can range from abouttwo weeks to several months. The rabies virus canbe found in animal saliva days before any obvioussymptoms develop. However, all animals thathave the virus will develop symptoms and eventuallydie of the disease. Most animals can be infectedby the virus and can transmit the disease to humans.Infected bats, raccoons, foxes, skunks, dogsor cats provide the greatest risk to humans. For immunizationwe used vaccine strain of the rabies virus.Finally we got quite a number of MAbs, whichwere characterized by the specificity to the glyco-Ordering information:protein (GP) and nucleoprotein (NP) of the rabiesvirus. MAbs 4G4, 4F1, 7E3 and 9A10 are specificto glycoprotein and they have neutralizing activity.MAbs 1E9 and 5B12 are RNP–specific and theyare working in Immunofluorescence (IF) and in Immunohistochemistrywith infected cells. MAb 1C5against GP is of special interest. This MAb is verygood in ELISA with the native RV and the purifiedGP and in immunofluorescence with intact infectedBHK cells. Hybridoma cells, when inoculated intomice, protect them from RV infection and the MAb1C5 demonstrates a therapeutic effect on the infectedmice. MAb 1C5 has a virus-neutralizing activity inrespect to the majority of standard RV and rabiesrelativeviruses: CVS, Lagos bat, Mokola, Flury lep,Duvenhage and the field isolates. This antibody maybe used for the detection of rabies virus and for creationof new vaccine anti-rabies preparations.Product Cat. # MAb Isotype RemarksAnti-Rabies virus 3R7 1C5 IgG2a EIA, IHCAnti-Rabies virus 3R7 4G4 IgG2b EIAAnti-Rabies virus 3R7 5B12 IgG2a EIA, WB, IFAnti-Rabies virus 3R7 1E9 IgG2a EIA, WB, IFAnti-Rabies virus 3R7 4F1 IgG2b EIA, WB, IFAnti-Rabies virus 3R7 7E3 IgG2b EIA, WB, IFAnti-Rabies virus 3R7 9A10 IgG1 EIA, WB, IF1.5. Echinococcus granulosisEchinococcus granulosis, also called the Hydatidworm, is a cyclophyllid cestode that parasitizes thesmall intestine of canids as an adult, but which hasimportant intermediate hosts such as livestock andhumans, where it causes hydatid disease. In canids,E. granulosis causes a typical tapeworm infection.Adult worms mature in the intestine of the dog (definitivehost) and the eggs are released in the foeces.By an accidental ingestion in humans, oncosphereshatch in the duodenum, penetrate the intestine andare carried via the bloodstream to various organs. Hydatidcysts form in organs like liver, lungs and brain.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Echinococcus granulosis 3EG3 EHG IgG1 EIA, WB80 INFECTIOUS DISEASE REAGENTS

2. Bovine2.1. RotavirusThe incubation period for rotavirus disease is approximately2 days. The disease is characterized byvomiting and watery diarrhea for 3 - 8 days, and feverand abdominal pain occur frequently. As with allviruses, some rotavirus infections cause few or nosymptoms, especially in adults. Rotavirus spreadsby fecal-oral transmission.We used purified bovine rotavirus as an immunogenand MAb 3C10 is specific for mammalian group Arota viruses. Antibody is cross-reacting with monkeyrotavirus (SA-11), porcine rotavirus (PP) and with numeroushuman rotaviruses. Antibody is reacting inWestern blotting with p42 major inner capsid antigenand it is working also in ELISA and immunohistochemistry.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Rotavirus Group Specific Antigen 3R10 3C10 IgG2a P42 Antigen, EIA, IHC, WB2.2. Bovine coronavirusBovine coronavirus infects neonatal calves andpresents as an acute diarrhea. It frequently leads todeath.MAb 5A4 was produced by immunization with bovinecoronavirus. MAb recognizes bovine coronavirussurface antigen (peplomer).Ordering information:Product Cat. # MAb Isotype RemarksAnti-Bovine coronavirus 3BCV1 5A4 IgG1 EIA, HIT2.3. Brucella abortus (Brucellosis)Brucellosis is an infectious disease caused by thebacteria of the genus Brucella. Cows are the sourceof Brucella abortus but other species of Brucellacan be contracted from other animals and can alsocause brucellosis. The disease may be