Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies.
Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL.
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes / Q000032:analysis; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D005779:Immunodiffusion; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D010954:Plasmacytoma / Q000276:immunology; D013552:Swine; D013696:Temperature
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D008077:Lipoproteins, LDL