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目的从单链大容量噬菌体抗体库中筛选特异性的抗HIF-1α肺腺癌人源单链抗体(scFv),并进行初步鉴定。方法先后以肺腺癌细胞A549、HIF-1α为抗原,通过“吸附-洗脱-扩增”从大容量噬菌体抗体库中筛选特异性抗体,感染大肠杆菌HB2151,实现可溶性抗体的表达,ELISA、免疫细胞化学检测抗原-抗体结合活性,SDS-PAGE和Western blot检测可溶性抗体表达和相对分子质量。结果经筛选后,单链抗体得到富集。随机选取10个克隆,ELISA检测5个与HIF-1α抗原呈阳性反应,阳性率为50%。免疫细胞化学表明抗体与A549细胞特异性结合。SDS-PAGE、Western blot证明单链抗体成功可溶表达,且相对分子质量在30×103左右,ELISA显示其有较好的特异性。结论利用单链大容量噬菌体抗体库技术成功获得特异性抗HIF-1α的肺腺癌人源单链抗体。
Objective To screen specific single-chain human scFv against HIF-1α lung adenocarcinoma from a single-stranded large-scale phage antibody library and make a preliminary identification. Methods The specific antibodies were screened from the large-scale phage antibody library by using the "lung adenocarcinoma cell line A549 and HIF-1α as the antigen and then were infected with Escherichia coli HB2151 to obtain soluble antibody expression. ELISA, immunocytochemistry antigen-antibody binding activity, SDS-PAGE and Western blot detection of soluble antibody expression and relative molecular mass. Results After screening, single chain antibodies were enriched. Ten clones were randomly selected and tested positive for HIF-1α antigen by ELISA. The positive rate was 50%. Immunocytochemistry showed that the antibody specifically binds to A549 cells. SDS-PAGE and Western blot showed that the single-chain antibody was successfully expressed in soluble form, and its molecular weight was about 30 × 103. ELISA showed that it had good specificity. Conclusion The human single-chain antibody of lung adenocarcinoma with specific anti-HIF-1α was successfully obtained by the single-chain high-capacity phage antibody library.