either subclinical,acute and subacute, relapsing, or chronic.The incubation period may be weeks to months. Itmay be mild or an explosive, toxic illness. Symptomsare non-specific and few localizing physical signsdevelop. Diagnosis is usually from blood or bonemarrow cultures, or a rise in anti-brucella antibodiesof 4 fold or greater.We used two antigens for anti-B. abortus MAb production:crude cell lysate and purified LPS. Respectively2 panels of MAb were obtained: BA35, BA37,BA39 and Bx85, Bx87, Bx88. Selection of the optimalpair for B. abortus detection in sandwich-ELISAshowed that MAb Bx85 is suitable for capture andMAb Bx88 works well and specifically as HRP-conjugate.Detection limit when using purified LPS as astandard is lower than 100 pg/ml, sensitivity is improvedat sample boiling before the analysis.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Brucella abortus 3BR11 BA35 IgG2a EIA, WBAnti-Brucella abortus 3BR11 BrF11 IgG1 EIA, WBAnti-Brucella abortus 3BR11 BrG11 IgG2a EIA, WBINFECTIOUS DISEASE REAGENTS81

2.4. Alpha-1 – Acid Glycoprotein (AGP)The Alpha-1 – Acid Glycoprotein (AGP) level in 152Holsteins from 1 year to 12 years is 283 μg/ml. Thereare no differences between male and female or betweenbreeds. The upper limit of normal AGP inhealthy bovine is 450 μg/ml. The serum value of AGPin a calf immediately following birth is 300 to 1750μg/ml, but this drops to normal by the end of the thirdweekn following birth. Levels of AGP that remainshigh longer than the 3-d week are indicative of abackground health problem that will adversely affectgrowth and productivity. AGP is elevated in stressedcattle that are clinically normal. In particular the levelsare elevated with hepatitis, pericarditis, arthritis,mastitis and pneumonia. With successful treatmentor stress removal levels return to normal rapidly.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Alpha-1-Acid Glycoprotein, Bovine (Orosomucoid) 5AG1 GPB2 IgG1 EIA, WB2.5. Foot-and-mouth disease (FMDV)MAbs against virulent serotype O1 of foot-andmouthdisease (FMDV) were screened in differentserological reactions like ELISA and gel precipitation,in which both antibodies were active. Antibodiescan be used to detect FMDV in the field isolatesfor differentiating type “O” from other types, for viruspurification and for antibody screening in competitiveELISA, etc.MAbs against the non-structural protein (NSP) offoot-and-mouth disease can be used for examplefor antigen purification and for antibody screeningin the competitive ELISA, etc. Combination of anELISA test, that detects antibodies directed againstFMDV viral non-structural proteins (NSPs) and a liquidphase blocking competitive ELISA for the detectionof antibodies against the viral structural proteins(SPs strain specific), can be used in analysis of fieldsamples allowed for a clear differentiation betweeninfected and uninfected animals, with high specificityand sensitivity, regardless of the animal’s vaccinationstatus.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Foot-and-mouth disease 3FM2 2D2 IgG2a virulent serotype O1, EIA, IDAnti-Foot-and-mouth disease 3FM2 3G8 IgG2a virulent serotype O1, EIA, IDAnti-Foot-and-mouth disease 3FM2 1G2 IgG2a non-structural proteins, EIAAnti-Foot-and-mouth disease 3FM2 1H4 IgG2a non-structural proteins, EIA82 INFECTIOUS DISEASE REAGENTS

3. Equine3.1. Burkholderia (Pseudomonas)mallei (Glanders)Burkholderia (Pseudomonas) mallei is a gram-negative,non-sporing bacilli and it is the etiologic agentof glanders. Glanders is a highly contagious diseaseof solipeds and it is characterized by nodular lesionsof the lungs and other organs, as well as ulcerativelesions of the skin and mucous membranes of thenasal cavity and respiratory passages. Ingestionof the pathogen, present in secretions from infectedanimals, constitutes the major route of infectionin glanders. Carnivores are susceptible to diseaseif they consume glandered meat. Humans also aresusceptible to infection with glanders, which is animportant occupational disease of veterinarians, farriers,and other animal workers. We have used cellextract of Pseudomonas mallei to develope a monoclonalantibody for detection of Pseudomonas mallei.MAb 3D11 MAb reacts with LPS of Burkholderiamallei in ELISA and Western blotting.