title: Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.
abstract: Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by all anti-PR3 mAbs. In conclusion, rPR3 expressed in insect cells, HMC-1 and 293 cells is recognized by anti-PR3 antibodies, whereas conformational epitopes recognized by anti-PR3 mAbs and PR3-ANCA are not well preserved on rPR3 expressed in E. coli or P. pastoris.
authors.affiliation: Department of Internal Medicine, University Hospital Groningen, Groningen, The Netherlands. y.m.van.der.geld@med.rug.nl
authors.forename: Y M
authors.initials: YM
authors.lastname: van der Geld
authors.forename: W
authors.initials: W
authors.lastname: Oost-Kort
authors.forename: P C
authors.initials: PC
authors.lastname: Limburg
authors.forename: U
authors.initials: U
authors.lastname: Specks
authors.forename: C G
authors.initials: CG
authors.lastname: Kallenberg
mesh_terms: D000328:Adult; D000368:Aged; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D016367:Baculoviridae / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D014890:Granulomatosis with Polyangiitis / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008407:Mast Cells / Q000378:metabolism; D008875:Middle Aged; D053146:Myeloblastin; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D009195:Peroxidase / Q000276:immunology; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology*; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000276:immunology*
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D009195:Peroxidase; D012697:Serine Endopeptidases; D053146:Myeloblastin