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目的从大容量甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)人源单链抗体(single chain variable fragments,sc Fv)库中筛选特异性抗ATC单链抗体并进行生物特性鉴定。方法对该抗体库进行4轮筛选后,挑取抗ATC阳性克隆感染E.coli HB2151;并用异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导该sc Fv抗体可溶性表达,ELISA法检测其可溶性表达;经HiTrap~(TM) Anti E-tag亲和柱层析纯化抗体;细胞ELISA鉴定可溶性抗体与细胞结合的特异性,分别设置TT细胞、ARO细胞、人肝癌Hep G2细胞及PBS空白对照组,用酶标仪检测各组在450 nm处的吸光度。SDS-PAGE法及Western blot检测抗体的表达和相对分子质量;流式细胞术观察细胞凋亡情况。结果经4轮筛选后抗体实现了显著富集,并得到了纯化抗体。ELISA结果表明筛选出的抗体与甲状腺未分化癌细胞能够特异性结合。SDS-PAGE和Western blot检测结果显示ATC抗体的相对分子质量约为2.9×10~4。流式细胞术结果显示ARO细胞经sc Fv特异性作用后细胞明显凋亡。结论成功获得抗ATC特异性人源单链抗体,鉴定结果显示抗体活性较好。
OBJECTIVE: To screen specific single-chain anti-ATC single chain variable antibody (scFv) from anaplastic thyroid carcinoma (ATC) and to identify its biological characteristics. Methods After four rounds of screening of this antibody library, anti-ATC positive clones were picked and used to infect E. coli HB2151. The cells were infected with IPTG (isopropyl β-D-thiogalactopyranoside) The soluble antibody was detected by ELISA. The purified antibody was purified by HiTrap ~ (TM) Anti-E-tag affinity column. The specificity of soluble antibody and cell binding was identified by ELISA. TT cells, ARO cells, human Hep G2 cells and PBS control group. The absorbance at 450 nm of each group was detected by microplate reader. The expression and relative molecular weight of the antibody were detected by SDS-PAGE and Western blot. The apoptosis was observed by flow cytometry. Results After 4 rounds of screening, the antibodies were significantly enriched and the purified antibodies were obtained. ELISA results showed that the selected antibodies and thyroid undifferentiated cancer cells can bind specifically. SDS-PAGE and Western blot results showed that the relative molecular mass of ATC antibody was about 2.9 × 10-4. Flow cytometry results showed that ARO cells were significantly apoptosis induced by scFv. Conclusion The anti-ATC specific human single-chain antibody was successfully obtained. The identification results showed that the antibody activity was good.