Conservation of the low density lipoprotein receptor-binding domain of apoprotein B. Demonstration by a new monoclonal antibody, MB47.
The fact that low density lipoprotein (LDL) from multiple animal species binds to the human LDL receptor suggested that the LDL-receptor binding domain of apoprotein (apo)B must be evolutionarily conserved. To determine if a common receptor domain epitope existed on apo B, we generated a monoclonal antibody that was specific for the LDL-receptor domain of apo B. This was accomplished by using a screening procedure that selected for a hybridoma supernatant that could block specific cellular uptake and degradation of LDL. Western blots showed that this antibody, termed MB47, was specific for apo B-100. Fluid phase assays indicated a high binding affinity (Ka = 4 X 10(9) M-1) and demonstrated that all human LDL particles expressed the MB47 epitope. Scatchard analysis indicated that a maximum of one MB47 molecule bound to each LDL particle. In solid phase assays, antibody MB47 bound to plasma or LDL of multiple mammalian species, including guinea pig, rabbit, pig, dog, cat, seal, whale, bear, and lion, but it did not bind to mouse or rat LDL. In contrast, a rabbit antiserum to LDL and two other anti-apo B monoclonal antibodies, MB3 and MB19, which do not bind to the receptor domain, were specific only for human LDL. LDL from multiple species, including mouse LDL, competed effectively with 125I-human LDL for binding to human fibroblasts. MB47 effectively inhibited uptake and degradation of labeled human, guinea pig, and rabbit LDL by both human and guinea pig fibroblasts. We conclude that antibody MB47 binds to a single receptor domain on LDL and identifies a vital region conserved through mammalian evolution.
D000818:Animals; D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B / Q000378:metabolism*; D001057:Apolipoproteins E / Q000378:metabolism; D001667:Binding, Competitive; D004285:Dogs; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008045:Lions; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism; D011817:Rabbits; D011863:Radioimmunoassay; D011973:Receptors, LDL / Q000378:metabolism*; D001503:Ursidae
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D007140:Immunoglobulin Fab Fragments; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D011973:Receptors, LDL