抗P-选择蛋白人源性单克隆抗体的制备

The Preparation of Human Anti-P-Selectin Monoclonal Antibodies

目的:获得人源性抗P-选择蛋白(selectin)特异性抗体,为相关疾病治疗奠定基础。方法:在HEK293细胞中真核分泌表达人P-选择蛋白功能性片段,以此蛋白片段作为抗原,利用本室构建的大容量全合成人源性噬菌体抗体库进行筛选,经过3轮固相筛选后,阳性克隆得到富集;将其中富集效果最好的一株单链抗体A1改造成全抗体(IgG1),重组质粒转染H293细胞后,抗体得到表达;表达后在全抗体水平上用ELISA和Western印迹分别验证了A1抗体的特异性,并通过非竞争ELISA方法初步测定这株抗体的亲和力。结果:3轮筛选得到3株特异性噬菌体抗体,其中富集效果最好的单链抗体A1改造成全抗体形式后特异性良好,抗体亲和力Kd=2×10-8 mol/L。结论:筛选得到一株特异性较好的抗P-选择蛋白人源性单克隆抗体A1,其特异性和亲和力较好,有继续开发的价值。

Objective： To obtain anti-human P-selectin monoclonal antibodies,and lay a basis for the therapy of P-selectin related diseases.Methods： P-selectin functional segment was expressed secretly by HEK293 cells,which was used as antigen for antibody panning.And then screening from the large fully synthetic human antibody library constructed in our lab,several candidates of antibodies specially binding to P-selectin were obtained,after 3 rounds of solid panning,the positive clones were enriched.Next,one candidates A1,which was enriched suffi-ciently,was transferred from scFv to whole antibody（IgG1）.At last,using ELISA and Western blot to identify the specificity of A1.Also using non-competitive ELISA method to detecte the affinity of A1.Results： After panning,we got 3 different candidates,and changed one of them into whole antibody form.The ELISA and Western blot results showed that the specificity of A1 was excellent.Furthermore,the affinity of A1 was detected by non-com-petitive ELISA method and the affinity constant Kd was 2×10-8 mol / L.Conclusion： We got an excellent anti-hu-man Pselectin antibody candidant,which proved to have well specificity and affinity,so it has potential value to go on study.