Preparation And Characterization Of Monoclonal Antibodies Against Aflatoxin B₁

Aflatoxin (AFT) is a group of structurally similar secondary metabolites, produced by Aspergillus flavus and A.parasiticu, aflatoxin B1 (AFB₁) is the most serious occurring chemical carcinogens and is one of the most toxic known to a fungal toxin. Investigation shows that aflatoxin contamination is widespread in the world. Therefore, the most widely attention pays to aflatoxin, governments have enacted very stringent standards to limit amount of aflatoxin in food. Since the 1950s, it gradually explored many ways to detect AFB₁, these methods have made some contribution in various historical periods for detecting aflatoxin, however, it also has shortcomings, such as complicated operating, pollution, high equipment requirement, the high false positive rate, long detection time; with the development of the times, it has been unable to meet the requirements of contemporary society that small pollution, high sensitivity, short detection time, simple testing. The accurate, rapid, clean, intelligent detection technology on the basis of immunoassay has become the hot of the rapid test to AFB₁. Because of the high extraction efficiency, high degree of purification, immunoaffinity chromatography technology to aflatoxin is increasingly respected by the research staff, this method is recognized as the most effective purification methods. High affinity, high sensitivity, stable quality antibody is the premise which should be solved firstly to establish the ideal testing method.In this study, monoclonal antibodies (McAb) against aflatoxin B1 was prepared and its characterizations were identified, then we preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.Objective: Prepared the monoclonal antibodies against aflatoxin B1 by hybridoma technique, identify its characterizations and preliminary apply the monoclonal antibodies.Method: (1) BALB/c mice were immunized with AFB₁-BSA; (2) Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells from the immunized BALB/c which was immune best; (3) Supernatant of the growing hybirdoma was used to screen for the presence of anti-AFB₁-BSA antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma; (4) Explored the method and conditions to prepare the large number of antibodies and antibodies purification; (5) Identified monoclonal antibodies'characterizations; (6) Preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.Result: (1) One hybirdoma secreting McAb against AFB₁ was produced and its subclass of the McAb was the IgG₂b, its affinity constant was 4.79×10⁸M^-1, and its cross-reaction rates with AFB₂, AFG₁, AFG₂, AFM₁ were <26.06%,63.68%,26.06%,39.26% ; (2) Freund's Incomplete adjuvant retreated 10 to 12 weeks mice to prepare the mice ascites, then purified by the method of CA-AS; (3) AFB₁ immunoaffinity column's procedures were 5mL 0.01M PBS (pH7.4) balanced the column, methanol concentration of the sample buffer was 10%, then 2mL ultrapure water wash, 1mL 80% methanol-water elute, 20mL0.01M PBS (pH7.4) regenerate; column capacity and antibodies'concentration relation formula is y=85.63x-0.043, R2=0.9732; the column that the column capacity was 40ng desend to 43.6% after one test.Conclusion: The cell line 5E10 could secret the purpose McAb, AFB₁ immunoaffinity column prepared by the McAb needs to be further optimized.