Phage display cloning and characterization of an immunogenetic marker (perinuclear anti-neutrophil 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 study utilized phage display technology to clone and characterize pANCA, which has resisted conventional isolation strategies. Since spontaneous pANCA-secreting B cells are detectable in UC lamina propria lymphocytes, this cell source was used to construct a complete IgG1-kappa Ig library. Selection of phage by panning with fixed neutrophils yielded a 195-fold enrichment after five cycles of panning. BstN1 fingerprinting of the enriched library revealed two predominant clones, and DNA sequencing 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 two recombinant Fabs, NANUC-1 and -2, validated their identity with serum pANCAs by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. NANUC-1 and -2, like serum UC-pANCA, lack reactivity with previously characterized ANCA-reactive neutrophil proteins and thus detect a novel Ag(s). This study demonstrates the feasibility of selecting phage display Ab libraries on uncharacterized biologic substrates to isolate marker Abs of pathogenic importance.
Eggena
M
M
Department of Pathology and Laboratory Medicine, University of California-Los Angeles 90024, USA.
Targan
S R
SR
Iwanczyk
L
L
Vidrich
A
A
Gordon
L K
LK
Braun
J
J
D000595:Amino Acid Sequence; D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies / Q000276:immunology* / Q000302:isolation & purification; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001666:Binding Sites, Antibody; D015415:Biomarkers / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D003093:Colitis, Ulcerative / Q000175:diagnosis / Q000276:immunology*; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies; D015415:Biomarkers; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins