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[目的]基于抗原抗体特异性结合的原理,制备一种针对全氟辛酸(PFOA)小分子的特异性抗体,以用于PFOA的现场快速检测。[方法]采用碳二亚胺法将PFOA与牛血清白蛋白(BSA)偶联,将此完全抗原作为包被抗原,用重链抗体噬菌体文库进行筛选获得阳性克隆,聚合酶链反应扩增重链抗体片段并与含有人抗体Fc段的质粒连接,采用原核表达系统进行抗体蛋白表达和纯化,间接竞争酶联免疫吸附试验(ELISA)进行验证。[结果]成功合成完全抗原PFOA-BSA,偶联比为8。采用重链抗体库进行4轮筛选,获得7株阳性克隆,经进一步的原核表达系统进行蛋白表达和纯化,得到5种与预期大小相符的、含有人抗体Fc段的抗体蛋白。经间接竞争ELISA方法初步检测验证,获得1个可与PFOA特异结合的单域重链抗体。[结论]最终获得针对PFOA的特异性抗体,有望用于建立快速检测不同介质中PFOA的免疫学检测方法。
[Objective] Based on the principle of antigen-antibody specific binding, a specific antibody against perfluorooctanoic acid (PFOA) small molecule was prepared for rapid on-site detection of PFOA. [Method] The PFOA was coupled with bovine serum albumin (BSA) by carbodiimide method. The complete antigen was used as coating antigen, and the positive clones were screened by the phage display library of heavy chain antibody. The PCR amplification amplified Antibody fragments were ligated with plasmids containing the Fc region of human antibody. The prokaryotic expression system was used to express and purify antibody. The indirect competitive enzyme-linked immunosorbent assay (ELISA) was used to validate the results. [Result] The complete antigen PFOA-BSA was successfully synthesized with a coupling ratio of 8. Four heavy chain antibody libraries were screened and seven positive clones were obtained. After further prokaryotic expression, the proteins were expressed and purified. Five antibody fragments containing the Fc region of human antibody were obtained according to the expected size. Indirect competitive ELISA was used to detect and identify a single domain heavy chain antibody that could specifically bind to PFOA. [Conclusion] The specific antibodies against PFOA were finally obtained and could be used to establish immunological detection methods for rapid detection of PFOA in different media.