Quantitative enzyme-linked immunosorbent assay (ELISA) for non-enzymatically glycated serum protein.

A competitive ELISA for quantitative determination of glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine was developed. We applied it to measure non-enzymatically glycated serum proteins. The antiserum obtained by immunizing guinea pigs with reductively glycated human albumin was capable of identifying and quantitating glucitollysine residues of serum proteins in normal and diabetic subjects after reduction of the proteins with sodium borohydride. The ELISA assay developed here had satisfactory reproducibility as judged by the intra-assay precision of 2.3-7.6% and the interassay precision of 6.7-9.8%. Results from this assay procedure correlated well with those from the radioimmunoassay and the boronate affinity chromatography procedure. The data suggested that diabetic serum proteins contained at least three times as much immunochemically detectable glucitollysine residues as normal serum proteins after reduction of the proteins with sodium borohydride. This method allows to quantitate glucitollysine residues on any of the proteins that have been implicated in the pathological sequelae of diabetes.