A simple high yield procedure for purification of human proteinase 3, the main molecular target of cANCA.
Proteinase 3, the antigen commonly recognized by classical anti-neutrophil cytoplasmic antibodies (cANCA) in patients with Wegener's granulomatosis was purified from neutrophil azurophilic granules. Proteinase 3, a serine protease with an apparent molecular mass of 29 kDa, was extracted with Triton X-100 from the azurophilic granule fraction of neutrophils after nitrogen bomb cavitation and Percoll gradient centrifugation. Anion exchange chromatography removed many proteins, which were bound to the column. The unbound proteins, which contained most of the proteinase 3, were then separated by gel filtration. All chromatography steps were done in the presence of detergent. SDS polyacrylamide gel electrophoresis of this preparation only revealed three bands migrating closely together at the position of a 29-31 kDa protein, characteristic of proteinase 3. Affinity-purified polyclonal antibodies raised against proteinase 3 were used for immunoblotting studies and demonstrated that the purified protein was proteinase 3. Antibodies to elastase, cathepsin G, myeloperoxidase, lactoferrin or lysozyme did not react in ELISA assays with the isolated protein. The proteinase 3 prepared by this procedure was found to be suitable as an antigen for detecting PR3-ANCA both in ELISA and in immunoblotting experiments.
Stummann
L
L
Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark.
Wiik
A
A
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood*; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D014890:Granulomatosis with Polyangiitis / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D053146:Myeloblastin; D012697:Serine Endopeptidases / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification*
D019268:Antibodies, Antineutrophil Cytoplasmic; D001324:Autoantigens; D007074:Immunoglobulin G; D012697:Serine Endopeptidases; D053146:Myeloblastin