Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes

Jiayan Sun, David N. Fass, Jay A. Hudson, Margaret A. Viss, Jörgen Wieslander, Henry A. Homburger, Ulrich Specks

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)111-123
Number of pages13
JournalJournal of Immunological Methods
Volume211
Issue number1-2
DOIs
StatePublished - Feb 1 1998

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Myeloblastin
Antineutrophil Cytoplasmic Antibodies
Enzyme-Linked Immunosorbent Assay
Antigens
Epitopes
Monoclonal Antibodies
Indirect Fluorescent Antibody Technique
Immune Sera
Rabbits
Granulomatosis with Polyangiitis

Keywords

  • ANCA
  • Capture ELISA
  • Human
  • Mast cell
  • Proteinase 3
  • Wegener's granulomatosis

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes. / Sun, Jiayan; Fass, David N.; Hudson, Jay A.; Viss, Margaret A.; Wieslander, Jörgen; Homburger, Henry A.; Specks, Ulrich.

In: Journal of Immunological Methods, Vol. 211, No. 1-2, 01.02.1998, p. 111-123.

Research output: Contribution to journalArticle

Sun, Jiayan ; Fass, David N. ; Hudson, Jay A. ; Viss, Margaret A. ; Wieslander, Jörgen ; Homburger, Henry A. ; Specks, Ulrich. / Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes. In: Journal of Immunological Methods. 1998 ; Vol. 211, No. 1-2. pp. 111-123.
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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.",
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AU - Hudson, Jay A.

AU - Viss, Margaret A.

AU - Wieslander, Jörgen

AU - Homburger, Henry A.

AU - Specks, Ulrich

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N2 - 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.

AB - 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.

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