Analytical Methods For Determination Of Ochratoxin A In Cereals, Pig Feeds And Milk

Ochratoxin A (OTA) are mainly produced by several species of Aspergillus Ochraceus and Penicillium Verrucosum moulds, providing high toxicology which threatens the health of human and animal. In this study, a monoclonal antibody (mAb) and a single chain antibody (scFv) were produced. Based on the mAb, enzyme linked immunosorbent assay (ELISA) and chemiluminescence enzyme immunosorbent assay (CLEIA) were developed. An immunoaffinity chromatography (1AC) for clean-up OTA from matrices was developed. Moreover, an IAC-UPLC-MS/MS method was developed. These methods were used for determination of OTA in samples.In this study, a mAb was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1(κ-light chain) isotype. The titer of mAb was1:1.28×106and the affinity constant was5.31×109mol L-1of the mAb. A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was used to characterize the mAb. The concentrations causing50%inhibition (IC50) of binding of mAb to OTA-ovalbumin (OTA-OVA) by free OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were1.29,4.78, and0.94ng mL-1,respectively. Aflatoxin (AF) B1, B2, G1, G2, M,, M2, fumonisin B1(FB1, zearalenone (ZEN), deoxynivalenol (DON) and T-2toxin did not inhibit the binding of mAb to the marker antigen in ciELISA. The relative cross-reactivities of mAb to OTA, OTB and OTC were calculated to be100%,26.98%and137.23%, respectively. Based on the hybridoma cell line, a scFv was produced. The relative cross-reactivities of scFv to OTA, OTB and OTC were calculated to be100%,15.25%and124.34%, respectively. AFB,, B2, G1, G2, M1, M2, FB1, ZEN, DON, and T-2toxin did not inhibit the binding of scFv to the marker antigen in ciELISA.The ciELISA method using the mAb for determination of OTA in maize, soybean, wheat and pigfeed were developed. The linear range of ciELISA was0.3～3.0μg kg-1. The limit of detection (LOD) of the optimized ciELISA method was0.15μg kg-1in maize, soybean and wheat. Meanwhile, the LOD in pigfeed was0.6μg kg-1. At1.0～100μg kg-1fortified levels in maize, soybean, wheat and pigfeed, the mean recoveries of OTA ranged from80.5%to99.1%with coefficients of variations (CVs)6.6-13.6%. The accuracy and precision of the method at this level fall within the EU and China regulatory limits. This method can be used for determination of OTA in maize, soybean, wheat and pigfeed.Based on the mAb, an IAC for clean-up OTA from matrices was developed. The maximum OTA-binding capacity of the IAC was approximately5592ng of OTA per milliliter of gel (13.85nmol mL-1gel) which was equivalent to about978ng of OTA per milligram of antibody (2.4nmol mg-1Ab). An IAC-UPLC-MS/MS method using IAC for determination of OTA in cereals and pigfeeds was developed. This methodology has been validated in eight different matrices (millet, maize, soybean, wheat, rice, oat, buckwheat and pigfeed) with highly satisfactory results. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N=10) ranging from0.5μg kg-1to1.0μg kg-1and a limit of detection (LOD, S/N=3) ranging from0.2μg kg-1to0.3μg kg-1in cereals and pigfeeds. The IAC-UPLC-MS/MS method offers a good LOQ and LOD. The accuracy and precision at this level fall within the EU and China regulatory limits. The IAC-UPLC-MS/MS method can be used for determination and confirmation of OTA in cereal and pigfeed.A ELISA and a high sensitive chemiluminescence enzyme immunoassay (CLEIA) method using the4H10mAb for determination of OTA in milk was developed. The linear range of ELISA was0.3～3.0μg L-1and the LOD of ELISA was0.15μg L-1. At0.5～5.0μg L-1fortified levels in milk, mean recoveries ranged from97.1～103.2%with coefficients of variations (CVs)7.9-13.8%. The linear range of CLElA was0.064～0.67μg L-1and the LOD of CLEIA was0.03μg L-1. At0.1-5.0μg L-1fortified levels in milk, mean recoveries ranged from98.3～104.1%with coefficients of variations (CVs)11.2～15.4%. The accuracy and precision at this level fall within the EU and China regulatory limits. The ELISA and CLEIA methods can be used for determination of OTA in milk.