Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B.
Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D001055:Apolipoproteins B / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007158:Immunologic Techniques; D008077:Lipoproteins, LDL / Q000276:immunology*; D008079:Lipoproteins, VLDL / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D000939:Epitopes; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D010446:Peptide Fragments