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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。
OBJECTIVE: To screen anti-MAGE-A1 specific single-chain antibody from whole human single-chain phage antibody library and identify its immunological activity. Methods: The purified anti-MAGE-A1 phage antibody was screened from the single-stranded phage antibody library by five cycles of adsorption-elution-amplification using purified MAGE-A1 as antigen. The antigen binding ability of MAGE-A1 was detected by ELISA and DotBlotting Specific clones were extracted to express soluble antibodies. Western Blotting, DotBlotting and immunocytochemistry were used to detect the antigen binding activity, and non-competitive ELISA was used to detect the affinity constants. Results: The phage antibody was screened from the single-stranded phage antibody library and enriched, and was identified as a specific phage antibody against MAGE-A1. The molecular weight of anti-MAGE-A1 soluble antibody was about 30kD, which was specifically bound to MAGE-A1 with an affinity constant of about 1.432 × 106L / mol. CONCLUSION: The obtained full-human anti-MAGE-A1 scFv retains the specificity of intact antibody binding antigen and has weak immunogenicity. It is an ideal carrier for tumor-directed therapy.