Document - FAVV

Evaluation of immunoassay kits for determination of ochratoxin A in cereals4.1 Materials and methods4.1.1 Kits usedThe following kits were assessed: AgraQuant® Ochratoxin from Romerlabs, ROSA OCHRATOXIN GRAIN byCharm, OCHRATOXIN-A ELISA by Europroxima and RIDASCREEN® Ochratoxin A 30/15 by R-Biopharm.The kit of Aokin arrived too late to be included in this assessment (Table 39).Table 39: Summary of kitsProvider Concerned kit Batch Format Expire dateRomerlabs AgraQuant® Ochratoxin 200831-09-04 96 wells August, 31st 2010Charm ROSA OCHRATOXIN GRAIN 010C 100 strips May 2010Europroxima OCHRATOXIN-A ELISA KN6733 96 wells February 2011R-Biopharm RIDASCREEN® Ochratoxin A 30/15 4209 96 wells Octobre 20104.1.2 Standards and reagentsAcetonitril and methanol (HPLC-MS grade) were purchased from Biosolve (Valkenswaard, The Netherlands).Deionised water was delivered by a Milli-Q system (Millipore, MA, USA). Certified reference solutions (Table 40) ofOTA, OTB, OTα, Citrinin, Zearalenone and AFLA B1 in acetonitril were purchased from Biopure (Tulln, Austria).Unfortunately, no commercial supplier of OTC Could be found. Pure crystalline forms of Coumarin, 4-HydroxyCoumarin and L-phenylalanine were purchased from Sigma Chemical Co. (St Louis, MO, USA).Table 40: Standard solutionsOTA OTB OTα CIT ZEA AFLA B1 Coumar 4-hydrCou PhAProvider Biopure Biopure Biopure Biopure Biopure Biopure Sigma Sigma Sigmaμg/ml 10.01 10.3 11.9 100.3 10 2.02 / / /Quant 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml / / /Solvent ACN ACN ACN ACN ACN ACN MeOH MeOH H2OStock solutions of 800 ng/ml of OTA, OTB, OTα, Citrinin, Zearalenone and AFLA B1 were prepared in pureacetonitril and stored at 4°C. Coumarin and 4-HydroxyCoumarin were dissolved in methanol whilst L-phenylalaninewas dissolved in water. The former were stored at 4°C, L-phenylalanine was aliquoted to 2 ml vials and stored at -20°C. Daily working dilutions were prepared by diluting the stock solutions in the appropriate extraction solvent ofthe kit to a concentration of 8 ng/ml. Successive dilutions for direct application on the kit were made in theappropriate extraction solvent.CODA-CERVA | Experimental evaluation 4-39