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目的从全人源单链噬菌体抗体库中筛选出抗前列腺特异性膜抗原(PSMA)特异性单链抗体并进行免疫活性鉴定。方法以合成的PSMA多肽为抗原,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PSMA噬菌体抗体,ELISA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和免疫组织化学检测其抗原结合性,非竞争ELSIA法检测其亲和常数。结果从单链噬菌体抗体库中筛选出的噬菌体抗体,经ELISA鉴定为抗PSMA的特异性噬菌体抗体。抗PSMA可溶性抗体相对分子质量约为3.0×104,与PSMA特异性结合,其亲和常数约为5.077×106L/mol。结论所得全人源抗PSMA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 out specific anti-prostate specific membrane antigen (PSMA) scFv from whole human single-chain phage antibody library and identify its immunological activity. Methods The specific anti-PSMA phage antibody was screened from the single-stranded phage antibody library by five cycles of adsorption-elution-amplification using the synthesized PSMA peptide as an antigen. The antigen binding ability of the anti-PSMA peptide was detected by ELISA. Clones were extracted for expression of soluble antibodies. Antigen binding was detected by Western Blotting and immunohistochemistry. The non-competitive ELSIA method was used to detect the affinity constants. Results The phage antibodies screened from the single-stranded phage antibody library were identified as anti-PSMA specific phage antibodies by ELISA. The relative molecular mass of the anti-PSMA soluble antibody was about 3.0 × 104, which was specifically bound to PSMA with an affinity constant of about 5.077 × 106 L / mol. Conclusion The obtained full-human anti-PSMA single-chain antibody retains the specificity of intact antibody molecule-bound antigen and has low immunogenicity and is an ideal carrier for tumor-directed therapy.