Preparation And Characterization Of High Speciality Monoclonal Antibodies For Aflatoxin B 1

Aflatoxins are one of most important mycotoxins, which contaminate agri-products and foods. In 1993, they are listed as groupâ… carcinogens by the International Agency for Research on Cancer (IARC). Aflatoxin B₁ is not only the most toxic, but also account for above 70 % of aflatoxins total. Therefore, development of a high specificity immunoassay is very important for monitoring aflatoxin B₁ contamination. uce.In the study, considering epitopes, cross-reactivity of antibodies induced and synthesis of antigen, hapten was designated. AFB₁ was transformed to aflatoxin hemiacetal B2a, and then conjugated to protein by a reduction of sodium borohydride. After immunization, titers of three Balb/c mice serum were over 1/250000, and the rate of secreting antibodies was no difference. After selection of fused cells on the semi-solid medium, about 678 strains of hybridomas were obtained. 58 strains were positive in non-competitive ELISA and 4 strains were positive in competitive ELISA, namely 2A11, 3A12, 3G1 and 5D7. It showed the conjugation AFB2a-BSA could induce bodies to secret antibodies, especially high specificity antibodies against AFB₁.After characterization of hybridomas, the affinity constant of 3G1 was 1.8×108 L/M. Its sensitivity was better than others, upto 1.6±0.066 ng/mL. Its cross-reactivity with B2 was 6.4 % and with G1and G2 below 1 %, and better than the reported. The minimal inhibition concentration was 0.19±0.027 ng/mL. The supernate of culturing hybridomas was tested in 7 weeks by competitive ELISA. It showed 3G1 was stable. A high specificity antibody was the key to develop a high specificity immunoassay, so 3G1 which performs low cross-reactivity and well stability will be able to use to test AFB₁ and prodELISA was essential for high specificity, sensitivity and though-out immunoassays such as immunostrips, liquid chips and biosensors. In the paper, ELISA for AFB₁ was developed using antibody 3G1. The concentration of methanol influenced sensitivity sharply. For instance, the sensitivity of 10 % methanol was three time of one of 40 % methanol. The sensitivity was stable in the solution which contains high concentration ions, but interaction of immobilized antigen and antibody was disturbed. The acidic surrounding was not benefit to the interaction, but the weak alkaline surrounding. Matrix could affect the activity of enzyme, but an indirect ELISA was adopted to decrease the effect, although the effect of interaction of antigen and antibody was able to avoid. Spiked three concentrations, 80 ng/mL, 20 ng/mL and 8.0 ng/mL, the recoveries were between 90 % and 120 %. It showed the assay would be able to test AFB₁ in peanuts.