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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 a monoclonal antibody to monomeric C-reactive protein (mCRP). METHODS: BALB / c mice were immunized with synthetic human C-reactive protein terminal octapeptide amino acids conjugated with bovine serum albumin. The spleen cells were fused with mouse myeloma cells SP2 / 0 in a 5: 1 ratio , Indirect competitive ELISA and limiting dilution method for screening monoclonal hybridoma cells; preparation of ascites antibody; using Protein G affinity chromatography purification of antibodies by indirect ELISA and Western blot determination of antibody specificity, and the use of the monoclonal antibody To investigate the protective effects of mCRP on neonatal rat cardiomyocytes. Results: A hybridoma cell line secreting monoclonal antibodies was obtained. The purity of the purified monoclonal antibody was 98%, and the antibody activity was good. The antibody could reduce the expression of TGF-β in mCRP-induced cardiomyocytes. CONCLUSION: One hybridoma cell that can stably secrete monoclonal antibody was successfully obtained and its primary application was identified. It was confirmed that it could neutralize the damage of mCRP-induced cardiomyocytes.