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Advanced Glycated Human Serum Albumin as AGE-Carrier Protein in Enzyme-Linked Immunosorbent Assay

Competitive ELISAs for advanced glycation end product (AGE) measurements are based on anti-AGE-antibody and in vitro prepared AGE-carrier competitive antigen. AGE-human serum albumin (HSA) prepared by non-enzymatic reaction between protein and glucose is often used as a competitive antigen. The aim of the study was to examine the impact of pH on HSA preparation. The sets of AGE-HSA analyzed by gel filtration chromatography, IEF and UV/VIS showed significant modifications of native albumin. A competitive ELISA was developed by polyclonal-AGE-antibody and in vitro prepared AGE-HSA at pH 4.5-8.0. AGE-HSA preparations showed impact on the ELISA ability to recognize AGE-immunoreactivity of serum proteins. The highest AGE-immunoreactivity was recorded with the antigen prepared at pH 4.5 ; detection limit declined by ~50% with antigens prepared at pH 6.5, 7.5 or 8.0. Study results confirmed the impact of pH on the glycation adduct formation, thus significantly modifying the results of competitive ELISA measurements.

Advanced glycation end product; Human serum albumin; Enzyme-linked immunosorbent assay

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