Intended use

The Wieslab^? Capture MPO-ANCA test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of IgG antibodies to myeloperoxidase (MPO) in human sera. The results of the assay are to be used as an aid to the diagnosis of microscopic polyangiitis (MP). The assay is intended for use in patients with signs and symptoms consistent with MP. It is not intended for screening a healthy population. The analysis should be performed by trained laboratory professionals.

FOR IN VITRO DIAGNOSTIC USE.

Background

ANCAs (anti-neutrophil cytoplasmic antibodies) are a family of autoantibodies related to vasculitis and inflammatory disorders. When c-ANCA was shown to relate to granulomatosis with polyangiitis (Wegener's granulomatosis (WG)), interest in ANCAs has increased steadily, and today these antibodies are considered to be a major diagnostic tools for the investigation of systemic vasculitis.

The first method to detect ANCA was indirect immunofluorescence (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 purified proteins.

The granulocyte is full of granules each with many different proteins. 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.

Approximately 80-90% of WG patients manifest PR3-ANCA and 5-15% MPO-ANCA. One category of vasculitis is microscopic polyangiitis (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^? Capture MPO-ANCA is calibrated against the AF-CDC international standard (Reference Human Serum 15, code IS2720).

Technical information

The wells of the microtitre plate are coated with purified anti-MPO monoclonal antibody and 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.