B族黄曲霉毒素抗原合成设计及特异性、广谱性抗体制备与特性分析

为制备B族黄曲霉毒素(BGAFs)特异性强、广谱性好的抗体,本研究根据黄曲霉毒素B₁(AFB₁)的分子结构和活性位点,采用肟化活泼酯法(OAE)、氨甲基化法(MOA)、混合酸酐法(MA)、半缩醛法(SA)、环氧化物法(EP)和烯醇醚衍生物法(EED)6种方法合成BGAFs人工抗原AFB₁-BSA,并通过UV和SDS-PAGE进行鉴定;采用AFB₁-BSA免疫新西兰白兔制备多克隆抗体(AFB₁ pAb),间接ELISA检测AFB₁ pAb效价,间接竞争ELISA(icELISA)分析其敏感性,交叉反应试验(CR)分析其特异性和广谱性。结果表明,AFB₁-BSA合成成功,在BGAFs抗原合成的6种方法中OAE效果最好,AFB₁与BSA的分子结合比为8.46∶1;AFB₁ pAb的间接ELISA效价为1∶(1.28×10⁴),IC₅₀为10.32 μg·L^-1,与AFB₂、AFG₁、AFG₂、AFM₁、AFM₂的CR分别为75.21%、44.13%、14.72%、16.36%、1.44%。本研究制备出了高效价、敏感、特异、广谱的AFB₁ pAb,为BGAFs免疫检测方法的建立奠定了物质和技术基础。

Design of Antigen Synthesis and Characterization of Specific and Eurytopic Antibodies Against B-group Aflatoxins

The aim of this study was to prepare B-group aflatoxins(BGAFs) antibody with strong specificity and goodeurytopicity. According to the molecular structure and active site of aflatoxin B₁ (AFB₁), the BGAFs artificial antigen AFB₁-BSA was prepared by 6 methods including oxime active ester(OAE),methylation of ammonia(MOA),mixed anhydride(MA),semi acetal(SA),epoxide(EP) and enol ether derivative(EED) and identified by UV and SDS-PAGE. Polyclonal antibodies against AFB₁(AFB₁ pAb) were prepared by immunizing New Zealand rabbits with AFB₁-BSA, and the titers of AFB₁ pAb was detected by indirect ELISA, the sensitivity of AFB₁ pAb was analyzed by indirect competitive ELISA(icELISA) and the specificity and eurytopicity of AFB₁ pAb was analyzed by cross-reactivity(CR) test. The results showed that AFB₁-BSA was synthesized successfully and the best one was OAE method among the six synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB₁ to BSA was 8.46∶1. The immune efficacy of OAE method was the best, that its AFB₁ pAb had the highest titers of 1∶1.28×10⁴ by indirect ELISA, a good sensitivity with the 50% inhibition concentration(IC₅₀) of 10.32 μg·L^-1 to AFB₁ by icELISA and showed a high CR to AFB₂ of 75.21%, AFG₁ of 44.13%, AFG₂ of 14.72%, AFM₁ of 16.36% and AFM₂of 1.44%, respectively. In this study, AFB₁ pAbs with high titer, sensitivity, specificity and eurytopicity were prepared, proving a technical support for the establishment of BGAFs immunoassay.