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(epsilon)-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(epsilon)-carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N(epsilon)-carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N(epsilon)-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(epsilon)-carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N(epsilon)-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(epsilon)-carboxymethyllysine) and 0.1-2.4% (imidazolone), and 8.3-13.4% (N(epsilon)-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(epsilon)-carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.

Evaluation of Nonenzymatic Posttranslational Modification–Derived Products as Biomarkers of Molecular Aging of Proteins

“…Immunogenic properties of AGEs have also been used as an alternative solution to develop ELISA methods, for example for CML (50 ). However, these homemade techniques, which reduce sample pretreatment procedures, are subject to serious analytical problems, such as poor specificity of the antibodies and interferences from …”

On-Line Haemodiafiltration versus Haemodialysis: Stable Haematocrit with Less Erythropoietin and Improvement of Other Relevant Blood Parameters

“…AOPP were determined by a spectrophotometric assay. CML was quantifi ed by an enzyme-linked immunosorbent assay (ELISA) using the CMLspecifi c monoclonal antibody 4G9 (research assay provided by Roche Diagnostics GmbH, Penzberg, Germany) [17] . Free and protein-bound pentosidine were determined by an HPLC assay [18] .…”

Elevation of Nε-(carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis

“…Streptavidin-coated microtiter plates were coated with biotinylated BSA-AGE at room temperature for 1 h. Proteolytic pretreatment of the serum sample with proteinase K (final concentration 1 mg/mL) (Roche, Mannheim, Germany) for 3 h at 37°C followed by addition of phenylmethylsufonyl fluoride (PMSF) (final concentration 1 mmol/L) and incubation for 30 min (Roche, Mannheim, Germany) at 37°C resulted in an optimal exposure of the CML epitopes. A 50 μL aliquot of standard, positive control (CML-modified human serum albumin prepared as previously described [22]) and serum samples in assay buffer and 50 μL of horseradish-peroxidase-labeled monoclonal CML antibody (Roche, Penzberg, Germany; research only, not commercially available) with a dilution of 1:1500 were added. The plate was incubated for 1 h at room temperature and washed.…”