ANTINEUTROPHIL CYTOPLASMIC ANTIBODY MEASUREMENT BY CAPTURE PR3 ELISA - ADVANTAGES AND DISADVANTAGES

Background: Detection of antineutrophil cytoplasmic antibodies (ANCA) is now integral to the diagnosis of Wegener's granulomatosis and quantification may be important in monitoring disease activity. There is continuing debate about the best method to measure PR3 ANCA.Objectives: To compare the performance of a capture proteinase 3 enzyme linked immunosorbent assay (PR3 ELISA) with a direct PR3 ELISA in the measurement of PR3 ANCA.Methods: The clinical sensitivity of a capture PR3 ELISA using a novel PR3 specific mouse monoclonal antibody was defined using serum from patients suffering from Wegener's granulomatosis (WG). Clinical specificity was defined using serum from a well characterised patient cohort each member of which gave a false positive PR3 ANCA by direct ELISA. The cut-offs for positivity of the capture and direct ELISA were defined using three sets of non-WG sera: sera from normal blood donors, sera containing high titre autoantibodies to double stranded DNA, Ro, La nuclear antigens or rheumatoid factor and sera containing high titre antibodies to a spectrum of infectious agents. The performance of both direct and capture PR3 ELISA assays in monitoring WG activity was determined using serial serum samples from seven patients with WG.Results: The direct PR3 ELISA and the capture PR3 ELISA had similar intra and inter assay variability. As a result of heterophil antibody or rheumatoid factor binding the cut-off for positvity of the capture PR3 ELISA was over three times that of the direct PR3 ELISA. Using the assay specific cut-offs the clinical sensitivity of the direct PR3 ELISA was 92% and that of the capture ELISA 84%. The corresponding clinical specificities were 27% and 75% respectively. The mean concentration of PR3 ANCA in WG patient sera measured by the capture ELISA was more than twice than that measured by the direct PR3 ELISA. This improved analytical sensitivity and wider analytical range of the capture PR3 ELISA was also reflected in PR3 ANCA concentrations measured in serial serum samples from WG patients and the higher mean ANCA concentration in the WG cohort.Conclusion: In clinically defined control groups our capture PR3 ELISA offers equivalent diagnostic capability to our direct ELISA for the measurement of ANCA. An evaluation of the diagnostic performance of the capture PR3 ELISA in a routine setting, currently underway, will enable a fuller comparison of the assay setups. At the moment the main advantage appears to be its wider analytical range and improved analytical sensitivity. In the absence of rheumatoid factor or heterophil antibodies this latter attribute may make it a more valuable method for monitoring serial PR3 ANCA concentrations than currently used techniques.Citation: Ann Rheum Dis, volume 65, supplement II, year 2006, page 370Session: Vasculitis