MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY MAPPINGOF HUMAN IMMUNODEFICIENCY VIRUS-GP120 EPITOPES RECOGNIZED BY A LIMITED POLYCLONAL ANTIBODY

In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recogni zed by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. Ln the epito pe excision method, gp120 in the native form is first bound to the ant ibody immobilized on sepharose beads and cleaved with endoproteinase e nzymes. In the epitope extraction method, the gp120 was first proteoly tically cleaved and then allowed to react with the immobilized antibod y. The fragments that remain bound to the antibody, after repeated was hing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody b y matrix assisted laser desorption ionization/mass spectrometry. In th is study we have carried out epitope excision and extraction experimen ts with three different enzymes and have identified residues 472-478 a s a major epitope. In addition, antigenic regions containing minor epi topes have also been identified.