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Pseudomonas mallei LPS 3PM15 3D11 IgG1 EIA, WB4. Porcine4.1. Transmissible Gastroenteritis(TGE) virus of PigsTransmissible gastroenteritis (TGE) is an acute highlycontagious disease of pigs caused by virus fromthe Coronaviridae family. It is a common viral diseaseof the small intestine that causes vomiting andprofuse diarrhea in pigs of all ages. There is highmorbidity and mortality in piglets.MAb 1E11 is specific to peplomer of TGE virus. MAbcan be used in Immunometric detection of the TGEvirus in stool and cell culture fluids. The same MAbcan be used as capture and detection MAb. MAbcan also be used in haemagglutinin inhibition.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Transmissible Gastroenteritis Pig Virus 3TG1 1E11 IgG1 EIA (capture, detection), HIT5. Piscine5.1. Infectious Salmon Anemia virusInfectious salmon anemia virus (ISAV) is the causativeagent of ISA, which is a highly infectious diseaseof farmed Atlantic salmon in the Northernhemisphere. This virus is a member of the family Orthomyxoviridae,genus Isavirus. We have used twosynthetic peptides of Infectious Salmon Anemia Virusfrom putative haemagglutinin sequence, conjugatedto KLH to generate monoclonal antibodiessuitable for EIA ans WB. All MAbs recognize peptidesof putative haemagglutinin of Infectious SalmonAnemia Virus (8-23 a.a.r. for MAbs 16C7 and 16F4and 296-312 a.a.r. for MAbs 18D8 and 18F9).Ordering information:Product Cat. # MAb Isotype RemarksAnti-Infectious Salmon Anemia Virus, Putative Haemagglutinin 3SA1 16C7 IgG1 8-23 a.a.r., EIAAnti-Infectious Salmon Anemia Virus, Putative Haemagglutinin 3SA1 16F4 IgG2a 8-23 a.a.r., EIAAnti-Infectious Salmon Anemia Virus, Putative Haemagglutinin 3SA1 18D8 IgG2b 296-312 a.a.r., EIAAnti-Infectious Salmon Anemia Virus, Putative Haemagglutinin 3SA1 18F9 IgG1 296-312 a.a.r., EIAINFECTIOUS DISEASE REAGENTS83

6. Avian6.1. Newcastle disease virus (NDV)Newcastle disease (ND) is a highly contagiousand sometimes fatal illness affecting many domesticand wild bird species. The causal agent,Newcastle disease virus (NDV), is a negative-sensesingle-stranded RNA virus. NDVaffects the respiratory, nervous, and digestive systems.Clinical signs are extremely variable dependingon the strain of virus, species and age of bird,concurrent disease, and pre-existing immunity.NDV is so virulent that many birds die without showingany clinical signs. Transmission occurs by exposureto foecal and other excretions from infectedbirds, and through contact with contaminatedfood, water, equipment and clothing. Virus-bearingmaterial can be picked up on shoes and clothing andcarried from an infected flock to a healthy one. Exposureof humans to infected birds (for example inpoultry processing plants) can cause mild conjunctivitisand influenza-like symptoms, but NDV otherwiseposes no hazard to human health. MAbs arenegative with parainfluenza type 3 and avian influenzahaemagglutinins. All the MAbs listed under theordering information are Haemagglutinin-NeuraminidaseAg specific antibodies.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Newcastle disease virus 3ND5 9F7 IgG1 EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 11F12 IgG2a EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 13H3 IgG2a EIA, WB, HITAnti-Newcastle disease virus 3ND5 9C6 IgG2a EIA, WB, HITAnti-Newcastle disease virus 3ND5 1C10 IgG2a EIA (detection), WB, HITAnti-Newcastle disease virus 3ND5 2H4 IgM EIA (capture), HITAnti-Newcastle disease virus 3ND5 8H2 IgG2a EIA (capture)New! Anti-Newcastle disease virus 3ND5 6H12 IgG2a EIA, IHC, IF6.2. Marek disease virus (MDV)Marek disease (MD) is a highly contagious, lymphoproliferativedisease of chickens. It is caused