Mapping discontinuous epitopes recognized by human enhancing antibodies In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of proteinprotein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in proteinprotein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. We have previously identified the epitope recognized by the human monoclonal anti-HIV gp120 antibody as a sequence found in the fusogenic region of gp140. We are now in the process of developing an antigen containing the extended peptide sequence containing the epitope for development of antibodies. We have provided a peptide containing the core sequence of gp140 that we found to be bound in our epitope excision experiments for production of rabbit antibodies. The antibodies received from the company were not reactive with any of the peptides that we showed bound to the 4E10 antibody. The implication of this is that the sequence recognized by the mAb is highly conformational in nature and that antibodies are not produced against the linear sequence.

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Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2009
Total Cost
$233,717
Indirect Cost
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Hager-Braun, Christine; Hochleitner, Elisabeth O; Gorny, Miroslaw K et al. (2010) Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis. J Am Soc Mass Spectrom 21:1687-98
Dhungana, Suraj; Williams, Jason G; Fessler, Michael B et al. (2009) Epitope mapping by proteolysis of antigen-antibody complexes. Methods Mol Biol 524:87-101