Two immunochemical assays to measure advanced glycation end-products in serum from dialysis patients

Abstract Advanced glycation end-products are uremic toxins that accumulate in the serum and tissues of patients with chronic renal failure. Here, we established two enzyme-linked immunosorbent assays (ELISAs) for N ε-carboxymethyllysine and imidazolone to analyze advanced glycation end-products in human serum. Both ELISAs detected advanced glycation end-products bound to human serum albumin in a dose-dependent way. Whereas the formation of imida-zolone was independent of the presence of oxygen, concentrations of N ε-carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N ε-carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N ε-carboxymethyllysine, whereas the imidazolone antibody showed slightly higher affinity toward peptide-bound compared to protein-bound imidazolone. In human serum, linear dilution ranges from 1:10 to 1:40 (N ε-carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N ε-carboxymethyllysine from serum was 101±10% and 94±12%, respectively, and 93±15% and 97±12% for imidazolone. The coefficients of variation for intra-assay variability were 0.26–2.7% (N ε-carboxymethyllysine) and 0.1–2.4% (imidazolone), and 8.3–13.4% (N ε-carboxymethyllysine) and 7.8–12.5% (imidazolone) for inter-assay variability. In serum samples from hemodialysis patients (n=20) and controls (n=20), an approximately two-fold increase was detected in the patient group (p<0.001). The combination of the N ε-carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.