Immunologic 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 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.
E A
EA
Shea
M P
MP
Cohen
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B / Q000032:analysis* / Q000378:metabolism; D001161:Arteriosclerosis / Q000503:physiopathology; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D017127:Glycation End Products, Advanced; D006031:Glycosylation; D006801:Humans; D008077:Lipoproteins, LDL / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012680:Sensitivity and Specificity; D000093844:Glycated Proteins
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D017127:Glycation End Products, Advanced; D008077:Lipoproteins, LDL; C031797:glycated lipoproteins, LDL; D000093844:Glycated Proteins