STANDARDIZING THE IMMUNOLOGICAL MEASUREMENT OF ADVANCED GLYCATION ENDPRODUCTS USING NORMAL HUMAN SERUM

Advanced glycation endproducts (AGEs) have been linked to many sequela e of diabetes, renal disease and aging. To detect AGE levels in human tissues and blood samples, a competitive enzyme-linked immunosorbent a ssay (ELISA) has been widely used. As no consensus or standard researc h method for the quantitation of AGEs currently exists, nor a universa lly defined AGE unit available, the comparative quantitation of AGEs b etween research laboratories is problematic and restricts the usefulne ss of interlaboratory clinical data. By comparing the cross-reactiviti es of five different anti-AGE antisera with five different in vitro AG E-modified proteins, we found that the immunological recognition of AG Es by competitive ELISA is both AGE-carrier protein-and anti-AGE antib ody-dependent. This suggests that in vitro AGE-modified proteins might not be appropriate standards for AGEs that occur naturally in vivo. B ased on our observation that serum AGE levels in the normal human popu lation are consistently within a narrow range and several folds lower than in diabetics, we propose a method to standardize AGE units agains t normal human serum (NHS). In this new method, one AGE unit is define d as the inhibition that results from 1:5 diluted NHS in the competiti ve AGE-ELISA; thus the AGE value in NHS is 5 units/ml. This NHS method requires a competitive AGE-ELISA with reasonable sensitivity such tha t 1:5 NHS produces a 25 to 40% inhibition of anti-AGE antibody binding to immobilized AGE-proteins. By using this standardized method we fou nd that the AGE levels in normal human serum (5.0 +/- 2.2 units/ml; me an +/- SD, n = 34) fit a normal distribution (chi(2)-test, p < 0.01), and the serum AGE levels in diabetic patients (20.3 +/- 3.8 units/ml, n = 7) are significantly higher than that of the normal population (p < 0.0001). Since AGE units can now be defined against a universally av ailable standard, NHS, the results of quantitative AGE measurements us ing this method should be comparable between assays and between differ ent laboratories. Taken together, standardizing the AGE-ELISA protocol as described here provides a simple and quantitative method that shou ld facilitate the expanded application of clinical AGE data. (C) 1997 Elsevier Science B.V.