A promt diagnosis is essential for patients with medical conditions such as Goodpasture Syndrom, granulomatosis with polyangiitis (Wegener's granulomatosis) or microscopic polyangiitis. Fast and simultaneous detection of autoantibodies against GBM, Proteinase 3 (PR3-ANCA) and Myeloperoxidase (MPO-ANCA) is possible with Vasculitis screen Wieslab® using capture technology. It has been described that the capture technique for PR3-ANCA has a higher sensitivity and correlates to severity of disease.
The Wieslab® Vasculitis 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.
A positive result should always be confirmed by a quantitative assay.
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, but there are several other autoimmune diseases which may present with similar symptoms and less than one third of patients with reno-pulmonary syndromes have antibodies against the Goodpasture antigen, the majority having either proteinase 3 (PR3)-ANCA or myeloperoxidase (MPO)-ANCA.
Thus patients with reno-pulmonary syndromes can be identified by three autoantibodies: i.e. anti-GBM, PR3-ANCA or MPO-ANCA.
The molecular nature of the Goodpasture antigen is the NC1 domain of the alpha 3 chain of type IV collagen. 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. 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 most important antigens are 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.
Antibodies to PR3 and MPO can be detected either by a direct ELISA method or capture technique where monoclonal antibodies capture the antigens. The capture technique is therefore an alternative method to detect ANCA. It has been described that the capture technique for PR3-ANCA has a higher sensitivity and correlates to severity of disease. It is uncommon with relapse of disease if the capture ELISA is negative.
Approximately 80-90% of WG patients manifest PR3-ANCA and 5-15% MPO-ANCA. In microscopic polyangiitis (MP) most patients with active MP are characterised by positive ANCA test results, MPO-ANCA being more frequent than PR3-ANCA.
The wells of a microtiterplate are coated with purified GBM antigen (Bovine) (row A and B), anti-PR3 monoclonal antibody and purified proteinase 3 (Human Neutrophil) (row C and D), anti-MPO monoclonal antibody and purified myeloperoxidase (Human Neutrophil) (row E and F), monoclonal mouse antibody (row G) and HSA (Human Serum Albumin) (row H). G and H are control 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. Addition of stop solution terminates the reaction, resulting in a coloured endproduct. The amount of bound antibodies correlates to the colour intensity and is measured as absorbance (optical density (OD)).