title: Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes.
abstract: Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids.
authors.affiliation: Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: D N
authors.initials: DN
authors.lastname: Fass
authors.forename: J A
authors.initials: JA
authors.lastname: Hudson
authors.forename: M A
authors.initials: MA
authors.lastname: Viss
authors.forename: J
authors.initials: J
authors.lastname: Wieslander
authors.forename: H A
authors.initials: HA
authors.lastname: Homburger
authors.forename: U
authors.initials: U
authors.lastname: Specks
mesh_terms: D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000097:blood* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D014890:Granulomatosis with Polyangiitis / Q000097:blood* / Q000276:immunology; D006801:Humans; D053146:Myeloblastin; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000276:immunology*
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001324:Autoantigens; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin