Measurement of MPO-specific ANCA is an important adjunct to clinical findings in the evaluation of clinical subtypes within the systemic vasculitides spectrum. The Wieslab® MPO-ANCA is standardized against the AF-CDC international standard (Reference Human Serum 15, code IS2720).
The Wieslab® MPO-ANCA test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of IgG antibodies to myeloperoxidase 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 microscopic polyangitis. The analysis should be performed by trained laboratory professionals.
FOR IN VITRO DIAGNOSTIC USE.
ANCAs (anti-neutrophil cytoplasmic antibodies) are a family of autoantibodies related to vasculitis and inflammatory disorders. Since 1985, when c-ANCA was shown to be related to granulomatosis with polyangiitis (Wegener’s granulomatosis (WG)), interest in ANCAs has increased steadily, and today these antibodies are considered to be the major diagnostic tools for the diagnosis and follow up of systemic vasculitis.
The first method to detect ANCA was indirect immuno-fluorescence (IIF) performed on ethanol fixed granulocytes. This method yields two patterns, a cytoplasmic staining of the granulocyte denoting the presence of c-ANCAs, and a perinuclear staining denoting the presence of p-ANCAs. IIF was followed by ELISAs using the purified proteins.
The granulocyte is full of granules each with many different proteins. It was early shown that antibodies from systemic vasculitis patients bind to the alpha fraction containing the azurophil granules. The most important proteins were proteinase 3 (PR3) and myeloperoxidase (MPO). Thus antibodies to proteinase 3 are termed PR3-ANCA, and antibodies to myeloperoxidase are termed MPO-ANCA. PR3 is a serine protease with a molecular weight of 29kD, and MPO is a dimer with a molecular weight of 140kD.
Approximately 80-90% of granulomatosis with polyangiitis (WG) patients manifest PR3-ANCA and 5-15% MPO-ANCA. One category of vasculitis is microscopic polyangitis (MP). Most patients with active MP are characterised by positive ANCA test results, MPO-ANCA being more frequent than PR3-ANCA.
An international workgroup has developed an international standard for MPO-ANCA serology. The Wieslab® MPO-ANCA is standardized against the AF-CDC international standard (Reference Human Serum 15, code IS2720).
The wells of the mikrotiter plate are coated with purified myeloperoxidase. 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 this 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 MPO.