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  • Title: Immunochemical detection of Nepsilon-(carboxyethyl)lysine using a specific antibody.
    Author: Nagai R, Fujiwara Y, Mera K, Yamagata K, Sakashita N, Takeya M.
    Journal: J Immunol Methods; 2008 Mar 20; 332(1-2):112-20. PubMed ID: 18242632.
    Abstract:
    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.
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