Anti-MPO pANCA ELISA Assay

Additional Information

Assay Background

Anti-neutrophilic-cytoplasm antibodies (ANCA) represent a group of autoantibodies directed towards the cytoplasmatic components of the neutrophilic granulocytes and monocytes. The classical methods for the determination of ANCA are the immunofluorescent methods. With these indirect immunofluorescence techniques, two main patterns are recognized, a cytoplasmatic (c-ANCA) and a perinuclear (p-ANCA) type.

The main antigen for the c-ANCA is the proteinase 3 (PR3), which is a serine proteinase of the present in primary granules. Antibodies of p-ANCA positive sera are mainly directed to myeloperoxidase (MPO). Antibodies to other antigens e.g. lactoferrin, elastase, cathepsin-G and also lysozyme often result in a similar p-ANCA pattern. Beside different untypical variants of p- ANCA IF patterns granulocyte specific antinuclear antibodies (GS-ANA) is indistinguishable from p-ANCA. This makes a clear interpretation and classification of the IF patterns difficult.

Therefore every positive IF-ANCA findings esp. p-ANCA should be differentiated by ELISA techniques using purified antigens. A survey of documented clinical indications of specific ANCA is given in the table below. PR3- ANCA and MPO-ANCA are reliable serologic markers in the diagnostics of vasculitides.

PR3- ANCA is the classical autoantigen in Wegener´s granulomatosis with a clinical specificity of more than 95%. c-ANCA is documented to be present in different diseases. Anti-MPO antibodies are highly specific for idiopathic and vasculitis associated crescentic glomerulonephritis and also for classic polyarteritis nodosa, Churg-Strauss syndrome and the polyangiitis overlap syndrome without renal involvement.7-10 With respect to sensitivity, either MPO or PR-3 antibodies were found in 77 to 100% of patients with idiopathic and vasculitis associated crescentic glomerulonephritis. In WG, anti-MPO antibodies were detected only occasionally and generally in patients negative for PR-3 antibodies.

The MPO and PR-3 specific ELISA methods can provide an important confirmatory result for two of the more important of the identified antigens. ELISA is also useful for interpreting “difficult” samples by IFA such as those which exhibit several antibodies simultaneously or those with high background fluorescence.

Assay Principle

Anti MPO (p-ANCA) ELISA Assay Kit is based on the binding of the antibodies in the sample to the human neutrophil myeloperoxidase coated on the microplates. In the first step, the antibodies in calibrators, controls or prediluted samples bind to the inner surface of the wells. After 60 minutes of incubation, the microplate is washed with a wash buffer to remove the non-reactive serum components. In the second step, an anti-human-IgG horseradish peroxidase-conjugated solution recognizes the IgG class antibodies bound to the immobilized antigens. After 30 minutes of incubation, any excess of enzyme conjugate which is not specifically bound is washed away with the wash buffer. Then a chromogenic substrate solution containing TMB is dispensed into the wells. After 15 minutes of incubation, the color development is stopped by adding the stop solution. The solutions color change into yellow. The amount of color is directly proportional to the concentration of IgG antibodies present in the original sample. The concentration of IgG antibodies in the sample is calculated through a calibration curve.