High-sensitivity Detection of Autoantibodies Against Proteinase-3 by a Novel Third-generation Enzyme-linked Immunosorbent Assay

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1 CONTEMPORARY CHALLENGES IN AUTOIMMUNITY High-sensitivity Detection of Autoantibodies Against Proteinase-3 by a Novel Third-generation Enzyme-linked Immunosorbent Assay Dirk Roggenbuck, a Thomas Buettner, a Lars Hoffmann, b Helmuth Schmechta, b Dirk Reinhold, c and Karsten Conrad d a GA Generic Assays GmbH, Dahlewitz/Berlin, Germany b Seramun Diagnostica GmbH, Heidesee, Germany c Institute of Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany d Institute of Immunology, Technical University Dresden, Dresden, Germany The aim of this study was to evaluate a novel third-generation enzyme-linked immunosorbent assay (ELISA) for the high-sensitivity detection of autoantibodies to proteinase-3 (PR3) in patients with Wegener s granulomatosis (WG). First- and secondgeneration ELISA for the detection of antineutrophil cytoplasmic antibodies (ANCA) frequently demonstrate insufficient sensitivity due to inadequate presentation of autoantigenic epitopes. Human PR3 was immobilized on the solid phase of ELISA plates by anchoring technique. Anti-PR3 reactivity was measured in 34 C-ANCA positive patients with WG, 11 MPO-ANCA positive patients with other autoimmune vasculitides, 65 patients with systemic lupus erythematosus (SLE), and 137 healthy blood donors. Thirtythree of 34 patients with WG (97.1%) showed positive anti-pr3 IgG antibody reactivity. None of 11 MPO-ANCA positive vasculitis patients, none of 137 blood donors, and 3 of 65 SLE patients expressed elevated IgG reactivity to PR3 (specificity: 98.4%). Comparison with another third-generation ELISA did not reveal different qualitative results. However, there was no significant correlation between quantitative results of both assays. Receiver operating characteristic (ROC) curve analysis revealed a significantly better assay performance compared with first (direct)- and second (capture)-generation assays (P = and P = 0.001, respectively). Third-generation (anchor) anti-pr3 ELISA exhibit significantly higher sensitivity than previous generation assays. Anchoring of PR3 renders the granulocyte protein more autoantigenic compared with direct or capture immobilization. Key words: ANCA; proteinase-3; autoantibody; Wegener s granulomatosis; ANCAassociated vasculitis; ELISA Introduction Autoantibodies against the neutrophil autoantigen PR3 are specific markers for the Address for correspondence: Dr. Karsten Conrad, MD, Institute of Immunology, Technical University Dresden, Fetscherstraße 74, Dresden, Germany. Voice: ; fax: k_conrad@mail.zih.tu-dresden.de serological diagnosis of Wegener s granulomatosis (WG) and can also be found less frequently in patients with microscopic polyangiitis (MPA) and Churg-Strauss syndrome. 1 Due to the occurrence of ANCA in these systemic vasculitides the term ANCA-associated vasculitis (AASV) has been coined for these clinical entities. ANCA typically exhibit two different staining patterns of ethanol-fixed granulocytes Contemporary Challenges in Autoimmunity: Ann. N.Y. Acad. Sci. 1173: (2009). doi: /j x c 2009 New York Academy of Sciences. 41

