IMMUNOLOGICAL DETECTION AND MEASUREMENT OF GLYCATED APOLIPOPROTEIN-B WITH SITE-SPECIFIC MONOCLONAL-ANTIBODIES

Nonenzymatic glycation of apolipoprotein B (apo B) is a post-secretory modification of low density lipoprotein (LDL) that affects its athero genic potential and is implicated in the accelerated atherosclerosis a ssociated with diabetes. To facilitate assessment of apo B glycation, we produced hybridomas secreting monoclonal antibodies specific for gl ycated apo B. SP 2/0 myeloma cells were fused with spleen cells from B ALB/c mice immunized with purified apo B glycated non-reductively in v itro. 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 nongl ycated apo B or nonglycated LDL. Immunoblotting of human plasma with E S12 monoclonal antibody yielded an approx. 180,000 molecular weight co mponent showing co-identity with apo B, indicating site specificity fo r glycated epitopes residing in apo B of the LDL complex and absence o f reactivity with other nonenzymatically glycated plasma proteins. Thi s reactivity of ES12 with the physiologic form of glycated apo B that occurs in vivo differs from properties of other antibodies raised agai nst glycated lipoproteins, which recognize glycated residues only afte r reductive conversion to glucitol-lysine and which do not discriminat e between different glycated proteins. In a competitive ELISA, mean co ncentration of glycated LDL, measured as apo B equivalents, in eight s eparate plasma samples was 19.7 +/- 1.9 mug/ml, representing 3.5 +/- 0 .3% of total apo B. The ES12 monoclonal antibody allows specific deter mination of plasma glycated LDL concentrations, which may have diagnos tic and pathogenetic importance.