中文摘要：

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。

英文摘要：

Objective To prepare monoclonal antibody （McAb） against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration. Methods McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [ CD3 × Pgp ] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [ CD3 × Pgp ] without E-tag, and Diabody [ CD3 × Pgp ] purified by anti-CD3 affinity chromatography column or anti-E- tag affinity chromatography column against KS62/A02 cell and Jurket cells were detected by fluorescence activated cell sorting （FACS） method. ELISA was used to identify the specificity of the McAb. Results MeAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [ CD3 × Pgp ]without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [ CD3 × Pgp] without E-tag decreased to 56.30% and 43.78%, respectively. Conclusion The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.