目的制备抗3-硝基酪氨酸(3-NT)单克隆抗体并鉴定其免疫学特性。方法用化学合成方法将3-NT与OVA用戊二醛偶联合成的人工抗原3硝基酪氨酸-戊二醛-鸡卵白蛋白(3NT-G-OVA)作为免疫原,皮下注射免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA法筛选阳性杂交瘤细胞株,扩大培养并对抗体进行免疫学鉴定与分析。结果获得1株稳定分泌抗3-硝基酪氨酸单克隆抗体的杂交瘤细胞株,所分泌的单抗亚型为Ig G2a;纯化后的抗体效价为1∶(6.14×106);单抗亲和力常数为7.81×107(L·mol^-1);Western blot检测证明该单抗与含有3-硝基酪氨酸的蛋白能特异地结合。结论制备了1株具有较高免疫学活性和特异性的抗3-NT单克隆抗体,并可用于Western blot检测病人样本中的蛋白硝基化水平。
This study aimed to prepare monoclonal antibodies(m Ab) against 3-nitrotyrosine(3-NT) and analyze its immunological traits. With synthesized 3NT-G-OVA as antigen, Balb/c mice were immunized and the splenocytes of the immunized mice were fused with the SP2/0 myeloma cells by hybridoma technique. Results showed that a hybridoma cell line 2F6 was established, which could stably secret monoclonal antibody against 3-NT. The secreted antibody was analyzed for titer, isotype, affinity and specificity. The isotype of the mc Ab was Ig G2a/κ, the indirect ELISA showed the titers of m Ab was 1∶(6.14×106), and the affinity constant was 7.81×107(L·mol^-1). Furthermore, indirect ELISA/Western blot indicated that the mc Ab could recognize the peptide/protein containing 3-NT. From all the results, we presume that the mc Ab prepared in this paper has high immune-activity and specificity, and might be useful for diagnosis of diseases related to 3-nitrotyrosine.