Glycated LDL concentrations in non-diabetic and diabetic subjects measured with monoclonal antibodies reactive with glycated apolipoprotein B epitopes

Abstract

The potential importance of the non-enzymatic glycation of low density lipoproteins (LDL) in atherogenesis and in the accelerated atherosclerosis associated with diabetes is well recognized. However, it has been difficult to evaluate LDL glycation in the clinical setting because of the lack of suitable methods. To approach this problem, we produced monoclonal antibodies, designated ES12, that recognize glycated apolipoprotein B epitopes in the LDL complex in human plasma. Here we report the use of these antibodies in a competitive enzyme-linked immunosorbent assay (ELISA) to measure glycated LDL concentrations in plasma from non-diabetic and diabetic subjects. In this assay, glycated LDL in the soluble phase inhibits binding of the ES12 antibody to glycated LDL immobilized to microtitre wells, whereas other glycated proteins and non-glycated LDL do not compete. A linear dose-response relationship for 10-125 ng glycated LDL per well allows the construction of standard curves, from which the concentration of glycated LDL in human plasma can be determined. The mean concentration of glycated LDL in samples from non-diabetic subjects was 21.8 +/- 0.9 mg/l, increasing to 40.8 +/- 2.6 mg/l in samples from patients with type II diabetes, comprising 1.9-4.8% and 3.2-14.8%, respectively, of total apolipoprotein B. Glycated LDL concentrations in samples from diabetic patients correlated positively and significantly with other indices of glycaemic status. The results indicate that circulating glycated LDL, which may have diagnostic and pathophysiologic importance, is increased in diabetes with attendant hyperglycaemia. The results further indicate that the described monoclonal antibody-based competitive ELISA affords a simple and reproducible method for quantitative measurement of glycated LDL.