Antitrombina III id ANTITROMBINA III - Linear

Antithrombin III idREF3307005CONTENTSAntithrombin III1 x 15 testsANTITHROMBIN IIIQuantitative determination of Antithrombin III by radialimmunodiffusionFor in vitro diagnostic use onlyRI33008-1/0404R1.PRINCIPLEThe examined protein, diffusing in agarose gel containing a specific antibodywill form an immuno-complex, visible as a ring around the well. The ringdiameter is in proportion to the concentration of the analyzed protein. Theproportion corresponds to the diffusion time. In fact, at the end (72 h), thesquare of the diameter will be in linear proportion to the concentration(Procedure 1), while after a shorter period of time the square of the diameterwill be in a logarithmic proportion to the concentration (Procedure 2). In bothcases, a calibration curve should be constructed, using at least threecalibration points. However, a reference table is provided showing therelation between any concentration and the diameter at the end of theanalysis time.REAGENT COMPOSITIONPlate with agarose gel containing the antiserum for the specific protein to beanalyzed.STORAGE AND STABILITYStore at 2-8ºC.The plates are ready to be used.The Reagents are stable until the expiry date stated on the label.SAMPLESSerum or plasma. Stable six days at 2-8 ºC.ASSAY PROCEDUREAllow the plate to come to room temperature, open it and, if moisture ispresent on the wells, wait until it has evaporated. Withdraw 5 µL of samplesand/or controls and wait until it has been completely absorbed beforehandling the plate. Cover it tightly and place it in a moist chamber during theincubation time:- 72 hours for the Procedure 1 or 3.- 18 hours for the Procedure 2.READING TEST RESULTSMeasure the precipitation ring to the nearest 0.1 mm, after the requiredperiod according to used assay procedure, 72 h for Procedure 1 or 3, 18 hfor Procedure 2.Procedure 3Read on the enclosed Reference Table, the concentration valuecorresponding to the precipitation ring diameter. The ring value obtained forthe control should have a confidence limit of 0.2 mm (from the referencetable).NOTESThe diffusion time and the reading time depend on the concentration and thespecific diffusing protein. After 72 hours, the diffusion of the protein, at anyconcentration, is completed. For lower concentrations, it is possible to read atlower times (ie 36 h). However, in such cases it is advisable to read againafter 3-5 hours. If the diameter is still the same, it is possible to set theconcentration, on the contrary, if the diameter is different, the ring should beremeasured after a further 3-5 hours.QUALITY CONTROLIt is suggested to perform an internal quality control. Not included in the kit.ANALYTICAL PERFORMANCE- Measure´s limit. 7-80 mg/dL.- Precisionmg/dL Intra-assay Inter-assayMean 35.61 67.8 35.5 67.95SD 0.54 0.63 0.44 0.68CV% 1.52 0.93 1.26 1.01N 10 10 20 20Replicates: 10 for each level.Replicates: 20 for each level.- Correlation. This assay (x) was compared with a similar comercialmethod (y). The results were:N = 70 r = 0.9992 y = 0.98x + 0.77REFERENCE VALUESProcedure 1Reproduce on a plotting paper, the square of the precipitation rings and theconcentration of the controls. A line with an intercept should be in the range10-12 mm 2 . Sample values are determined by interpolation.Procedure 2Reproduce on a plotting paper, the square of the precipitation rings againsttheir concentration logarithms. The graph will be a straight line only for a lowvalues. For high values may curve a little. Sample values are obtained byinterpolation.Antithrombin III20-40 mg/dLREFERENCESFahey et al., J. Immunol., 94: 84 (1965)Mancini et al., Immunochemistry, 2: 235 (1965)RI33070-1/0404R1.QUALITY SYSTEM CERTIFIEDISO 9001 ISO 13485LINEAR CHEMICALS S.L. Joaquim Costa 18 2ª planta. 08390 Montgat, Barcelona, SPAINTelf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es