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.
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.
Lee
Augustine S
AS
Thoracic Disease Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Finkielman
Javier D
JD
Peikert
Tobias
T
Hummel
Amber M
AM
Viss
Margaret A
MA
Specks
Ulrich
U
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
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