Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope.
Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001054:Apolipoproteins A / Q000097:blood / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001054:Apolipoproteins A; D000939:Epitopes; D008075:Lipoproteins, HDL