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目的制备抗抗CD3ScFv单克隆抗体,用以抗CD3抗体的亲和层析纯化及血清中该类抗体浓度的检测。方法采用常规免疫学方法制备抗抗CD3ScFv单克隆体并制备抗抗CD3ScFv单克隆抗体免疫亲和层析柱,用于抗CD3ScFv蛋白和去除E-tag的Diabody[CD3×Pgp]的分离纯化。采用FACS法测定分别经抗抗CD3ScFV抗体免疫亲和层析柱及抗E-tag亲和层析柱纯化的抗CD3ScFv蛋白和Diabody[CD3×Pgp]对K562/A02和Jurkat细胞特异结合活性。采用间接ELISA法进行抗抗CD3ScFv抗体特异性结合活性的检测。结果抗抗CD3ScFV单克隆抗体能特异性地与抗CD3ScFv蛋白结合而不与血清发生反应。经抗抗CD3ScFV抗体免疫亲和层析柱和经抗E-tag亲和层析柱纯化后的抗CD3ScFv蛋白均能与Jurkat细胞特异结合。经抗抗CD3ScFV抗体免疫亲和层析柱纯化的去除E-tag的Diabody[CD3×Pgp]与K562/A02和Jurkat细胞结合的阳性率分别为89.87%和83.95%;与其亲代抗体竞争结合K562/A02和Jurkat细胞后,结合率分别下降为56.30%和43.78%。结论制备了抗抗CD3单克隆抗体和亲和层析柱,可用于抗CD3抗体的亲和层析纯化及血清中该类抗体浓度的检测。
Objective To prepare anti-CD3ScFv monoclonal antibody for the purification of anti-CD3 antibody by affinity chromatography and the detection of the concentration of such antibodies in serum. Methods Anti-CD3ScFv monoclonal antibody was prepared by routine immunological method and anti-CD3ScFv monoclonal antibody immunoaffinity column was prepared for the isolation and purification of anti-CD3ScFv protein and Diabody [CD3 × Pgp] with E-tag removed. The specific binding activity of anti-CD3ScFv protein and Diabody [CD3 × Pgp] purified by anti-CD3ScFV antibody immunoaffinity column and anti-E-tag affinity column to K562 / A02 and Jurkat cells were determined by FACS. Indirect ELISA was used to detect the anti-CD3 ScFv antibody specific binding activity. Results The anti-CD3 ScFV monoclonal antibody specifically binds to the anti-CD3 ScFv protein without reacting with serum. Anti-CD3ScFV antibody immunoaffinity column and anti-CD3ScFv protein purified by anti-E-tag affinity column can all specifically bind to Jurkat cells. The positive rate of binding of Diabody [CD3 × Pgp] to K562 / A02 and Jurkat cells purified by anti-CD3ScFV antibody immunoaffinity column was 89.87% and 83.95%, respectively. The competitive antibody binding K562 / A02 and Jurkat cells, the binding rate decreased to 56.30% and 43.78% respectively. Conclusion Anti-CD3 monoclonal antibodies and affinity chromatography columns were prepared for purification of anti-CD3 antibodies by affinity chromatography and detection of the concentration of such antibodies in serum.