从半合成噬菌体抗体库中克隆抗地高辛人单链抗体

Cloning of human ScFv against digoxin from a semi-synthetic phage antibody library

目的 :从半合成噬菌体抗体库中克隆抗地高辛 (Dig)人单链抗体 (ADAScFv) ,为Dig中毒的诊断和治疗提供人源性抗体。方法 :①以改良法制备Dig BSA偶联物 ;②以固相化的Dig BSA偶联物对人噬菌体ScFv抗体库进行了 4轮筛选 ,通过ELISA筛选出能结合Dig的抗体克隆并鉴定其活性 ,通过ELISA竞争抑制实验分析其特异性 ,对阳性克隆的DNA进行测序分析。结果 :①在对抗体库的筛选过程中可见有明显的富集 ;②获得一株可结合Dig及其类似物的阳性克隆 ;③阳性克隆的可变区分别属于VH5和Vλ1亚群。结论 :利用噬菌体抗体库技术获得了能与Dig及其它洋地黄类药物结合的人ADAScFv ,经进一步分析及抗体工程改造后 ,有可能为临床上诊断及治疗洋地黄类药物中毒提供具有实用价值的人源性抗体。

Objective:To clone human anti digoxin ScFv from a human semi synthetic phage antibody library.Methods:①Digoxin BSA conjugate(Dig BSA) was preparaed by a modified periodate oxidation method.②A semi synthetic phage antibody library was panned against immobilized Dig BSA.Collected clones were analysed by ELISA,inhibition ELISA and DNA Sequencing.Results:①During the four rounds panning against Dig BSA,the enrichment of the eluted phage particles was observed;②Analysis of the eluted clones identified one clone that could bind Dig as well as other digitalis.③Sequencing analysis showed that the variable genes of the positive clone belonged to VH5 and Vλ1 subgroup respectively.Conclusion:Human Dig specific ScFv that could bind Dig and other digitalis has been cloned from a semi synthetic phage antibody library which may provide a potential reagent for the diagnosis and therapy of Dig toxication.