Intended use

The Wieslab^® Anti-GBM, ANCA Screening Test Kit is an enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of antibodies to glomerular basement membrane (GBM), Proteinase-3 (PR3) and Myeloperoxidase (MPO) 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 reno-pulmonary syndromes and rapidly progressive glomerulonephritis, especially Goodpasture syndrome (GP), Wegener’s granulomatosis (WG) (granulomatosis with polyangiitis) and microscopic polyangiitis (MP). The assay is intended for use in patients with signs and symptoms consistent with GP, WG, and MP. It is not intended for screening a healthy population.

FOR IN VITRO DIAGNOSTIC USE.

Background

Goodpasture syndrome is characterised by lung haemorrhage, renal failure and the presence of anti-GBM antibodies. The diagnosis is based on clinical signs of lung haemorrhage and rapidly progressive glomerulonephritis combined with findings of anti-GBM antibodies. As there are several other autoimmune diseases which may present with similar symptoms, this kit is a useful aid in differentiation. Less than one third of patients with reno-pulmonary syndromes have antibodies against the Goodpasture antigen, the majority having either proteinase 3-ANCA or myeloperoxidase-ANCA.

Indirect immunofluorescence was formerly used to detect anti-GBM antibodies. When the first ELISA based on a collagenase digest was published in 1981, assays using crude extracts were the only alternative. In 1984, the specific antigen was shown to derive from the C-terminal domain of type IV collagen (a3 chain) and sensitive and specific assays were subsequently developed. The molecular nature of the Goodpasture antigen is reviewed in. The antigen is characterised by a restricted tissue distribution, occurring mainly in kidney and lungs.

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), interest in ANCAs has steadily increased, and today these antibodies are considered to be the 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 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). PR3 is a serine protease with a molecular weight of 29kD, and MPO is a dimer with a molecular weight of 140kD. 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.

The Wieslab^® anti-GBM, ANCA screening kit has been clinically evaluated (see IFU).

Technical information

The wells of a double microtiterstrip are coated with purified (Bovine) GP antigen (A1, A2, B1 and B2), purified (Human Neutrophils) proteinase 3 (C1, C2, D1 and D2), purified (Human Neutrophils) myeloperoxidase (E1, E2, F1 and F2), HSA (Human Serum Albumin) (G1, G2) and empty (H1, H2). G and H are blank wells. 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 (goat) 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 as absorbance (optical density (OD)).