抗人SOCS3单克隆抗体的制备及其生物学特性鉴定

Preparation and characterization of a monoclonal antibody against human SOCS3

目的制备高效价的抗人SOCS3单克隆抗体并对其生物学特性进行鉴定。方法以重组GST-SOCS3融合蛋白免疫BALB/c小鼠,取其脾细胞与Sp2/0细胞融合,经多次筛选及克隆化,建立可稳定分泌抗SOCS3单克隆抗体的杂交瘤细胞株。用ELISA及Western blot鉴定单克隆抗体的特性,并测定其效价、Ig亚类及相对亲和力。结果筛选到一株可稳定分泌抗人SOCS3单克隆抗体的杂交瘤细胞株。Western blotting显示在相对分子质量为6.3×104处出现特异性反应带。杂交瘤细胞培养上清效价为1∶640,腹水效价为1∶25600,Ig亚类为IgG2a,相对亲和力达4.84×106L/mol。结论成功制备能特异性识别人SOCS3的单克隆抗体,为进一步研究人体细胞因子信号传导的负反馈调节及SOCS3在微生物感染中的免疫调节作用奠定了基础。

Objective To prepare and characterize the monoclonal antibody （mAb） against human SOCS3. Methods BALB/c mice were immunized with recombinant GST-SOCS3 protein, from which the spleen cells were isolated and fused with Sp2/0 cells. After several rounds of screening and cloning, the hybridoma cell strain secreting anti-SOCS3 mAb was obtained, whose specificity was evaluated using ELISA and Western blotting, and the titer, immunoglobulin subtype and affinity of the mAb were also measured. Results The hybridoma cell strain secreting anti-SOCS3 mAb was identified to belong to IgG2a subtype. The mAb titers in cultural supematant and ascetic fluid were 1：640 and 1：25600, respectively, as determined by ELISA with affinity reaching 4.84×^10^6 L/mol. Conclusion The success in anti-SOCS3 mAb preparation provides the basis for further study of the negative regulation of cytokine signal transduction and the immunoregulation in microorganism infections.