EPITOPE MAPPING OF THE GASTRIN-RELEASING PEPTIDE ANTI-BOMBESIN MONOCLONAL-ANTIBODY COMPLEX BY PROTEOLYSIS FOLLOWED BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

We have developed a method to rapidly identify the antigenic determina nt for an antibody using in situ proteolysis of an immobilized antigen -antibody complex followed by matrix-assisted laser desorption ionizat ion time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesi n monoclonal antibody was immobilized to agarose beads and then the an tigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct an alysis of the immobilized antigen-antibody complex by MALDI/TOF is dem onstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was su bjected to proteolysis with trypsin, chymotrypsin, thermolysin, and am inopeptidase M. Following proteolysis, the part of the antigen in cont act with the antibody and protected from proteolysis was identified di rectly by MALDI/TOF. Subsequently, the epitope was eluted from the imm obilized antibody with 0.1 M glycine buffer (pH 2.3), separated by rev ersed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was sh own to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.