by theMD virus (MDV), an oncogenic avian herpesvirus. Itis the most serious chronic concern to the poultry industry.Chickens are exposed at an early age to cellfreeMDV through inhalation of contaminated dust.MDV-infected lymphocytes in the peripheral blooddistribute the virus to other tissues. In susceptiblechickens second round of cytolytic infection occursafter about 2 weeks. After 3 weeks the chronic inflammationof the peripheral nerves is often seenand changes in lymphoid cells may induce malignanttransformation in this cell type. The disease ischaracterized by presence of T cell lymphoma aswell as infiltration of nerves and organs by lymphocytes.Infected birds can suffer from asymmetric paralysisof one or more limbs, difficulty of breathing,dilation of the crop, depression and paralysis. Deathoccurs usually in a large number of birds (up to 80percent).Ordering information:Product Cat. # MAb Isotype RemarksAnti-Marek disease virus 3MD8 14C8 IgG3 EIAAnti-Marek disease virus 3MD8 1G6 IgG2a EIAAnti-Marek disease virus 3MD8 5C2 IgM EIAAnti-Marek disease virus 3MD8 3G2 IgM EIAAnti-Marek disease virus 3MD8 3H9 IgG2b EIA6.3. Avian influenzaOf the 16 different influenza haemagglutinin typesonly strains within the H5 and H7 subtypes causehighly pathogenic avian influenza, which is highlycontagious and rapidly fatal in susceptible avianspecies. Find more information about antibodiesagainst H5 and H7 from pages 14 - 16.84 INFECTIOUS DISEASE REAGENTS

6.4. Infectious bursal disease virus(IBDV)Infectious bursal disease (IBD) is an acute, highlycontagious avian viral infection, which is disseminatedworldwide and brings major economic losses inthe poultry industry. Infectious bursal disease wasdescribed by Cosgrove in 1962, and since the firstoutbreaks occurred in the area of Gumboro, Delaware,“Gumboro disease” became a synonym forthis disease.The genome of IBDV consists of two segments (Aand B) of double-stranded RNA, which are packagedin a nonenveloped icosahedral shell about 60 nm indiameter. The segment A of the IBDV genome has atleast two partially overlapping open reading frames(ORF). The large continuous ORF encodes the precursorprotein which is subsequently processed intothe mature viral proteins: pVP2, VP3 and VP4. Duringvirus maturation in a host cell, pVP2 (48 kDa),also known as VPX, turns into 37 kDa outer capsidstructure protein by host proteases cleavage. VP2carries the determinants responsible for the causingof antigenic variation as well as for virus tropism tothe cell. VP3 (32 kDa) is the inner capsid structureprotein, while VP4 (28 kDa) is the nonstructural protein,which is involved in processing of the precursor.For detection and identification of the IBD virus infield isolates and in vitro studies along with differenttypes of reverse transcription-polymerase chainreactions, many methods are based on antigenantibodyinteraction principle. Among them: histochemistry,virus neutralization, sandwich (antigencapture)immunoassay, dot- and western blotting.Methods utilizing high affinity monoclonal antibodiesare much more sensitive and have lower nonspecificreactions than those which utilize polyclonalantisera. Pairs of high affinity MAbs could also beused in one-step diagnostic kits based on membranechromatography.HyTest offers a panel of MAbs, which could besucessfully used in dot- and Western blotting analsisof VP2 and VP3 virus proteins, for immunohistochemicalanalysis in diagnostics and for peroxidaseimmunostaining of infected cell cultures. Severalpairs of MAbs could be utilized for the designing ofsandwich immunoassays to be used for virus titration;for live and inactivated vaccine preparation aswell as in diagnostics.6.4.1. Immunodetection of VP2 and VP3 IBDVstructure proteins in Western blottingTwo structure proteins of IBDV capsid, VP2 (about37 kDa) and VP3 (32 kDa) are the major proteins ofinterest in Gumboro disease investigation, as theycarry neutralizing epitopes and play important role invirus evolution (4, 5, 10, 11, 