抗生物素化三聚氰胺人源单链可变区抗体筛选与鉴定

Screening and identification of biotinylated melamine in human single-chain variable region antibody

目的筛选生物素化三聚氰胺人源单链可变区(scFv)抗体,为研究相关快速检测试剂盒创造条件。方法本试验利用生物素化三聚氰胺为包被抗原,从半合成噬菌抗体库中筛选其特异性噬菌体抗体片段。即采用菌噬体表面展示技术,以链亲和素-生物素标记的三聚氰胺为固相包被靶抗原,把靶抗原包被在免疫平板上,加入噬菌体抗体库,则头部带有能与靶抗原特异性结合的抗体分子的噬菌体就被固定在免疫平板上,不能特异结合的噬菌体则被漂洗掉;将特异结合的噬菌体洗脱下来,侵染大肠杆菌,就可以得到含抗体基因的噬菌粒。结果从半合成的菌噬体单链可变区抗体库中经过3轮"吸附-洗脱-扩增"筛选过程后,获得抗原特异性和结合性较强的生物素化三聚氰胺scFv片段。结论酶联免疫吸附试验等进行鉴定后,筛选得到了抗生物素化三聚氰胺的噬菌体抗体基因片段,为下一步亲和力鉴定、蛋白表达及开发相关检查试剂盒奠定了基础。

Objective To screen biotinylated melamine in human single-chain variable region（scFv）antibody and to create conditions for the study of rapid detection kit.Methods Biotinylated melamine were used as coating antigen and antigen-specific phage antibody fragments were screened from semisynthetic phage antibody library.Using bacteria phage surface display technology,streptavidin-biotinylated melamine coated solid phase target antigen and the target antigen was coated on the immune flat.After phage library were added,the phages′head with antibody molecule which could bind with scFv-antigen specifically was fixed on the immune plates,which could not bind with the antigen specifically were rinsed.Then the eluted specific binding phages were used to infect E.coli,macrophages contained specific antibody genes bacteria tablets were obtained.Results After three semi-synthetic bacteria from phage single chain variable region antibody library ＂absorption-elution-amplification＂screening process,scFv fragments biotinylated melamine with stronger antigen binding specificity were obtained.Conclusion By enzyme-linked immunosorbent assay,fragments of biotinylated melamine in human single-chain variable region antibody could be identified,which may lay the foundation of the identification of affinity,the expression of protein and the development of the test kits.