Monoclonal antibodies distinguish between lipid-dependent and reversible conformational states of human...

Monoclonal antibodies (MAbs) have been prepared against apolipoprotein (apo) B which had been delipidated and resolubilized (apo Bsol). The MAbs were classified into nine groups according to their behavior in competitive binding assays and, following SDS electrophoresis and immunoblots, their corresponding epitopes were assigned to apo B48, apo B74, apo B26 and to fragments of LDL apo B (apo BLDL) generated by limited tryptic proteolysis. In addition to their reactivity with soluble apo B all antibodies also reacted with LDL which had been adsorbed to polystyrene. Competitive binding of MAbs to insolubilized antigen also indicated conformational similarities between apo Bsol and apoBLDL. These similarities were, however, less apparent when the respective antigens were in solution. In competitive radioimmunoassays the majority of anti-apo Bsol MAbs reacted better with apo Bsol than with LDL although the opposite was true for one group of MAbs. In immunoprecipitation studies the MAbs which preferentially recognize apo BLDL could precipitate 95% of 125I-LDL whereas the immunoprecipitation with those MAbs which preferentially recognize soluble apo B varied between 0 and 95%. Between 65 and 82% of 125I-apo Bsol was immunoprecipitated under the same conditions. Thus, epitopes defined by MAbs prepared against apo Bsol may be expressed on apo BLDL when it is adsorbed to plastic but not necessarily when it is in solution. For those epitopes preferentially expressed on apo BLDL, reincorporation of soluble apo B into phospholipid-cholesteryl ester microemulsions or phospholipid-cholesterol liposomes increased their immunoreactivity, whereas, the reincorporation of apo B into lipid vesicles resulted in a decreased reactivity with those MAbs which recognized better apo Bsol than apo BLDL. Thus, while the respective conformations of apo Bsol and apo BLDL are only partially similar, they can be reversibly interchanged by delipidation and relipidation.