| Ochratoxin A(OA), a chlorinared dihydroisocoumarin derivative connected through an amide bond to L-beta-phenylalanine at the 7-carboxy group, is an important food-borne secondary metabolite produced by various species of the fungal genera Aspergillus and Penicillium, and pollutes the farm severely world wide. This toxin is a potent nephrotogen, teratogenen and carcinogen with strong kidney and liver toxicity, and it has immunosuppressive effects in BALB/c mice as well.For detection of OA, there are two main methods: confirmatory methods and enzyme immunoassay. However the former are time-consumption, costly and highly skilled which need some big machines such as TLC, HPLC, and GC/MS. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological method is gradually applied in this area. So this research synthesized the antigen, developed the antibodies and established the ELISA method for detecting OA.One Hybridoma cell lines named D6 producing monoclonal antibody against OA were established by fusions between SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with protein linked OA. According to the result of specificity-testing, Monoclonal antibody (McAb) D6 could only react a bit with Ochratoxin B in all other structurally related compounds in this assay. So we can conclude that this McAb has a high specificity against OA.OA-OVA, as the coating antigen, were prepared by activated ester method, and at last we established the indirect competitive ELISA for detecting OA. The method showed that the optimum concentrations of OA-OVA, monoclonal antibody against OA and goat anti-mouse IgG were1.5μg/hole, 1:500 and 1:10000 respectively. The sensitive range to detect OA varied from 1ng/mL to 100ng/mL. The minimum detectable was 1ng/mL. The variation coefficients of intra-assay and inter-assay were 1.6% and 6.08%, respectively. The established method is vital to rapidly detect the residues of OA in farm and animal product. |
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