Jens Lüdemann

Anticytoplasmic Autoantibodies: Their Immunodiagnostic Value in Wegener Granulomatosis

STUDY OBJECTIVE To determine disease specificity and sensitivity of anticytoplasmic autoantibodies (ACPA) for Wegener granulomatosis, as well as their value as a marker of disease activity. DESIGN Blind analysis of serum samples, retrospective analysis of clinical data on patients, and prospective follow-up of a subgroup of patients with Wegener granulomatosis. PATIENTS The study included 277 patients with Wegener granulomatosis (222 with biopsy-proven disease) and 1657 control patients. SETTING University hospital and academic medical center. LABORATORY INVESTIGATIONS: Analysis of 2653 serum samples from 1934 patients for ACPA. Antibody detection was by indirect immunofluorescence and a new type of enzyme-linked immunoadsorbent assay (ELISA). Prospective follow-up was on 172 patients with Wegener granulomatosis. MEASUREMENTS AND MAIN RESULTS Specificity of ACPA for Wegener granulomatosis measured by indirect immunofluorescence was 99% (CI, 98.9% to 99.7%) and 98% (CI, 97.4% to 99.2%) by ELISA. Sensitivity of ACPA depended on disease activity and extent: It was 67% (CI, 38% to 89%) by immunofluorescence and 60% (CI, 32% to 84%) by ELISA for patients with active locoregional symptomatology (n = 15); and 32% (CI, 14% to 54%) by immunofluorescence and 40% (CI, 21% to 61%) by ELISA for patients in full remission after initial locoregional symptoms (n = 25). The sensitivity was 96% (CI, 89% to 99%) by immunofluorescence and 93% (CI, 86% to 98%) by ELISA for patients with active generalized disease (n = 92). Serial testing was done; every patient with active generalized disease eventually had at least one positive serum sample. Sensitivity decreased to 41% (CI, 22% to 62%) by both immunofluorescence and ELISA for patients in full remission after active generalized disease (n = 27). Levels of ACPA expressed both as immunofluorescence titers and ELISA values (U/mL) correlated well with disease activity. CONCLUSIONS Testing for ACPA in serum of patients with Wegener granulomatosis is valuable for differential diagnosis; furthermore, APCA can be used as a marker to follow disease activity. A new type of ELISA yielded the same results as indirect immunofluorescence for the specificity, sensitivity, and correlation with disease activity of ACPA.

The value of indirect immunofluorescence and solid phase techniques for ANCA detection: A report on the first phase of an international cooperative study on the standardization of ANCA assays

This study describes the results of phase I of an international effort to develop and standardize assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). 12 sera, four of which were selected for their potential to cause problems in the detection of various ANCA specificities, were analyzed in the standard indirect immunofluorescence (IIF) test and in ELISAs for ANCA routinely performed in the seven participating laboratories. The IIF methodology differed with respect to the dilution of the serum being screened and the concentration of the conjugate used. Results from sera with high ANCA titers were similar, although the quantitative values could not be compared. In sera containing rheumatoid factor and anti-nuclear antibodies (ANA), ANCA-unrelated staining patterns were observed. Six antigen preparations were used in ELISA for the detection of cANCA. In ELISA with purified proteinase-3 all three cANCA sera were positive, but not anti-myeloperoxidase (MPO) or anti-lactoferrin (LF) positive sera. The other assays were less sensitive or gave inconsistent results. Various preparations of purified MPO and LF used in ELISA were readily recognized by anti-MPO and anti-LF positive sera. From this study it can be concluded that the IIF test, although performed with different methods, shows comparable results using strongly positive sera. In general solid phase assays for cANCA detection are not well standardized and need improvement although the purified proteinase-3 ELISA is possibly an exception. MPO and LF can be used in ELISA procedures for the detection of pANCA-related antibodies.

Elafin is a potent inhibitor of proteinase 3

Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C.

Detection and quantitation of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by ELISA using affinity-purified antigen

Autoantibodies directed against cytoplasmic components of neutrophil granulocytes and monocytes (ACPA) have previously been described as a disease-specific marker for Wegeners granulomatosis (WG). We have developed an ELISA for determining and quantifying ACPA using an affinity-purified antigen preparation. The antigen was purified from supernatants of human neutrophils stimulated with phorbol ester to induce degranulation, by means of affinity chromatography with naturally occurring human autoantibodies. The established ELISA was sufficiently specific and sensitive, and the ACPA concentrations obtained with it correlated significantly with the ACPA titres determined by an indirect immunofluorescence technique (Spearmans rank correlation coefficient = 0.85, P less than 0.001, n = 105 WG patients). This ELISA provides precise ACPA quantitation and should prove valuable for monitoring disease activity in WG.

Anti-Cytoplasmic Antibodies in Wegener’s Granulomatosis are Directed Against Proteinase 3

Wegener’s granulomatosis (WG), a systemic necrotizing granulomatous vasculitis, used to be considered a relatively rare disease, but in the past few years it has been diagnosed much more frequently. This is at least partially due to the study by Van der Woude et al. (1985), who showed that autoantibodies directed against a cytoplasmic antigen of human neutrophil granulocytes and monocytes (anti-cytoplasmic antibodies = ACPA, synonym: ANCA; C-ANCA) are a disease-specific marker of WG. The im-munodiagnostic value of ACPA has been confirmed by many investigators (Gross et al., 1986; Savage et al., 1987; Ludemann and Gross, 1987; Parlevliet et al., 1988). Moreover, it has been shown that changes in ACPA titer parallel changes in disease activity (Van der Woude et al., 1985; Parlevliet et al., 1988; Ludemann et al., 1988a; Specks et al., 1989; Nolle et al., 1989).

Inhibition of proteinase 3 activity by peptides derived from human epidermis.

Elafin and antileukoprotease are potent peptide-like inhibitors of human leukocyte elastase and have been isolated from human skin and bronchial mucus. Elafin proved to be a potent inhibitor of proteinase 3, whereas other inhibitors of human leukocyte elastase such as antileukoprotease and eglin C proved to be much less effective.