抗地高辛人源小分子抗体的获取

Preparation of Human Anti-Digoxin Antibody Fragments

目的 从大容量噬菌体抗体库中筛选人源性抗地高辛抗体，并构建双体抗体。方法 以固相化的地高辛对构建的大容量噬菌体抗体库进行筛选，3～4轮后挑取克隆用酶联免疫吸附测定方法鉴定其特异性，对抗地高辛阳性抗体克隆进行DNA指纹分析及测序分析。选取活性好的克隆进行改造，构建双体抗体。结果 在抗体库的筛选过程中可见到明显的富集现象，获得了4株可与地高辛特异性结合的人源抗体，经DNA指纹分析及测序分析证明为不同克隆基因，阳性克隆的可变区基因轻链均属于入第一亚群，重链分别属于第三和第四亚群。选取4号克隆进行基因改造，构建双体抗体表达载体，获得了活性较好的双体抗体。结论利用噬菌体抗体技术获得了人源性抗地高辛抗体，并改造成应用前景较好的双体抗体，可能为临床诊断及治疗洋地黄类药物中毒提供具有实用价值的人源抗体。

Objective To obtain human anti-Digoxin antibodies from a large human phage antibody library and to construct diabody vector. Methods A large human phage antibody library was panned against immobilized Dig for 3-4 times. The specificities of clones were identified by ELISA. Positive clones against Dig were analyzed by DNA fingerprint and sequencing analysis. A diabody vector was constructed with a selected clone with good specificity to Dig. Results There was obvious enrichment during four times panning against Dig. Four specific human anti-Dig antibodies were obtained. DNA fingerprint and sequencing analysis proved they were different clones. All the 4 VLs belong to λ, subgroupl and the VHs belong to subgroup 3 and 4 respectively. One clone was picked out to construct diabody vector. The diabody also bound Dig specifically. Conclusion Human anti-Dig antibodies were obtained by phage display. A diabody expression vector was constructed. These anti-Dig human antibodies may provide a potential reagent for the diagnosis and therapy of Dig toxication.