Immunochemical detection of Nε-(carboxyethyl)lysine using a specific antibody
Immunochemical detection of Nε-(carboxyethyl)lysine using a specific antibody
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DOI：
10.1016/j.jim.2007.12.020
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发表时间：
2008-03-20
影响因子：
2.2
通讯作者：
Takeya, Motohiro
中科院分区：
文献类型：
--
作者：
Nagai, Ryoji;Fujiwara, Yukio;Takeya, Motohiro
甲基乙二醛（MG）通过Embden-Meyerhof和多元醇途径产生，并迅速与蛋白质反应形成晚期糖基化终产物（AGE），如N-β-（羧乙基）赖氨酸（CEL）。本研究制备了CEL的多克隆抗体和单克隆抗体，用于检测经修饰蛋白中CEL的含量以及在人肾脏中的病理定位。利用N-羧甲基赖氨酸（CML）偶联亲和层析法，去除与AGE主要结构之一CML交叉反应的抗体，制备多克隆的细胞特异性抗体。以CEL-RNase阳性、CML-RNase阴性为筛选标准，用CEL-牛血清白蛋白免疫小鼠，获得单克隆CEL-SP抗体。非竞争性ELISA显示，多克隆和单克隆的细胞特异性抗体显着反应与细胞蛋白，但不与CML蛋白。竞争性ELISA也证明CEL-SP对CEL类似物如CML、羧甲基精氨酸（CMA）和S-羧甲基半胱氨酸（CMC）不显示交叉反应性，因此表明抗体能够识别羧甲基和羧乙基之间的一个甲基的差异。此外，CEL-SP与MG修饰的人血清白蛋白有显著的反应性，而与乙二醛或3-脱氧葡萄糖醛酮无反应性，其反应性与高效液相色谱法测定的CEL含量高度相关。免疫组化研究，使用CEL-SP提供的证据表明，细胞修饰的蛋白质积累在远端肾小管上皮细胞的糖尿病大鼠。这些结果表明，针对CEL的特异性抗体可以是用于在体外和体内检测CEL的有力工具。(C)2008 Elsevier B. V.保留所有权利。
Methylglyoxal (MG) is generated through the Embden-Meyerhof and polyol pathways, and it rapidly reacts with proteins to form advanced glycation end products (AGE) such as N-epsilon-(carboxyethyl)lysine (CEL). In the present study, polyclonal and monoclonal antibodies specific for CEL were prepared to estimate CEL content in aldehydes-modified proteins and the pathological localization in human kidneys. Polyclonal CEL-specific antibody was prepared by removing cross-reactive antibodies against N-epsilon-(carboxymethyl) lysine (CML), one of the major AGE structures, using CML-conjugated affinity chromatography. Monoclonal CEL-specific antibody (CEL-SP) was obtained by immunization with CEL-bovine serum albumin, followed by successive screening according to CEL-RNase-positive but CML-RNase-negative criteria. A non-competitive ELISA showed that both the polyclonal and monoclonal CEL-specific antibodies significantly reacted with CEL-proteins but not with CML-proteins. A competitive ELISA also demonstrated that CEL-SP does not show cross-reactivity against CEL analogues such as CML, carboxymethylarginine (CMA) and S-carboxymethylcysteine (CMC), thus indicating that antibody is able to recognize the difference of one methyl group between carboxymethyl group and carboxyethyl group. Furthermore, CEL-SP significantly reacted with human serum albumin modified with MG but not with glyoxal or 3-deoxyglucosone, and its reactivity was highly correlated with the CEL content, which was determined by high performance liquid chromatography. Immunohistochemical studies using CEL-SP provided evidence that CEL-modified proteins accumulate in distal tubular epithelial cells of the diabetic rat. These results demonstrate that a specific antibody against CEL can be a powerful tool for detecting CEL both in vitro and in vivo. (C) 2008 Elsevier B.V. All rights reserved.