Characterization of antibodies to advanced glycosylation end products on protein.
Antibodies directed against advanced glycosylation end products (AGEs) formed during a Maillard reaction have been generated and characterized. Since protein-bound AGEs recognized by the antibodies were labile to acid hydrolysis, the antibodies were further characterized by using the AGE-alpha-acetyl-L-lysine methyl ester (AGE-ALME) with a brown and fluorescent property as well as the AGE-proteins. The antibodies reacted with fluorescent compounds, rather than brown pigment compounds, in the AGE-ALME. The fluorescent compounds in the AGE-ALME were separated into four fluorescent compounds by reversed-phase thin layer chromatography (TLC). Of the fluorescent compounds tested, compound 3 (Rf = 0.63), as designated on a TLC plate, showed the highest affinity for the antibodies. In addition, the antibody recognition to the cross-linked oligomers with fluorescence in the AGE-protein was investigated by using bovine pancreatic ribonuclease A (RNase), which is known as a model protein for studying AGE-induced cross-linking. Fluorescence in the AGE-RNase existed in both of the oligomers and the monomer. The cross-linked oligomers exhibited higher affinity to the antibodies than did the monomer, which has a similar degree of fluorescent intensity. These results indicate that our antibodies against cross-linked protein-bound AGEs may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.
Nakayama
H
H
2nd Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
Taneda
S
S
Mitsuhashi
T
T
Kuwajima
S
S
Aoki
S
S
Kuroda
Y
Y
Misawa
K
K
Yanagisawa
K
K
Nakagawa
S
S
D000906:Antibodies / Q000032:analysis*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D008239:Lysine / Q000031:analogs & derivatives* / Q000276:immunology; D015416:Maillard Reaction
D000906:Antibodies; C069694:alpha-acetyllysine methyl ester; D008239:Lysine