Advanced Glycated Human Serum Albumin as AGE-Carrier Protein in Enzyme-Linked Immunosorbent Assay
Benko, B.; Turk, Z.
Clinical Laboratory 54(9-10): 331-339
2008
ISSN/ISBN: 1433-6510 PMID: 19097490 Document Number: 10807
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 AGE-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 using polyclonal-AGE-antibody and in vitro prepared AGE-HSA at pH 4.5-8.0. AGE-HSA preparations showed an 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.
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