Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system
Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system
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DOI：
10.1128/cdli.10.4.587-595.2003
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发表时间：
2003-07-01
期刊：
影响因子：
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通讯作者：
Morrow, KJ
中科院分区：
文献类型：
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作者：
Deng, XK;Nesbit, LA;Morrow, KJ
采用重组抗体克隆技术和噬菌体展示技术制备艰难梭菌毒素B单链抗体（scFv），以小鼠B细胞杂杂瘤细胞系5A8为起始材料，产生抗该毒素的单克隆抗体。Krebber等人的克隆、筛选和噬菌体展示集成系统[j]。方法（201:35- 55,1997）使我们能够从杂交瘤细胞系中快速获得毒素b结合的scFv序列。根据初步酶联免疫吸附试验（ELISA）筛选数据，将最佳候选scFv序列亚克隆到兼容表达载体中。重组单链抗体在大肠杆菌中表达。在聚丙烯酰胺凝胶电泳上观察到一条29 kda的条带，与预测一致。表达产物经免疫印迹和抗flag抗体检测鉴定。采用夹心ELISA法检测单链抗体与毒素b的结合功能。该抗体对毒素B具有高度特异性，并且不会与毒素B阴性艰难梭菌菌株分离的物质发生交叉反应。ELISA结果表明，该可溶性单链抗体的灵敏度明显高于原单克隆抗体，最低可检出10 ng的毒素B。竞争性elisa结果表明，5A8亲本抗体与最佳代表单链抗体（克隆10）亲和度相似，且在10(-8)m范围内，提示重组抗体技术是开发下一代免疫诊断试剂的快速有效途径。
Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.