MEASUREMENTS OF PROTEINASE-3 AND ITS COMPLEXES WITH ALPHA(1)-PROTEINASE INHIBITOR AND ANTINEUTROPHIL CYTOPLASM ANTIBODIES (ANCA) IN PLASMA

Wegener's granulomatosis (WG) is a systemic vasculitis which is diagno sed on clinicopathological findings. The diagnosis may be aided by the presence of anti-neutrophil cytoplasm antibodies (ANCA). In WG, ANCA are primarily directed to proteinase 3 (PR3), a serine protease of the azurophilic granules of the neutrophilic granulocyte. The main plasma inhibitor of PR3 is alpha(1)-proteinase inhibitor (PI). To study if f ree PR3 or complexes between the enzyme and PI or PR3 and ANCA could b e found in the plasma from patients with WG we have developed three EL ISA systems for the detection of these complexes and free PR3. In all three assays monoclonal antibodies against PR3 were used as capture an tibodies. After incubation with plasma, free PR3 was detected by affin ity purified rabbit anti-PR3 followed by alkaline phosphatase-labelled swine anti-rabbit IgG. Serial dilutions of purified PR3 was used as s tandard. The detection limit was 3 ng/ml. PR3 complexed with PI was me asured by rabbit anti-PI antibodies and alkaline phosphatase-labelled swine anti-rabbit IgG. Pre-formed in vitro complexes of PR3/PI in seri al dilutions were used as standard. The detection limit of this assay was 1 ng/ml. PR3/IgG-ANCA complexes were detected by alkaline phosphat ase labelled goat anti-human IgG. A positive plasma sample in serial d ilutions was used as standard. Plasma samples from nine patients with WG, eight patients with fever of infectious origin without evidence of vasculitis and ten healthy donors were examined by these methods. Fre e PR3 could not be found in any of the plasma samples. PR3/PI complexe s were detected in healthy donors at levels between 41-85 ng/ml. All W G patients, both active and inactive, had PR3/PI concentrations above this level, and so had all patients with fever. PR3/IgG-ANCA was found in three of the patients with WG, two being ANCA negative with inacti ve disease andone was ANCA positive with active disease. Thus, the dev eloped methods can be useful for future studies of the clinical releva nce of these complexes in patients with WG and possibly other vasculit ides.