DEFINITION OF A NONLINEAR CONFORMATIONAL EPITOPE FOR THE APOLIPOPROTEIN B-100-SPECIFIC MONOCLONAL-ANTIBODY, MB47

The apolipoprotein (ape) B-100-specific monoclonal antibody MB47 has b een widely used in lipoprotein metabolism and atherosclerosis research . When bound to apoB-100 on low density lipoproteins (LDL), antibody M B47 completely blocks the binding of LDL to the LDL receptor. The epit ope for antibody MB47 has previously been mapped to the vicinity of ap oB-100 amino acid (aa) residue 3500. To map the epitope for antibody M B47 more precisely, we used recombinant bacterial fusion proteins. Ant ibody MB47 bound strongly to a fusion protein containing apoB-100 aa 3 214-3728, but no specific binding was observed to fusion proteins cont aining aa 3214-3351, 3214-3506, 3351-3506, or a fusion protein contain ing aa 3214-3351 and 3506-3728. Although antibody MB47 did not bind to aa 3214-3506, it did bind to aa 3214-3510. Further fusion protein stu dies revealed that antibody MB47 bound to aa 3429-3510, but bound only very weakly to aa 3453-3510, indicating that aa 3429-3453 constitute an important part of the MB47 epitope. Subsequent fusion protein studi es revealed that MB47 bound much more strongly to aa 3429-3523, 3429-3 544, 3429-3565, and 3429-3590 than to aa 3429-3510. Thus, aa 3507-3523 also constitute an important part of the MB47 epitope. In summary, th e fusion protein data indicated that two nonlinear domains of apoB-100 separated by similar to 53 aa (the 25 residues from aa 3429 to 3453 a nd the 17 residues from aa 3507 to 3523) form key parts of the MB47 ep itope. Antibody MB47 failed to bind to any of 15 different synthetic a poB peptides that spanned aa 3415-3510, a finding that is consistent w ith the fusion protein data indicating that the MB47 epitope is formed by the conformational alignment of discontinuous amino acid sequences .