Identification of prostate specific membrane antigen (PSMA) as the target of monoclonal antibody 107-1A4 by proteinchip; array, surface-enhanced laser desorption/ionization (SELDI) technology.
Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. The identity of the antigen has been confirmed by Western blots using MAbs of known specificity. MAb 107-1A4 is not reactive on Western blots. The conformational epitope for 107-1A4 is on the extracellular domain of PSMA. In competition studies, the binding of MAb 107-1A4 to LNCaP cells is inhibited by itself but not by any other of several other anti-PSMA MAbs, suggesting that the epitope may be unique. These results suggest that 107-1A4 is reactive to a conformational epitope in the external domain of PSMA that is unique among the panel of anti-PSMA MAbs in this study. Furthermore this work demonstrates the ability of mass spectroscopy to elucidate antibody-ligand interaction.
Wang
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S
Department of Urology, University of Washington Medical Center, Seattle, WA, USA.
Diamond
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Hass
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Sokoloff
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Vessella
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D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D000954:Antigens, Surface; D001667:Binding, Competitive; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D043425:Glutamate Carboxypeptidase II; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
C429108:107-1A4 monoclonal antibody; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D008024:Ligands; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D014357:Trypsin