ES12

Nonenzymatic glycation of apolipoprotein B (apo B) is a post-secretory modification of low density lipoprotein (LDL) that affects its atherogenic potential and is implicated in the accelerated atherosclerosis associated with diabetes. To facilitate assessment of apo B glycation, we produced hybridomas secreting monoclonal antibodies specific for glycated apo B. SP 2/0 myeloma cells were fused with spleen cells from BALB/c mice immunized with purified apo B glycated non-reductively in vitro. Specificity of monoclonal antibodies secreted by the cloned cell line designated ES12 was demonstrated by immunoblotting and by direct ELISA, wherein the antibodies reacted with glycated epitopes residing in LDL but not in other plasma proteins, and did not react with nonglycated apo B or nonglycated LDL. Immunoblotting of human plasma with ES12 monoclonal antibody yielded an approx. 180,000 molecular weight component showing co-identity with apo B, indicating site specificity for glycated epitopes residing in apo B of the LDL complex and absence of reactivity with other nonenzymatically glycated plasma proteins. This reactivity of ES12 with the physiologic form of glycated apo B that occurs in vivo differs from properties of other antibodies raised against glycated lipoproteins, which recognized glycated residues only after reductive conversion to glucitol-lysine and which do not discriminate between different glycated proteins. In a competitive ELISA, mean concentration of glycated LDL, measured as apo B equivalents, in eight separate plasma samples was 19.7 +/- 1.9 micrograms/ml, representing 3.5 +/- 0.3% of total apo B. The ES12 monoclonal antibody allows specific determination of plasma glycated LDL concentrations, which may have diagnostic and pathogenetic importance.

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.