12). HyTest offers monoclonalantibodies selectively recognizing VP2 or VP3proteins of IBDV. MAbs IBDV67 and IBDV92 are VP2-specific, MAbs IBDV9, IBDV99, IBDV105 are VP3-specific.VP2VP3Figure 79. Immunodetection of major structure proteins of IBD viruscapsid in Western blotting by different monoclonal antibodies afterTricine-SDS-PAGE in reducing conditions.Lanes: 1- MAb IBDV9, 2- MAb IBDV67, 3- MAb IBDV92, 4- MAb IBDV99,5- MAb IBDV105, 6- molecular mass markers.6.4.2. Direct ELISAAll MAbs recognize IBD virus in direct ELISA.OD 4902.521.510.5IBDV92250 kDa130 kDa72 kDa55 kDa36 kDa28 kDa00.0001 0.001 0.01 0.1 1 10MAb concentration, ng/mlFigure 80. Titration curve for MAb IBDV92.Antigen: purified IBD virus, 0.5 µg/well.INFECTIOUS DISEASE REAGENTS85

6.4.3. HistochemistryTo determine if the virus is able to infect cells in vitroor in virus neutralization tests in cell culture, immunostainingof infected cells is usually used. We recommendour MAbs, conjugated with HRP for rapidand very specific detection of IBD virus in cell cultureexperiments (Fig. 81).ABcps1000000100000100001000IBDV9 - IBDV1051000,1 1 10 100 1000 10000Purified IBDV concentration, ng/mlFigure 81. Immunodetection of IBD virus in Vero cells.Vero cells were cultured in 96-well plate, than infected with S706 IBDV strainand fixed with ethanol-acetone mixture. Sites of non-specific binding wereblocked with 5% dry milk in PBS containing 0,1% Tween 20 (PBST).A. Early stage of virus infection. After blocking cells were incubated 1 hourwith MAb IBDV 9, conjugated with HRP, 1:500 dilution in PBST. After washing,3,3`-diaminobenzidine HRP substrate was added.B. Totally infected cell culture. After blocking cells were incubated 1 hourwith non-conjugated MAb IBDV105 in PBST (10 μg/ml). Polyclonal antimouseIgG conjugated with HRP was used for MAb-IBDV complex visualization.3-amino-9-ethilcarbozol was used as HRP substrate.Figure 82. Calibration curve for the assay IBDV9-IBDV105.Capture MAb: IBDV9, 1 µg/well.Detection MAb (Eu-chelate labeled): IBDV105, 0.4 µg/well.Incubation time – 30 min.100000001000000100000IBDV99 - IBDV1056.4.4. Sandwich immunoassay for IBD virus detectionAll MAbs were tested in sandwich type immunoassayas capture or detection antibodies. Best pairs weretested with different strains of IBDV (MB, D 78, GM 97,S 706, LC75) see Table 16. For IBDV immunoassaydevelopment following two-site MAb combinationsare recommended (capture-detection):cps1000010001000,1 1 10 100 1000 10000Purified IBDV concentration, ng/mlFigure 83. Calibration curve for assay IBDV99- IBDV105.Capture MAb: IBDV99, 1 µg/well.Detection MAb (Eu-chelate labeled): IBDV 105, 0.4 µg/well.Incubation time – 30 min.IBDV9 - IBDV105 (Fig. 82)IBDV99 - IBDV105 (Fig. 83)IBDV67 - IBDV92 (Fig. 84)Table 16. Detection of different IBDV strains by threepairs.IBDVstrainIBDV9-IBDV105IBDV99-IBDV105IBDV67-IBDV92MB - + ++S 706 +++ +++ ++GM 97 - - +D 78 - - +LC75 +++ +++ ++++++ very high response, ++ high responce; + low responce;- no interaction.10000000cps1000000100000100001000IBDV67 - IBDV921000,1 1 10 100 1000 10000Purified IBDV concentration, ng/mlFigure 84. Calibration curve for assay IBDV67- IBDV92.Capture MAb: IBDV67, 1 µg/well.Detection MAb (Eu-chelate labeled): IBDV92, 0.4 µg/well.Incubation time – 30 min.86 INFECTIOUS DISEASE REAGENTS

6.5. Infectious bursal disease virus(IBDV)Avian infectious bronchitis (IB) is a highly contagiousupper-respiratory disease in chickens. It was firstobserved in 1930 and it is disseminated worldwidecosting the poultry industry billions of dollars annually.The causative agent of IB, infectious bronchitisvirus (IBV), belongs to the Group 3 of Coronavirusgenus of Coronaviridae family.IBV is enveloped, lipid-containing positive-senseRNA virus with non-segmented, single-strandedgenome, polyadenylated at 3’-terminus. Like othercoronaviruses the IBV virus particle contains threemajor protein structures: the spike, membrane,and the nucleocapsid. The nucleocapsid (N-protein)of IBV is a phosphoprotein of 409 