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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。
Objective To prepare 3-nitrotyrosine (3-NT) monoclonal antibody and identify its immunological properties. Methods Three nitro-tyrosine-glutaraldehyde-chicken ovalbumin (3NT-G-OVA) synthesized by glutaraldehyde coupling of 3-NT and OVA were used as immunogens to immunize Balb / c mice were immunized with spleen cells and SP2 / 0 myeloma cells for cell fusion, using indirect ELISA screening of positive hybridoma cell lines, expanded culture and antibody immunological identification and analysis. Results A hybridoma cell line stably secreting anti-3-nitrotyrosine monoclonal antibody was obtained. The secreted McAb was Ig G2a. The purified antibody titer was 1: (6.14 × 106) The anti-affinity constant was 7.81 × 107 (L · mol -1). Western blot showed that the McAb specifically binds to 3-nitrotyrosine containing protein. Conclusion An anti-3-NT monoclonal antibody with high immunological activity and specificity was prepared and used to detect the level of protein nitration in samples by Western blot.