Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.
The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.
Allan
C M
CM
Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
Tetaz
T
T
Fidge
N H
NH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000302:isolation & purification*; D001667:Binding, Competitive; D003488:Cyanogen Bromide; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000302:isolation & purification*; D006801:Humans; D006868:Hydrolysis; D008075:Lipoproteins, HDL / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D000939:Epitopes; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D010450:Endopeptidases; D003488:Cyanogen Bromide