amino acidsand molecular mass about 50 kDa. N-proteinforms a protective shell that packages the viral genomicRNA and its phosphorylation is thought tocontrol the RNA binding activity, replication andtranscription. Nucleocapsid protein is highly con-6.5.1. Immunodetection of IBV nucleoprotein inWestern blottingN-protein250 kDa130 kDa95 kDa72 kDa55 kDa36 kDa28 kDaserved protein across various IBV strains within majorantigenic groups of Coronaviruses. It has immunogenicproperties and N-specific antibodies crossreactbetween different strains of the group.We developed the monoclonal antibody specific tothe N-protein of infectious bronchitis virus. This antibodyrecognizes the N-protein in Western blottingassay with excellent specificity. It could be used asthe capture antibody in sandwich-immunoassayswith polyclonal antibodies as the detection. It alsocould be utilized as the capture antibody in wholevirus based serology assays to increase sensitivityand specificity in case of low purity of the antigen.6.5.2. Serological sandwich-type immunoassayMAb IB95 could be used as capture antibody in serologyassay to detect anti-IBV antibodies in vaccinatedchickens when semi-purified IB virus is usedas an antigen. MAb presents nucleoprotein and providebetter sensitivity and low non-specific reactioncomparing direct surface virus adsorption; lessamount of the antigen is needed (Fig. 86).OD 45032.521.51120.517 kDa11 kDa010.10.010.001serum dilution0.00010.00001Figure 85. Immunodetection of IBV nucleoprotein in Western blotting byMAb IB95 after Tricine-SDS-PAGE in reducing conditions.N-protein specificity was confirmed by mass spectrometry. Molecular massmarkers are marked at the right side of the picture.Figure 86. Titration curve of serum sample from IBV-immunized chickenin two different immunoassays.1: Sandwich type assay with MAb IB95 as the capture antibody and semipurifiedIB virus as the antigen.2: Direct sorption of semi-purified IB virus onto the plate surface. In both casessemi-purified virus was diluted 1000-fold (about 1 µg/ml of total protein).Ordering information:Product Cat. # MAb Isotype RemarksAnti-IBDV 3BD5 IBDV9 IgG2a EIA (capture), WB, IHC, Inner capsid structure protein VP3Anti-IBDV 3BD5 IBDV67 IgG1 EIA (capture), WB, IHC, Outer capsid structure protein VP2Anti-IBDV 3BD5 IBDV92 IgG1 EIA (capture, detection), WB, IHC, Outer capsid structure protein VP2Anti-IBDV 3BD5 IBDV99 IgG1 EIA (capture, detection), WB, IHC, Inner capsid structure protein VP3Anti-IBDV 3BD5 IBDV105 IgG3 EIA (capture, detection), WB, IHC, Inner capsid structure protein VP3Anti-IBV 3BN1 IB95 IgG2a EIA (capture), WBINFECTIOUS DISEASE REAGENTS87

XII Miscellaneous1. Borrelia burgdorferi(Borreliosis, Lyme Disease)Lyme borreliosis is an infection caused by a bitefrom a tick infected with the bacterium Borrelia burgdorferia.It is an inflammatory disease characterizedby a skin rash, joint inflammation and flu-like symptoms.Tick bite results in a skin lesion and is followedby heart and nervous system involvement and lateron by arthritis. Late involvement of eye, nervous system,joints, and skin can also occur. The only signthat enables a reliable clinical diagnosis of borreliosisis erythema migrans. Microbial or serologicalconfirmation of borrelial infection is needed for allmanifestations of the disease except for typical earlyskin lesions. Treatment with antibiotics is beneficialfor all stages of Lyme borreliosis, but is most successfulearly in the course of the illness.Two different partially purified B. burgdorferi strains,sensu stricto and garinii, were used as immunogensto generate MAb panels. MAbs Bss42, and Bss98are specific for B. burgdorferi sensu stricto, whileMAbs Bg14 and Bg64 are directed against B. burgdorferigarinii. End-point titer in indirect ELISA for allthe MAbs is 7.2 x 10 6 reflecting high antibody aviditytowards corresponding antigens. B. burgdorferiUS isolated strain 39/40 is well recognized by MAbsBss42 and Bss98 in indirect ELISA and WB with asingle band in the region 30K (outer surface proteinA, OspA, 31kD), which is a highly specific protein forB. burgdorferi spp. MAbs Bg14, and Bg64 give noreactivity with B. burgdorferi strains Colic and Sidn.