Qu Li-Na, Yang Tang-Bin, Yuan Yan-Hong, Zhong Ping, Yang Bin, Zhao Hai
13th Department, Institute of Space Medico-Engineering, 1 West Yuanmingyuan Road, Beijing, China.
Hybrid Hybridomics. 2003 Dec;22(6):401-6. doi: 10.1089/153685903771797129.
3-Nitro-L-tyrosine (nitrotyrosine) has recently been considered to be useful as a biomarker of endogenous production of several reactive nitrogen species including peroxynitrite. In the present study, nitrotyrosine was coupled to human serum albumin (HSA) using a two-step glutaraldehyde method and immunized mouse with multifocal intradermal injections. Using a conventional immunization protocol, 12 stable monoclonal antibodies (MAbs) producing cell lines recognizing nitrotyrosine were obtained. Six MAbs were selected for further characterization. A study of cross-reactions with nitrotyrosine-like compounds showed that the antibodies had a high specificity for nitrotyrosine, but no detectable reactivity with L-tyrosine, p-nitro-L-phenylalanine, o-phospho-L-tyrosine or 3-amino-L-tyrosine. Using these high titer and affinity antibodies, a competitive inhibition ELISA was developed with a lower detection limit of approximately 20 nmol/L to detect both free and protein-bound nitrotyrosine in biological systems.
3-硝基-L-酪氨酸（硝基酪氨酸）最近被认为是包括过氧亚硝酸盐在内的几种活性氮物质内源性生成的有用生物标志物。在本研究中，使用两步戊二醛法将硝基酪氨酸与人血清白蛋白（HSA）偶联，并通过多部位皮内注射免疫小鼠。采用常规免疫方案，获得了12株产生识别硝基酪氨酸的稳定单克隆抗体（MAb）细胞系。选择6株单克隆抗体进行进一步表征。与硝基酪氨酸样化合物的交叉反应研究表明，这些抗体对硝基酪氨酸具有高特异性，但与L-酪氨酸、对硝基-L-苯丙氨酸、邻磷酸-L-酪氨酸或3-氨基-L-酪氨酸无明显反应性。使用这些高滴度和高亲和力抗体，开发了一种竞争性抑制ELISA，检测限约为20 nmol/L，用于检测生物系统中游离和与蛋白质结合的硝基酪氨酸。
