Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine
Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine
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DOI：
10.1016/j.freeradbiomed.2004.09.013
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发表时间：
2005-01-01
影响因子：
7.4
通讯作者：
Osawa, T
中科院分区：
文献类型：
--
作者：
Kato, Y;Kawai, Y;Osawa, T
在吸入过敏原和实验性败血症时，已报道溴化酪氨酸的形成。本研究首先检测了n -溴代琥珀酰亚胺（NBS）处理后的溴化蛋白的免疫原性。获得的免疫血清与溴化牛血清白蛋白（BSA）反应。酶联免疫吸附法测定了NBS剂量依赖性免疫反应性的形成，并通过液相色谱/四极杆串联质谱法（LC/MS/MS）测定了修饰后的牛血清蛋白中3,5-二溴酪氨酸（DiBrY）的形成与NBS剂量依赖性免疫反应性的增加一致。其次，利用免疫小鼠制备溴化抗体的单克隆抗体。从免疫小鼠中获得的两种新型单克隆抗体与DiBrY、3,5-二氯酪氨酸（DiClY）和3,5-二碘酪氨酸（DiIY）反应。此外，这些抗体还能识别3,5-二卤-4-羟基苯甲酸（3,5-二氯-4-羟基苯甲酸和3,5-二溴-4-羟基苯甲酸）。这些结果表明二卤代酪氨酸（DiBrY， DiClY和DiIY）是表位。最后，我们将该抗体用于免疫组织化学研究。小鼠腹腔注射脂多糖（LPS），并切除肝脏。估计lps处理小鼠肝组织抗二卤代酪氨酸抗体和抗髓过氧化物酶抗体均呈阳性染色，提示炎症组织损伤诱导体内二卤代酪氨酸形成。(C) 2004爱思唯尔公司版权所有。
During inhalation of allergens and experimental sepsis, formation of brominated tyrosine has been reported. In this study, we first examined the immunogenicity of brominated protein prepared by treatment of N-bromosuccinimide (NBS). The immunized serum obtained reacted with brominated bovine serum albumin (BSA). The NBS dose-dependent formation of immunoreactivity, which was estimated by enzyme-linked immumosorbent assay, was observed, and the increase coincided with 3,5-dibromotyrosine (DiBrY) formation in the modified BSA, which was chemically determined by liquid chromatography/quadrupole tandem mass spectrometry (LC/MS/MS). Second, by use of immunized mice, monoclonal antibodies to the brominated one were prepared. The two established novel monoclonal antibodies obtained from the immunized mice reacted with DiBrY, 3,5-dichlorotyrosine (DiClY), and 3,5-diiodotyrosine (DiIY). Moreover, 3,5-dihalo-4-hydroxybenzoic acids (3,5-dichloro-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzoic acid) were recognized by these antibodies. These results suggest that dihalogenated tyrosines (DiBrY, DiClY, and DiIY) are the epitopes. Lastly, we used the antibody in an immumohistochemical study. Lipopolysaccharide (LPS) was intraperitoneally administered to mice, and livers were removed. Positive staining of LPS-treated mouse liver tissues by both the anti-dihalotyrosine antibody and anti-myeloperoxidase antibody was estimated, suggesting that inflammatory tissue damage induces the formation of dihalotyrosine in vivo. (C) 2004 Elsevier Inc. All rights reserved.