Immunochemical detection of Nepsilon-(carboxyethyl)lysine using a specific antibody.
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.
Nagai
Ryoji
R
Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. nagai-883@umin.ac.jp
Fujiwara
Yukio
Y
Mera
Katsumi
K
Yamagata
Kazuya
K
Sakashita
Naomi
N
Takeya
Motohiro
M
D000447:Aldehydes / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003921:Diabetes Mellitus, Experimental / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000473:pathology; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D008297:Male; D011765:Pyruvaldehyde / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology
D000447:Aldehydes; D000911:Antibodies, Monoclonal; D012709:Serum Albumin; C048496:N(6)-carboxymethyllysine; D011765:Pyruvaldehyde; D008239:Lysine