Development and Evaluation of Novel ELISA for Determination of Urinary Pentosidine

Pentosidine is the most well-characterized advanced glycation end product (AGE). It has been measured by HPLC, although this approach cannot be adapted to analyze many clinical samples and is also time-consuming. Furthermore, the detection of pentosidine using a reported ELISA kit and HPLC system requires pretreatment by heating, which generates artificial pentosidine leading to overestimation. We developed a novel pentosidine ELISA system that don't require sample pretreatment for analyzing urine samples. We then analyzed the accuracy, precision, and reliability of this system. Urinary samples for analysis were obtained from healthy volunteers and stored urinary samples from the participants of the Nagano cohort study were also used. The LoB and LoD were 4.25 and 6.24 pmol/mL, respectively. Intra- and inter-assay coefficients of variation were less than 5%. The spiking and dilution recoveries were 101.4% and 100.5%, respectively. Analysis of the cross-reactivities against seven compounds representative of AGEs and structurally similar to pentosidine showed no significant cross-reactivity. The correlation coefficient between the concentrations of pentosidine obtained from HPLC and ELISA for the same urine samples was r=0.815. The urinary excretion of pentosidine upon overnight fasting was lower than that after a meal, suggesting the presence of diurnal variation in urinary pentosidine. In contrast, day-to-day variation was not observed. These results indicate that the ELISA system has sufficient reliability, accuracy, and precision for measuring urinary pentosidine. Sampling of fasting urine is suitable for minimizing variation. In conclusion, this ELISA system is promising to evaluate the effect of AGE on lifestyle-related diseases.