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Borrelia burgdorferi sensu stricto 3BS25 Bss42 IgG2a EIA, WB, IFAnti-Borrelia burgdorferi sensu stricto 3BS25 Bss98 IgG2b EIA, WB, IFAnti-Borrelia burgdorferi garinii 3BB24 Bg14 IgG1 EIA, WB, IFAnti-Borrelia burgdorferi garinii 3BB24 Bg64 IgG1 EIA, WB, IF2. Tick-borne encephalitis virus(TBEV)Tick-borne encephalitis (TBE) is a human viral infectiousdisease involving the central nervous system.It most often manifests as meningitis, encephalitis,or meningoencephalitis. It is most commonly recognizedas a neurological disorder but mild fever canalso occur.TBE is caused by tick-borne encephalitis virus(TBEV), a member of the genus Flavivirus in the familyFlaviviridae. It is usually transmitted by the biteof several species of infected ticks, including Ixodesscapularis, Ixodes ricinus and Ixodes persulcatus.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Tick born encephalitis virus (TBEV) 3TBE1 TBE-F2 IgG2a EIA (peptide)88 INFECTIOUS DISEASE REAGENTS

3. Cyclosporin ACyclosporin A (CsA) is a fungal metabolite fromTolypocladium inflatum, an undeca - cyclic peptide, itis a potent immunosuppressive agent, it inhibits primarilyT lymphocytes and interleukin 2 production.CsC-BSA was used as immunogen, MAb CSZ22has according to Biacore the following Kd: 4.45 x 10-10, 5.93 x 10-8 and 1.54 x 10-9M for CsC-BSA, CsAand CsC-Biotin respectively. In competitive assayMAb CSZ22 are well correlated with Sandimmun-kitat CsA determination in blood.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Cyclosporin A 3C13 CSZ22 IgG1 EIA4. Helicobacter pylori CagAHelicobacter pylori is a Gram-negative, microaerophilicbacterium that can inhabit various areas of thestomach, particularly the antrum. It causes a chroniclow-level inflammation of the stomach lining andis strongly linked to the development of duodenaland gastric ulcers and stomach cancer. Over 80% ofindividuals infected with the bacterium are asymptomatic.The CagA protein is the product of the cagAgene carried among virulent H. pylori strains and isassociated with severe disease outcomes, most notablygastric carcinoma. CagA is injected from theattached H. pylori into gastric epithelial cells and undergoestyrosine phosphorylation. The phosphorylatedCagA binds and activates SHP-2 phosphataseand thereby induces a growth factor-like cellularmorphological change termed the “hummingbirdphenotype.”, which is characterized by dramatic cellelongation.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Helicobacter pylori CagA-protein 3HE70 HP-317 IgG EIA (capture)Anti-Helicobacter pylori CagA-protein 3HE70 HP-387 IgG EIA (detection)Anti-Helicobacter pylori CagA-protein 3HE70 HP-1811 IgG EIA (capture), WB, IP5. Hamster prion proteinPrions are infectious agents composed of protein.They propagate by transmitting a mis-folded proteinstate; the protein does not itself self-replicate and theprocess is dependent on the presence of the polypeptidein the host organism. The mis-folded form ofthe prion protein has been implicated in a numberof diseases in a variety of mammals, including BSE(“mad cow disease”) and Creutzfeldt-Jakob diseasein humans. All prion diseases affect the structure ofthe brain or other neural tissue, and all are currentlyuntreatable and are always fatal.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Prion protein, hamster 3PP3 PrPA5 IgM N-terminal, WB, indirect EIA, N/cr wtih human PrPAnti-Prion protein, hamster 3PP3 PrPB7 IgG1 C-terminal, WB, indirect EIA, N/cr wtih human PrPAnti-Prion protein, hamster 3PP3 PrPH8 IgG1 C-terminal, WB, indirect EIA, N/cr wtih human PrPINFECTIOUS DISEASE REAGENTS89

