Advanced glycation endproducts (AGEs) have been linked to many sequelae of diabetes, renal disease and aging. To detect AGE levels in human tissues and blood samples, a competitive enzyme-linked immunosorbent assay (ELISA) has been widely used. As no consensus or standard research method for the quantitation of AGEs currently exists, nor a universally defined AGE unit available, the comparative quantitation of AGEs between research laboratories is problematic and restricts the usefulness of interlaboratory clinical data. By comparing the cross-reactivities of five different anti-AGE antisera with five different in vitro AGE-modified proteins, we found that the immunological recognition of AGEs by competitive ELISA is both AGE-carrier protein- and anti-AGE antibody-dependent. This suggests that in vitro AGE-modified proteins might not be appropriate standards for AGEs that occur naturally in vivo. Based on our observation that serum AGE levels in the normal human population are consistently within a narrow range and several folds lower than in diabetics, we propose a method to standardize AGE units against normal human serum (NHS). In this new method, one AGE unit is defined as the inhibition that results from 1:5 diluted NHS in the competitive AGE-ELISA; thus the AGE value in NHS is 5 units/ml. This NHS method requires a competitive AGE-ELISA with reasonable sensitivity such that 1:5 NHS produces a 25 to 40% inhibition of anti-AGE antibody binding to immobilized AGE-proteins. By using this standardized method we found that the AGE levels in normal human serum (5.0 +/- 2.2 units/ml; mean +/- 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 available standard, NHS, the results of quantitative AGE measurements using this method should be comparable between assays and between different laboratories. Taken together, standardizing the AGE-ELISA protocol as described here provides a simple and quantitative method that should facilitate the expanded application of clinical AGE data.
Adult Aged