全氟辛酸特异性单域重链抗体的制备

Preparation of Specifi c Single-Domain Heavy-Chain Antibody for Perfl uorooctanoic Acid

[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 ] To generate a specific antibody for perfluorooctanoic acid （PFOA） based on antigen-antibody specific binding, which can be used to develop an immunoassay for on-site fast detection of PFOA. [ Methods ] At first, a PFOA- bovine serum albumin （BSA） complete antigen was synthesized by carbodiimide method. Then an varible domain of heavy china of heavy-chain antibody （VI-IH） phage display library was used for isolating positive clones. The DNA region of the specific antibodies by polymerase chain reaction amplification was ligated to pET22b-Fc vector, and the antibody was expressed using a prokaryotic expression system. The specificity of the antibody was confirmed by indirect competitive enzyme-linked immunosorbent assay （ELISA） assay. [ Results ] The complete antigen PFOA-BSA was synthesized in a coupling ratio of 8. Using the VHH antibody library, 7 positive clones were isolated after four rounds of screening. Five antibodies containing human Fc fragment were obtained with the molecular weights in line with our expectations. Finally, one VHH antibody was verified by indirect competitive ELISA assay showing specific binding to PFOA. [ Conclusion ] A specific VHH antibody for PFOA is obtained, which might be used to develop immunoassays for fast detecting PFOA in various media.