Development Of Indirect Competitive ELISA For Detection Of Aflatoxin B₁ And The Preparation Of Monoclonal Antibody Affinity Chromatography Column

Aflatoxin B₁ (AFB₁) is a highly toxic secondary metabolite produced predominantly by some fungal strains. Because it is extremely toxic, potent carcinogenic and wide spread in the environment, many countries legislated to monitor the AFB₁ contamination in food and feed stuff. It is difficult to completely prevent food and feed stuff from the contamination of AFB₁. Much efforts were made to control AFB₁ contamination, So development of quick, exact and sentitive detection method is very important to prevent food chains from AFB₁ contamination.In this study, AFB₁ artificial antigen was prepared by 3-mercaptopropionic acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) catalytic reaction, and AFB₁ monoclonal antibody immunoaffinity chromatography column was also prepared.During the artifical antigen preparation, room temperature oximation reaction monitored by thin layer chromatography (TLC) assay was used to get AFB₁-Oximation (AFB₁-O) instead of heating flux oximation reaction, the yield was up to 95%. In order to prevent cross linking reaction happening between BSA moleculars, the modified bovine serum albumen (M-BSA) was used in the EDC catalytic coupled reaction between AFB₁ and BSA, the coupled reaction was monitored by ELISA assay. The initial molar ratio of AFB₁-O to M-BSA influence on the molarratio of the coupled reaction production was studied mainly. The appropriate reaction conditions were: oximed for 24 h at 25 °C, the initial molar ratio of AFBrO to M-BSA was 30:1, coupled for 24 h, and the molar ratio of the conjugation of them were 5:1 in the coupled reaction production.The conditions that effects on indirect competitive enzyme-linked immunosorbent assay such as coating, blocking, antigen, antibody, methanol concentration and reaction model were optimized. The method with sensitivity 1.5 ng/mL, minimum detectability 0.1 ng/mL was establishedThe monoclonal antibody affinity chromatography column was prepared for treatment of the samples tested. When preparing the column, the monoclonal antibody concentration was more than lmg/ mL, coupling reacted for 3 h at 25 "C, the couple rate is more than 90%. When the column was used to purified AFB^ Methanol-Phosphate Buffere (20:80, v/v; containing 0.2 % NaCl) was used as the working solution, flow rate was 10 mL /min. Then the column was washed with 20 mL 20% methanol, eluted with 1 mL methanol for 4 min. The elutant collected was detected by HPLC with post column derivation. The detection results indicated that the recovery of the standard AFB^as more than 85%.