Development Of Gold Immunochromatography Assay For Rapid Detection Of Aflatoxin B1

Preparation and identification of AFB1 monoclonal antibody for. AFB1 was conjugated to keyhole limpet hemocyanin(KLH) and bovine serum albumin (BSA).AF-KLH was used as immune antigen and AF-BSA as coating antigen .Female BALB/C(6⁸ wk old) were immunized six or seven times, the spleen was removed and the cells were fused with SP2/0 myeloma cells with PEG-1400(sigma). The 4 isolated hybridoma cell lines were specific for AF by indirect ELISA, named 2F5,4G6,1F4 and 5F8. The ascites titer was 5×10^-5,5×10^-5, 5×10^-4,5×10^-4, respectively.The antibody isotypes of 4G6 and 1F4 were IgG2b and IgG1, and the others were IgM. By IC50 analysis, Ka values of 4G6 and 1F4 were 8.99×108M-1, and 7.63×108M-1, respectively.4G6 and 1F4 were administered to mice via the tail vein 30 min prior to ip AFT challenge (20μg/kg). All mice injected with 150μg and 200μg of 4G6 and 1F4 were protected,and the non-specific McAb did not have protection against AFT lethality. The mice which administered 500μg 1F4 and 400μg 4G6 via the tail vein 15²0 min after orally given a lethal dose of AFT in PBS2F5,4G6,1F4å’Œ5F8, were prevented from death, and the control ones died within 30 min.Polyclonal rabbit anti-idiotypic antibodies were raised by immunization with a protein A-purified McAb 4G6, and the rabbit IgG were purified to immunize common mice for the induction of anti-Aflatoxin antibody responses.The immunized mice which were challenged with AFT(20μg/kg) were protected by anti-Id, and the control mice died within 3⁵ min.On the basis of competitive ELISA, immunoassay and test kit were developed, the direct assay format with standard AFT competitor concentrations was a range of 1 to 32 ng/mL, the detection curve equation was y = -0.2533x + 0.8073, (R2 = 0.982) and variation coefficient was 2.3%³.6%. As expected, the recoveries of AFT spiked in shellfish meat were about 90%. The intra-assay CV% was below 10%.A rapid immunochromatographic assay for AFT was developed. McAb of 4G6 was labeled with the 20 nm prepared colloidal gold particles which served as a detection reagent, and the conditions for immobilizating the antibodies no nitrocellulose membrane (NC) were tested. The limit of the test strip detection was 5ng/ml, the expending time of detection was 5 to 10 min.