PR3 IgG (c-ANCA) capture ELISA

Inteded use

Enzyme immunoassay for the qualitative and quantitative determination of IgG antibodies against Proteinase 3 (c-ANCA) (capture version) in human serum or plasma (EDTA, citrate or heparin).

Introduction and background

Anti-neutrophil cytoplasmic antibodies (ANCA), originally identified by immunofluorescence assays (IFA), are directed against cytoplasmic components of neutrophil granulocytes and monocytes. They have proven to be a useful serologic marker for a number of systemic, autoimmune mediated vasculitides (1, 2, 3).

A granular cytoplasmic (c-ANCA) staining pattern of the neutrophil substrate is indicative for autoantibodies against Proteinase 3 (PR3), a 29 kDa serine proteinase present in the azurophilic granules of human granulocytes and monocytes (4, 5).

PR3 antibodies occur in patients with Wegener's granulomatosis (WG), a systemic vasculitis affecting the respiratory tract (5). Their specificity is about 95 %, their sensitivity depends on the phase and activity of the disease (6).

The present enzyme-linked immuno sorbent assay (ELISA) is intended for the quantitative or qualitative determination of IgG antibodies in human serum or plasma (cf. section 7), directed against PR3. It is calibrated against the international standard for PR3-serology, AF-CDC (human reference serum 16, code IS2721). The immobilised antigen is a highly purified preparation of PR3, isolated from human granulocytes.

During recent years it has been shown that capture technique of antigen immobilisation achieves improved sensitivity, as compared to conventional (adsorptive) fixation (7, 8, 9). The present ELISA takes advantage from this technique, with the additional feature that the PR3 molecule is exposed in two distinctly different orientations.

The test is fast (incubation time 30 / 30 / 30 minutes) and flexible (divisible solid phase, ready-to-use reagents). Six calibrators allow quantitative measurements; a negative and a positive control check the assay performance.

Principle of the test

The wells of the solid phase are coated with PR3 by a special capture technique. On this surface, the following immunological reactions take place:

1st reaction: PR3-specific antibodies present in the sample bind to the immobilised antigen, forming the antigen antibody complex. Then, non-bound sample components are washed away from the solid phase.

2nd reaction: A second antibody, directed at human IgG antibodies and conjugated with horse-radish peroxidase (HRP), is added. This conjugate binds to the complex. Then, excess conjugate is washed away from the solid phase.

3rd reaction: The enzyme-labelled complex converts a colourless substrate into a blue product. The degree of colour development reflects the concentration of PR3 IgG in the sample.