Journal of Lipid Research, Vol 32, 595-601, Copyright © 1991 by Lipid Research, Inc.
Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay
CM Allan, T Tetaz and NH Fidge
Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
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.
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