具有中和功能的抗单体C-反应蛋白单克隆抗体的制备及其初步应用

Characterization and potential application of monoclonal antibody specific to monomeric C-reactive protein

目的:制备单体C-反应蛋白(monomeric C-reactive protein,mCRP)的单克隆抗体。方法:用合成的人C-反应蛋白末端八肽氨基酸,与牛血清白蛋白偶联后免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1的比例融合,间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;制备腹水抗体;采用Protein G亲和层析法纯化抗体,用间接ELISA和Western blot方法测定抗体特异性,并采用该单抗研究其对mCRP干预的乳大鼠心肌细胞的保护作用。结果:得到1株能稳定分泌单克隆抗体的杂交瘤细胞株,纯化后的单克隆抗体纯度达98%,抗体活性良好,并且该抗体可以降低mCRP导致的心肌细胞TGF-β的表达。结论:成功获得1株能稳定分泌单克隆抗体的杂交瘤细胞,并对其初步应用进行了鉴定,并证实其可以中和mCRP引起的心肌细胞的损伤作用。

Objective：To prepare monoclonal antibody（MAb）against monomeric C-reactive protein.Methods：BALB/c mice were immunized with mCRP-BSA and the spleen cells of the mice were fused with myeloma cells SP2/0 at the ratio of 5∶1.Monoclonal hybridomas were screened by competitive indirect enzyme-linked-immunoadsorbent assay（ELISA）and limited dilution.Mouse ascites were prepared by intraperitoneal injection of hybridoma cells.MAbs to mCRP was purified by Protein G Affinity chromatography methods.The specificity and activity of ascites antibody were determined by Western Blot and indirect ELISA.The protective effects of this antibody influenced on cardiomyocytes were further studied.Results：Strain of hybridoma cell line could secrete MAb stably. After purification,the obtained monoclonal antibody can decrease mCRP induced TGF-β expression on cardiomyocytes.Conclusion： The obtained strain of hybridoma cell line could secrete MAb stably,and purified MAb had protective effect on cardiomyocytes.