Development Of An Indirect Competitive Enzyme Immunoassay For Anatoxin B1

After screening the hybridoma cell lines which were fused by our laboratory,we acquired three anti-aflatoxin B1monoclonal antibody cell lines. The subclassand specificity of three cell lines are identified by using indirect competitiveenzyme-linked immunosorbent assay. Finally, we choose the10D12cell line toproduce the monoclonal antibody as much as possible. The results of purificationby caprylic-ammonium sulfate combined with HiTrap Protein G HPchromatography showed that the main antibody subclass is IgG1. The affinityconstant is2.85×10¹⁰L/mol, which represents a high affinity of antibody. Thepurified antibody was analyzed by SDS-PAGE and displaying heavy chain andlight chain and no other protein. The activity which determined by indirectenzyme-linked immunosorbent assay was1:16000. All results indicated that theantibody was in good conditions.An indirect competitive chemiluminescence enzyme immunoassay for aflatoxinB1was developed. Luminol, p-iodophenol, hydrogen peroxide were used as signaldetecting system, in which the optical concentration of luminol, p-iodophenol andhydrogen peroxide was1mM, respectively. The effects of several factors wereoptimized and the standard curve was established as well. The linear equation wasLogit（Y）=1.5602-4.1389log（X）. The half inhibitory concentration was2.4314μg/L. The detection limit of AFB1was0.09μg/L and the linear range of proposedmethod was0.31²0μg/L. The established CLEIA method shows a good correlationwith the commercial available ELISA kit for AFB1indicating that it can be used todetermine AFB1in real samples.We used the same antigen and antibody to develop an indirect competitive enzyme-linked immunosorbent assay for aflatoxin B1. The detection limit ofproposed method was0.56μg/L and the linear range was0.63¹0μg/L. Thecross-reactive rate for AFG1was14.765%and for others were less than3%. Thecross-reactive rates show that proposed method is good specificity.The sensitivity of proposed chemiluminescence enzyme immunoassay foraflatoxin B1was six-fold higher than that of ELISA and there is a better linear range.The components of CLIEA and ELISA were stored at4℃for three months at least.