A novel monoclonal antibody against methylglyoxal–arginine adduct

Abstract

Methylglyoxal (MG), a precursor of advanced glycation end products, reacts rapidly with arginine residue in proteins. Three products, one fluorescent and two non-fluorescent adducts, have been so far identified. The fluorescent MG–arginine adduct, putatively identified as 5-methylimidazolone, has been shown to be identical to argpyrimidine. The non-fluorescent products were identified as the 5-hydro-5-methylimidazol-4-one and a novel MG adduct, which we named tetrahydropyrimidine (THP). THP was found to be formed from the reaction of guanidino group of arginine with two-MG molecules under physiological conditions. In the present study, to detect the THP adduct in vivo, we developed a monoclonal antibody (mAb5A3) directed to protein-bound THP. During preparation of the monoclonal antibody, hybridomas were selected by comparing the reactivities of the culture supernatant to THP using a competitive enzyme-linked immunosorbent assay. It was found that mAb5A3 specifically recognized THP as the dominant epitope. The presence of THP adduct was attested by immunohistochemical analysis of human atherosclerotic lesion, in which macrophage-derived foam cells were positively stained by the antibody. Our present results suggest that accumulation of THP occurs in vivo and may contribute to the progression of vascular diseases.