Intended use
The Wieslab® Capture PR3-ANCA test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of IgG antibodies to proteinase 3 (PR3) in human sera. The assay is used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of Wegener’s granulomatosis. The analysis should be performed by trained laboratory professionals.
FOR IN VITRO DIAGNOSTIC USE.
Principle of the Wieslab® Capture PR3-ANCA
The wells of the microtitre plate are coated with purified anti-PR3 monoclonal antibody and proteinase 3. During the first incubation, specific antibodies in diluted serum, will bind to the antigen coating.
The wells are then washed to remove unbound antibodies and other components.
A conjugate of alkaline phosphatase-labelled antibodies to human IgG binds to the antibodies in the wells in the second incubation.
After a further washing step, detection of specific antibodies is obtained by incubation with substrate solution. The amount of bound antibodies correlates to the colour intensity and is measured in terms of absorbance (optical density (OD)). The absorbance is then calculated against a calibrator curve and the results are given in IU/mL adapted to the AF-CDC international standard for PR3.
Kit components and storage of reagents
- One frame with 96 wells (red coloured) coated with monoclonal anti-proteinase 3/proteinase 3 with lid, sealed in a foil pack with a dry pack.
- 1.5 mL negative control (NC) containing human serum in diluent.
- 1.5 mL positive control (PC) containing human serum in diluent.
- 13 mL conjugate containing alkaline phosphatase-labelled antibodies to human IgG in PBS (blue colour).
- 2 x 32 mL Diluent (Dil) containing PBS (red colour).
- 13 mL Substrate pNPP.
- 13 mL Stop solution
- 30 mL wash solution 30x concentrated.
- Six calibrators (five calibrators, Cal 2-6, containing human serum) in diluent. 1.5 mL Cal 1 = 0 IU/mL, 1.5 mL Cal 2 = 2 IU/mL, 1.5 mL Cal 3 = 10 IU/mL, 1.5 mL Cal 4 = 30 IU/mL, 1.5 mL Cal 5 = 100 IU/mL, 1.5 mL Cal 6 = 200 IU/mL.
All reagents in the kit are ready for use except wash solution and should be stored at 2-8°C.
Remove only the number of wells needed for testing, resealing the aluminium package carefully.
References
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Biological Markers of Disease.
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2. Jenne DE. Et al. Wegeners autoantigen decoded.
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3. Wieslander J, Wiik A. ANCA antigens: Myeloperoxidase, in van Venrooij W, Maini R (eds): Manual of Biological Markers of Disease.
Dordrecht, Kluwer; 1994:1-9.
4. Jennette C, Falk R, review. Small vessel vasculitis.
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5. Segelmark M. et al. Heterozygosity for the alpha 1-antitrypsin PiZ gene affects the outcome of PR3-ANCA positive vasculitis.
Kidney Int 1995, 48, 844-850.
6. Boomsma MM. et al. Prediction of relapses in Wegener’s granulomatosis by measurement of antineutrophil cytoplasmatic antibody levels: a prospective study.
Arthritis Rheum, 2000, 43 2025-33
7. Baslund B. et al. Screening for ANCA: is IIF the method of choice.
Clin Exp J Imm. 1995,99, 486-492.
8. Westman K. et al. Clinical evaluation of a Capture ELISA for detection of proteinase 3
anti-neutrophil cytoplasmic antibody.
Kidney Int 53: 1230-1236 1998
9. Westman K. et al. Relaps rate, renal survival and cancer morbidity in patients with Wegener’s granulomatosis or microscopic polyangiitis with renal involvment.
J Am Soc Nephrol 9: 842-852, 1998
10. Segelmark M. et al. How and why should we detect ANCA?
Clin Exp Rheumatol 2000, 18, 629-635.
11. Arranz O. et al. Comp
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