Anti-Myeloperoxidase
Enzyme-linked immunosorbent assay method for the semi-quantitative determination of specific IgG autoantibodies to myeloperoxidase in human serum.
Autoantibodies recognising antigens in the cytoplasmic granules of neutrophils (ANCA) were first described in a population of patients suffering from Wegener’s granulomatosis. Initial investigations were carried out using immunofluorescence and two major patterns were observed. One of these patterns, Peri-nuclear fluorescence (P-ANCA) was shown to be due to a number of antigens but primarily to the enzyme Myeloperoxidase (MPO), a 140kD dimer which makes up almost 5% of the cell protein.
In some sera, anti-myeloperoxidase antibodies give a more granular rather than peri-nuclear localisation. This is often due to their association to other ANCA’s. Other antigens recognised by ANCA’s and producing a P-ANCA pattern are Elastase, Cathepsin G and Lactoferrin. P-ANCA antibodies are found in patients with microscopic polyarteritis and other vasculitides e.g. Churg Strauss syndrome [6]. Up to 10% of patients with Wegener’s granulomatosis have anti-MPO rather than anti-PR-3 (C-ANCA) which is characteristic of the disease [7].
Anti-MPO are found in 65% of patients with idiopathic or vasculitis associated necrotising crescentic glomerulonephritis and also in patients with Goodpastures syndrome (30-40%)[7] where the antibodies are found in association with anti-Glomerular Basement Membrane (GBM) antibodies.
The AutostatII assay for detection of autoantibodies is a solid phase immunosorbent assay (ELISA) in which the analyte is indicated by a colour reaction of an enzyme and substrate. The AutostatII wells are coated with purified antigen (1).
On adding diluted serum to the wells the antibodies (2) present bind to the antigen. After incubating at room temperature and washing away unbound material, horseradish peroxidase conjugated anti-IgG monoclonal antibody (3) is added, which binds to the immobilised antibodies.
Following further incubation and washing, tetra-methyl benzidine substrate (TMB) (4) is added to each well. The presence of the antigen-antibody-conjugate complex turns the substrate to a dark blue colour. Addition of the stop solution turns the colour to yellow.
The colour intensity is proportional to the amount of autoantibodies present in the original serum sample.