6. FK 506 (Tacrolimus)FK-506, a macrolide antibiotic obtained from Streptomycesisukubaensis, strain No 9993, was isolatedfrom the soil of Tsuluba, northern Japan. Its structurehas been determined chemically and by X-ray crystallography.FK-506 has been shown to have strongimmunosuppressive activity against a mixed lymphocytereaction (MLR) and promises to be useful inorgan transplantation. Its mechanism is consideredto be a suppression of both interleukin 2(IL-2) and interleukin-2receptor expression on T cells. Its activityhas been reported to be 100 times more potent, thanthat of cyclosporin A (CsA).We used FK506-BSA conjugate as immunogen andfor screening. MAb FK1 interacts with free FK506,conjugate, but doesn’t recognize protein-carrier. Itworks in competitive ELISA and provides for the detectionlimit ca 1 ng/ml for free FK-506.Ordering information:Product Cat. # MAb Isotype RemarksAnti-FK 506 (Tacrolimus) 4FK42 FK1 IgM EIA7. Allergen fromDermatotophagoides farinaeIn the early 1920’s it was discovered that housedust causes allergic reactions and later during thatdecade the presence of mites in house dusts wasknown. Today we know that dust mites (Dermatotophagoidesfarinae) are one of the most commonsources of sensitisation in all parts of the world. Aconnection between dust mite allergy and asthmahas also been reported by many researchers. Mites’faeces seem to be the major source of allergenic exposure.They are about the size of a pollen grain andcould therefore very easily become airborne andpenetrate the lung alveolus. So far the only effectiveway to get a permanent reduction of house-dustmiteallergens seems to be lowering of air humidityand temperature together with efficient cleaning.We used recombinant 15K major allergen from excretionsof house dust mite Dermatophagoides farinaeto produce MAb Df10. MAb could be used inEIA and WB.Ordering information:Product Cat. # MAb Isotype RemarksAnti-15 K allergen of House Dust Mite Dermatophagoides farinae 3K15 Df10 IgG1 EIA, WB8. Streptavidin fromStreptomyces avidiniiMAb panel S8E4, S8C12, S10D4 and S3E11, wasgenerated against streptavidin from Steptomyces avidinii.MAbs are suitable for IHC and EIA.Ordering information:Product Cat. # MAb Isotype RemarksAnti-Streptavidin from Streptomyces avidinii 3ST10 S8E4 IgG1 EIA, WB, IHCAnti-Streptavidin from Streptomyces avidinii 3ST10 S8C12 IgG1 EIA, WB, IHCAnti-Streptavidin from Streptomyces avidinii 3ST10 S10D4 IgG1 EIA, WB, IHCAnti-Streptavidin from Streptomyces avidinii 3ST10 S3E11 IgG1 EIA, WB, IHC90 INFECTIOUS DISEASE REAGENTS

Cardiac MarkersInfectious Disease ReagentsMetabolic SyndromeAutoimmune ReagentsHuman Serum ProteinsNeuroscience ReagentsTumor MarkersBlood Coagulation ReagentsDiabetes and Obesity ReagentsFertility and Pregnancy Reagentswww.hytest.fiTroponinProBNP, BNP and NT-proBNPMyoglobinPregnancy Associated Plasma Protein A (PAPP-A)Myeloperoxidase (MPO)Soluble CD40 Ligand (sCD40L)Cystatin CD-Dimer and HMW Fibrin Degradation ProductsFatty Acid Binding Protein (FABP)High Sensitivity C-Reactive Protein (hsCRP)Retinol-Binding Protein 4 (RBP4)Adiponectin, LeptinProinsulin, Insulin, C-peptidewww.hytest.fiAsk for our other CatalogsGeneralProduct Catalog2010-2011Markers ofCardiovascularDiseases andMetabolic SyndromeFax back request form +358 2 512 0909To: HyTest LtdTel: +358 2 512 0900Intelligate 6 th floor, Joukahaisenkatu 6, FI-20520, Turku, FinlandFrom:Position:Company/Institute:Address:Country:Fax:Date:Phone:E-mail:Please send me more information on the following products:Catalog #: Catalog #:Catalog #: Catalog #:Catalog #: Catalog #:Please send me prices for the following products:Catalog #: Catalog #:Catalog #: Catalog #:Catalog #: Catalog #:Please send me following HyTest Catalogs:General Product CatalogMarkers of Cardiovascular Diseases and Metabolic SyndromeInfectious Disease ReagentsHyTest Catalogs, TechNotes and DataSheets can be downloaded from www.hytest.fiINFECTIOUS DISEASE REAGENTS91

Intelligate 6 th floor, Joukahaisenkatu 6FI-20520 Turku, FINLANDTel. +358 2 512 0900. Fax +358 2 512 0909E-mail: hytest@hytest.fiInternet: http://www.hytest.fiZeeland Turku Oy/Finepress Oy 06/2010HyTest 06/2011