ELISA of pentosidine, an advanced glycation end product, in biological specimens

Abstract

Abstract Pentosidine is a fluorescent protein cross-link and glycoxidation marker for the advanced glycation reaction in diabetes, aging, and uremia. We raised polyclonal antibodies in New Zealand White rabbits against this hapten coupled to keyhole limpet hemocyanin. The antibodies detected by ELISA reacted strongly with free pentosidine but not with pentosidine-like compounds. The working range of the competitive ELISA for standard pentosidine was 0.1-100 pmol. Pentosidine was detectable in bovine serum albumin incubated with ribose as a function of incubation time. Immunoblotting studies showed that pentosidine specifically stained in oligomers of lysozyme incubated with ribose. Digestion with protease (Pronase E, 20 g/kg) as well as acid hydrolysis enhanced the immunoreactivity of samples, the pentosidine values in digested human plasma correlating with those measured by HPLC (r = 0.98). Pentosidine in diabetic and uremic plasma digested with Pronase E was significantly higher than normal (P < 0.01; mean +/- SD): 1620 +/- 1940 and 2630 +/- 1320 [corrected] nmol/L, respectively, vs 151 +/- 55 nmol/L (normal). Amounts of pentosidine in hydrolyzed skin collagen increased with age and were increased in diabetes and uremia. This ELISA provides a new tool for assessing the role of the advanced Maillard reaction in aging and age-related diseases.