engleski

Autoantibodies against advanced glycation endproducts (AGEs) in human serum

Incubation of proteins with reducing sugar results in time-related production of advanced glycation endproducts (AGE). AGE modified proteins have antigenic properties. In the present study we develop an immunoenzyme assay for detection of autoantibodies against AGE in human serum. A blocking ELISA was chosen to test immunoreactivity against AGE epitope. Human serum albumin (AGE-HSA) glycated in vitro was used as a solid phase antigen. Multiple immunization of rabbits by AGE-rabbit albumin was used in the preparation of AGE polyclonal antiserum. An excess of AGE-HSA antigen was wall-fixed on the microtiter plate and during incubation with serum, autoantibodies bound to these antigens. This was followed by incubation with rabbit anti-AGE- antibodies to block the rest of free antigens. In parallel, native HSA served as a control antigen to measure non-specific reactions. The binding of autoantibodies against AGE for each serum sample was separately calculated as the percentage inhibition of antibody binding activity with correction for non-specific binding. Competitive ELISA measured total serum AGEs. AGE and anti-AGE antibodies were detected in diabetic (n=63) and control (n=20) serum. Total AGE level was significantly higher in diabetics (13.9 5.7 vs. 8.5 1.0  gEq/ml, p<0.001). We measured a broad range (3.1-87.4) of antibody binding activity in both groups. Unexpectedly, the mean percentage of inhibition measured in the control group (47.3 14.4%) significantly exceeded the values measured in the group of diabetic patients (26.0 11.8%, p<0.001). Thirty-one (48%) diabetic patient showed a mean value for free AGE antibodies lower by 2SD than that in the control subjects and they were considered as antibody negative. The antibody negative patients had a higher level of serum AGE (14.4 6.2 vs. 13.4 6.2, p<0.01). The presented method might be useful in the evaluation of advanced glycation consequences in diabetes and aging.

diabetes; advanced glycation; autoantibodies; immunoassay

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