Immunochemical Approaches to AGE-Structures—Characterization of Anti-AGE Antibodies

Abstract

Recent immunological approaches have greatly helped broaden our understanding of the biomedical significance of AGEs (advanced glycation end-products) in aging and age-enhanced disease processes. We previously prepared a monoclonal anti-AGE antibody (6D12) that recognized a common AGE-structure(s) as a major immunochemical epitope. Subsequently, Nɛ-(carboxymethyl)lysine (CML), one of the glycoxidation products of AGEs, was demonstrated to be a major immunological epitope among AGEs, and 6D12 turned out to recognize CML as an epitope. In the present study, 13 different polyclonal anti-AGE antibodies were characterized in order to obtain the other epitope structure(s), other than CML (non-CML). We used CML-bovine serum albumin as an authentic CML-protein and AGE-lysozyme as an authentic non-CML-protein. The results indicated that these antibodies were classified into 3 groups (Group I, II & III). Group I was specific for CML, but both Group II and Group III were unreactive to CML. Group II, but not Group III, recognized AGE-lysozyme, suggesting Group II and III were specific for non-CML but different epitopes. The epitope of Group II was formed much earlier than that of Group III during incubation of BSA with glucose in vitro. Furthermore, we made two monoclonal anti-AGE antibodies (M-1 and M-2) whose epitope structures appeared to be identical or closely similar to Group III and Group II, respectively. These results indicate that AGE-proteins express two major non-CML epitopes in addition to CML.