【摘要】 目的:从全人源单链噬菌体抗体库中筛选出抗MAGE鄄A1特异性单链抗体并进行免疫活性鉴定。方法:以纯化MAGE鄄A1为抗原,经过五轮吸附—洗脱—扩增,从单链噬菌体抗体库中筛选出特异性抗MAGE鄄A1噬菌体抗体,ELISA、DotBlotting检测其抗原结合能力,并对特异性较强的克隆提取质粒,表达可溶性抗体。WesternBlotting、DotBlotting、免疫细胞化学检测其抗原结合活性,非竞争ELISA法检测其亲和常数。结果:从单链噬菌体抗体库中筛选出噬菌体抗体并进行富集,经鉴定为抗MAGE鄄A1的特异性噬菌体抗体。抗MAGE鄄A1可溶性抗体分子量约为30kD,与MAGE鄄A1特异性结合,其亲和常数约为1.432×106L/mol。结论:所得全人源抗MAGE鄄A1单链抗体保留完整抗体分子结合抗原的特异性,免疫原性弱,是肿瘤导向治疗的理想载体。更多还原

【Abstract】 Objective:To screen and identify antibody against MAGE-A1 from a human scFv antibody library. Methods:Using purified MAGE-A1 as coating antigen, the scFv antibody library was screened by five times combining-eluting-amplifying. The positive phage antibodies were identified by ELISA and Dot Blotting. The soluble antibody was identified by Western Blotting, Dot Blotting and immunocytochemical study. The affinity constant of soluble antibody was measured by non-competitive ELISA. Results: The screened phage antibodies were specific for MAGE-A1. The soluble antibody was also specific for MAGE-A1; its molecular weight was about 30 kD by SDS-PAGE and its affinity constant was about 1.432× 106 L/mol. Conclusion: The screened scFv antibody is specific and with low immunoenicity. It can be further used in target treatments of malignant tumors.更多还原