Enzyme linked immunosorbent assay for the measurement of nonenzymatically glucosylated proteins in serum and in tissues.

We have developed an enzyme linked immunosorbent assay (ELISA) for glucosylated proteins. The polyclonal antiserum was prepared against reduced glucosylated lipoproteins and was specific for the glucose-lysine bond. The antiserum recognized, in a dose-dependent manner, all reduced glucosylated proteins tested, including albumin, fibrinogen, low density lipoprotein, high density lipoprotein and hemoglobin, yet had no affinity for native proteins or to glucosylated but nonreduced proteins. The sensitivity of the assay was in the order of 1-5 pmol glucosylated lysine/ml and half maximal displacement occurred at 8-24 pmol glucosylated lysine/ml. The inter- and intraassay variables were 10.8% and 13.5%, respectively. Serum proteins from diabetic patients (n = 30) contained 84 +/- 6 picomoles of glucosylated lysine/mg protein, compared to 28 +/- 3 in controls (n = 20), and the concentration of glycosylated proteins correlated with fasting blood glucose (r = 0.56, p less than 0.02), but not with glucosylated hemoglobin levels (r = 0.29, p greater than 0.1). Proteins from diabetic glomeruli and aortae similarly contained more glucosylated lysine residues than controls.