DiaMetra Anti PR3 (c-ANCA) Elisa Kit

CLINICAL SIGNIFICANCE
Anti-neutrophilic-cytoplasm antibodies (ANCA) represents 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 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 differenciated 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.

PRINCIPLE
Anti PR3 (c-ANCA) ELISA Assay Kit is a test is based on the binding of the antibodies in the sample with human neutrophil proteinase 3 coated into the microplates. In the first step the antibodies in calibrators, controls or prediluted patient samples bind to the inner surface of the wells. After a 60 minutes incubation the microplate is washed with wash buffer for removing 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 a 30 minutes incubation any excess 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 solution 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.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All Anti PR3 (c-ANCA) ELISA Assay Kit reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all Anti PR3 (c-ANCA) ELISA Assay Kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange Anti PR3 (c-ANCA) ELISA Assay Kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.

All serum or plasma samples must be prediluted 1:100 with sample diluent; for example 10 μL of sample should be diluted with 990 μL of sample diluent. The Controls are ready to use.

LIMITATIONS OF PROCEDURE
The presence of immune complexes or other immunoglobulin aggregates in the patient sample may cause an increased level of non-specific binding and produce false positives in this assay.