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 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 approximately 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.
Benko
Bojan
B
State Intellectual Property Office, Zagreb, Croatia.
Turk
Zdenka
Z
D000818:Animals; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017127:Glycation End Products, Advanced / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011817:Rabbits; D012260:Ribonucleases / Q000276:immunology; D012709:Serum Albumin / Q000737:chemistry* / Q000276:immunology; D000075462:Serum Albumin, Human
D017127:Glycation End Products, Advanced; D012709:Serum Albumin; C109748:advanced glycation end products-human serum albumin; D012260:Ribonucleases; D000075462:Serum Albumin, Human