title: A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.
abstract: Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.
authors.affiliation: Thoracic Disease Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: Augustine S
authors.initials: AS
authors.lastname: Lee
authors.forename: Javier D
authors.initials: JD
authors.lastname: Finkielman
authors.forename: Tobias
authors.initials: T
authors.lastname: Peikert
authors.forename: Amber M
authors.initials: AM
authors.lastname: Hummel
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Viss
authors.forename: Ulrich
authors.initials: U
authors.lastname: Specks
mesh_terms: D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000032:analysis / Q000097:blood*; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D019272:Leukocyte Elastase / Q000235:genetics / Q000276:immunology; D051379:Mice; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology*; D009195:Peroxidase / Q000235:genetics / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology*; D014162:Transfection
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D009195:Peroxidase; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin