Monoclonal antibody-based assay to identify compounds that inhibit Nδ-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) formation.

The advanced glycation end-product Nδ-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) accumulates during the progress of atherosclerosis with type 2 diabetes. Therefore, G-H1 might serve as early diagnostic marker of atherosclerosis, and its inhibition could become a strategy to prevent the pathogenesis of this disease. This study therefore aimed to determine the feasibility of an anti-G-H1 monoclonal antibody-based screen for G-H1 inhibitors. Mice were immunized with G-H1-keyhole limpet hemocyanin prepared with synthesized G-H1 standards to produce an anti-G-H1 monoclonal antibody (1E9). The specificity of 1E9 and levels of G-H1 were determined using an enzyme-linked immunosorbent assay (ELISA). The G-H1 levels correlated with those measured using liquid chromatography (LC)-tandem mass spectrometry (MS). We then mixed arginine with various glyoxal (GO) concentrations and applied GO-modified collagen and albumin to determine the conditions required for G-H1 formation. To screen for G-H1 inhibitors, an ELISA-based protocol was developed and leveraged with aminoguanidine to determine the inhibitory effect of screening compounds on G-H1 formation. The specificity of 1E9 for G-H1 was high. The abundance of G-H1 formation in glyoxal-modified arginine was maximal with 2 mM GO, which aligned with the LC-MS/MS and ELISA results. However, G-H1 was below the detection limit for the GO-modified proteins. Aminoguanidine inhibited G-H1 formation in ribose-modified arginine in a concentration-dependent manner. Overall, our findings emphasize the potential of 1E9 to specifically detect free G-H1 and its value for screening G-H1 inhibitors.