Isolation of a protein complex from purulent sputum consisting of proteinase-3 and alpha 1-antitrypsin reactive with anti neutrophil cytoplasmic antibodies.
The detection of ANCA (anti-neutrophil cytoplasmic antibodies) is of importance in the diagnosis of Wegener's granulomatosis (WG) and solid-phase assays for the detection of c-ANCA have been set up by various groups, using purified proteinase-3 (PR-3) in an ELISA or RIA. For the isolation of PR-3 large numbers of PMNs are needed. We therefore examined the possibility of isolating PR-3 from the purulent sputum of patients with chronic bronchitis or cystic fibrosis, since large numbers of PMNs and their degranulation products are present in such material. By a three-step chromatographic procedure (4-phenylbutylamine affinity chromatography, Biorex 70 cation exchange chromatography and monoclonal antibody anti-PR-3 affinity chromatography) we isolated a 53 kDa protein that was recognized on immunoblot by MoAbs directed against PR-3 and alpha 1-antitrypsin (alpha 1AT). We show that the 53 kDa protein is a complex of PR-3 and alpha 1AT. This complex is reactive with a selected set of c-ANCA positive sera from patients with Wegener's granulomatosis.
Ballieux
B E
BE
Department of Nephrology, University Hospital, Leiden, Netherlands.
Hagen
E C
EC
van der Keur
C
C
Zegers
N D
ND
van Es
L A
LA
van der Woude
F J
FJ
Daha
M R
MR
D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies / Q000097:blood*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D053146:Myeloblastin; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification*; D013183:Sputum / Q000737:chemistry*; D000515:alpha 1-Antitrypsin / Q000276:immunology / Q000302:isolation & purification*
D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies; D000515:alpha 1-Antitrypsin; D012697:Serine Endopeptidases; D053146:Myeloblastin