Anti-Proteinase-3
Enzyme-linked immunosorbent assay method for the semi-quantitative determination of specific IgG autoantibodies to Proteinase-3 in human serum.
Autoantibodies recognising antigens in the cytoplasmic granules of neutrophils (ANCA) were first described in a population suffering from Wegener’s granulomatosis. Initial investigations were carried out using immunofluorescence and two major patterns were observed: C-ANCA (cytoplasmic) and P-ANCA (peri-nuclear).
C-ANCA has been shown to be mainly due to a 28kD serine proteinase termed proteinase-3 and is found in the majority of patients with Wegener’s granulomatosis. Pr-3 is clinically significant as a diagnostic marker for Wegener’s granulomatosis, a disease characterised by a nectrotising vasculitis of the upper and lower respiratory tract and concomitant glomerulonephritis.
Previously, the diagnosis of this disease carried a very poor prognosis, however, like other autoimmune diseases, improvements in diagnosis, such as the measurement of ANCA, have shown the disease to be present in milder forms and appropriate treatment has improved patient prospects considerably. The sensitivity and specificity of Pr-3 as a disease marker for Wegener’s granulomatosis has been quoted as high as 96% and 99%, respectively, in severe and extended disease. A less severe or minimal Wegener’s with no renal involvement has been described: Pr-3 is present in 67-87% of these patients. C-ANCA is also found at levels of 40-50% in pauci-immune necrotising glomerulonephritis, a further variant of the disease.
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.