PHAGE DISPLAY CLONING AND CHARACTERIZATION OF AN IMMUNOGENETIC MARKER(PERINUCLEAR ANTINEUTROPHIL CYTOPLASMIC ANTIBODY) IN ULCERATIVE-COLITIS

Ulcerative colitis (UC) is genetically associated with a marker serum Ab (pANCA), identified by its reactivity with a neutrophil Ag. This st udy utilized phage display technology to clone and characterize pANCA, which has resisted conventional isolation strategies. Since spontaneo us pANCA-secreting B cells are detectable in UC lamina propria lymphoc ytes, this cell source was used to construct a complete IgG1-kappa Ig library. Selection of phage by panning with fixed neutrophils yielded a 195-foId enrichment after five cycles of panning. BstN1 fingerprinti ng of the enriched library revealed two predominant clones, and DNA se quencing demonstrated highly homologous heavy and light chain variable region segments. Clones were reengineered to express soluble Fab, and neutrophil binding was verified by ELISA. Detailed studies with the t wo recombinant Fabs, NANUC-1 and -2, validated their identity with ser um pANCAs by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. NANUC-1 and -2, like serum UC-pANCA, lack rea ctivity with previously characterized ANCA-reactive neutrophil protein s and thus detect a novel Ag(s). This study demonstrates the feasibili ty of selecting phage display Ab libraries on uncharacterized biologic substrates to isolate marker Abs of pathogenic importance.