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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 anti-Digoxin human single chain antibody (ADAScFv) from semi-synthetic phage antibody library to provide human antibody for the diagnosis and treatment of Dig poisoning. Methods: (1) The Dig BSA conjugate was prepared by modified method. (2) Four generations of ScFv phage were screened by immobilized Dig BSA conjugate. Antibodies capable of binding to Dig were screened by ELISA and their activities were identified , Its specificity was analyzed by ELISA competition inhibition assay, and the DNA of positive clones was sequenced and analyzed. Results: (1) Obvious enrichment was observed during the screening of antibody library. (2) A positive clone that could bind Dig and its analogue was obtained. (3) The variable region of positive clone belongs to VH5 and Vλ1 subgroup respectively. Conclusion: The human ADAScFv, which can bind to Dig and other digitalis, was obtained by the phage antibody library technology. After further analysis and antibody engineering, it is possible to provide valuable data for clinical diagnosis and treatment of digitalis intoxication Human antibodies.