人源性抗地高辛单链抗体(scFv)及diabody的获取

目的:从大容量噬菌体抗体库中筛选人源性抗地高辛单链抗体(scFv),并构建双价抗体(diabody)。方法:以固相化的Dig对构建的大容量噬菌体抗体库进行筛选,4轮后挑取集落,用ELISA法鉴定其特异性,并对抗Dig阳性抗体基因进行DNA指纹分析及测序分析。选取活性好的抗体基因进行改造,构建diabody。结果:在抗体库的筛选过程中可见到明显的富集现象,获得4株可与Dig特异性结合的人源单链抗体,经DNA指纹分析及测序分析证明为不同抗体基因,分泌抗Dig抗体的阳性克隆的可变区基因轻链均属于λ第1亚群,重链基因分别属于第3和第4亚群。选取4号克隆进行基因改造,构建diabody的表达载体,获得活性较好的diabody。结论:利用噬菌体抗体技术获得了人源性抗Dig的抗体,并改造成应用前景较好的diabody,为临床诊断及治疗洋地黄类药物的中毒提供了具有实用价值的人源抗体。

Preparation of humanized anti-digoxin scFv and diabody

AIM: To prepare humanized anti-digoxin(Dig) antibodies(single chain Fv, scFv) from a human phage antibody library and construct anti-Dig diabody vector. METHODS: A human phage antibody library was panned against immobilized Dig for four times, and the specificity of the selected scFv expression clones was identified by ELISA. Positive clones against Dig were analyzed by DNA fingerprint and sequencing. A clone which had high affinity to Dig was selected to construct a diabody vector. Diabody was secreted from E. coli by IPTG induction, and the specificity was also identified by ELISA. RESULTS: After four rounds of panning, four specific humanized anti-Dig antibodies(scFv) were obtained. DNA fingerprint and sequencing analysis proved that they had different Ab-encoding genes. The V(L) of the 4 expression clones belonged to lambda subgroup 1 and V(H) belonged to 3 and 4 subgroups, respectively. A scFv clone was picked out to construct diabody vector. The prepared humanized diabody reacted with Dig specifically. CONCLUSION: Humanized anti-Dig antibodies(scFv) have been got by using phage display. A diabody has been obtained from scFv. These humanized anti-Dig antibodies may be used for the diagnosis and therapy of Dig toxication.