琥珀酰辅酶A转移酶硝基化酪氨酸位点特异单克隆抗体的制备

Development of monoclonal antibody against nitrated Tyr^4 and Tyr^(76) in succinyl CoA:3-oxoacid CoA transferase

线粒体琥珀酰辅酶A转移酶(SCOT)含有4个酪氨酸残基,其4、76位点酪氨酸(Tyr4、Tyr76)的硝基化可导致其酶活性下降,制备特定的抗体可对其硝基化水平和位点进行检测。应用淋巴细胞杂交瘤技术,以化学合成的含琥珀酰辅酶A转移酶硝基化Tyr4和Tyr76肽段为半抗原,偶联钥孔血蓝蛋白(KLH)后,作为免疫原免疫Balb/c小鼠,分离免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,获得了多株融合细胞。经对融合细胞多次筛选和检测,最终获得4株单克隆抗体,分别对应SCOT Tyr4和Tyr76硝基化后表位,其间接ELISA检测效价分别达到1∶128 000、1∶32 000、1∶512 000和1∶4 096 000,表明抗体具有较强特异性和灵敏度。

There are 4 tyrosine residues in succinyl CoA： 3-oxoacid CoA transferase （SCOT）, in which nitration of Tyr4 and Tyr76 will lead to change of the structure and decrease of enzymatic activity. In this study artificial synthetic peptides covering the SCOT Tyr4 and Tyr76 region with the tyrosine nitrated were employed as hapten and conjugated to keyhole limpet hemocyanin （KLH） to immunize Balb/c mouse. The monoclonal antibodies against nitrated Tyr4 and Tyr76 in SCOT are developed. After screening of massive cell lines, 4 antibodies against nitrated Tyr4 and tyr76 epitope of SCOT respectively were selected and determined by indirect ELISA. The result showed that the titer of the antibodies were 1 ： 128 000, 1 ： 32 000, 1 ： 512 000 and 1 ： 4 096 000 respectively. The results indicated that the antibodies obtained showed good specificity and sensitivity which will lay a foundation for further study of SCOT nitrotyosine modification and its impact on physiology.