2 42 Annals of the New York Academy of Sciences in indirect immunofluorescence (IIF) a speckled cytoplasmic pattern (C-ANCA) and a perinuclear pattern (P-ANCA). C-ANCA, frequently found in patients with WG, are directed against PR3, while P-ANCA, occurring mainly in MPA, are directed against myeloperoxidase (MPO). 2 Determination of antibody levels is said to be helpful in the diagnosis and management of these clinical entities. Antibody values are usually associated with the activity of disease. 3 However, there have been other target antigens for ANCA described in IIF that can be found in patients with non-aasv. 4,5 Consequently, in accordance with recently established consensus guidelines, a different technique in addition to IIF for the detection of ANCA, such as ELISA, is recommended. 6 Commercially available ELISA employed in routine testing vary considerably in their performance characteristics, especially in terms of sensitivity of anti-pr3 evaluations. 6 8 Substantial differences in in-house ELISA even between reference laboratories have been reported. 9 Due to the fragility of this granulocyte autoantigen, sensitivity of ELISA depends upon presentation of PR3 in autoantigenic form to preserve its epitope structure. Direct immobilization of PR3 to ELISA plates employed in first-generation anti-pr3 ELISA proved to be inferior to the use of capture molecules like PR3-specific antibodies in second-generation ELISA. 9,10 However, even capture anti-pr3 ELISA lack sensitivity, so the search for better immobilization techniques have been proposed. 11 Anew technique to immobilize PR3 on the surface of ELISA plates by employing a nondisclosed anchor molecule as bridge has been described recently. 11 This novel anchor ELISA has been proven to be superior in sensitivity and specificity and may abolish the need to perform both IIF and ELISA, as discussed by the authors. This study describes the performance characteristics of an alternative novel third-generation anti-pr3 ELISA to prove the superiority of the anchor technique. Patients and Methods Patients Serum samples from 34 patients with WG positive for C-ANCA in IIF and 11 patients with other AASV positive for MPO-ANCA were collected and stored at 20 C. The diagnosis of WG was based on the Chapel Hill Consensus Definitions for WG. 12 Sixty-five patients fulfilling the ACR criteria of SLE were enrolled as disease controls in the study. 13 Sera from 137 healthy blood donors were used as controls. The study was approved by the local ethics committee. ANCA Detection by IIF ANCA were detected by running patient samples on ethanol- and formalin-fixed human granulocytes according to the recommendations of the manufacturer (GA Generic Assays GmbH, Dahlewitz, Germany). Briefly, fixed granulocytes were incubated in a moist chamber at room temperature (RT) for 30 minutes with 50 μl of serially diluted serum, starting at a concentration of 1:10. After washing, immune complexes were detected by incubating the samples with fluorescein-conjugated sheep anti-human IgG for 30 minutes at RT. Samples were subsequently washed, embedded, and analysed by the fluorescence microscope Axiovert 40 (Zeiss, Göttingen, Germany). Detection of MPO and PR3 Antibodies by ELISA Proteinase-3 and MPO autoantibodies in the patient sera were detected using ELISA, employing purified human PR3 and MPO as solid-phase antigen, respectively, according to the recommendations of the manufacturers (GA Generic Assays GmbH, Dahlewitz, Germany; Aesku.Diagnostics GmbH, Wendelsheim, Germany). For the detection of PR3 reactivity in patient sera, different assay generations were used; a commercial

3 Roggenbuck et al.: High-sensitivity Proteinase-3 Autoantibody Detection 43 first-generation anti-pr3 ELISA with direct immobilization of native PR3, a secondgeneration anti-pr3 ELISA employing capture molecules for the immobilization of native PR3, and two third-generation anti-pr3 ELISA with the anchor technique (Anti-PR3plus, GA Generic Assays GmbH, Dahlewitz, Germany; Anti-PR3 hs Orgentec Diagnostika GmbH, Wiesbaden, Germany). Anchor Anti-PR3 ELISA Proteinase-3 autoantibodies in the patient sera were detected using the Anti-PR3plus ELISA employing purified human PR3 as solid-phase antigen according to the recommendations of the manufacturer (GA Generic Assays GmbH, Dahlewitz, Germany). Briefly, 96-well microtiter plates (Nunc GmbH & Co. KG, Wiesbaden, Germany) were coated with a bridging molecule followed by incubation with purified human PR3 from neutrophil granulocytes (The Binding Site GmbH, Schwetzingen, Germany) in bicarbonate buffer (ph 9.5) for 24 h at 4 C. After blocking with 0.05 mol/l Tris/HCl, 1% bovine serum albumin (Tris- BSA, ph 7.4) at RT for one h, serum samples diluted 1:100 in dilution buffer (TrisBSA) were incubated at RT for one hour and subsequently washed. Horseradish peroxidase-conjugated antihuman IgG (Seramun, Heidesee, Germany) was added and developed with readyto-use H 2 O 2 /tetramethylbenzidine substrate (Seramun Diagnostica GmbH, Heidesee, Germany) after incubation for 30 minutes. The reaction was stopped with 0.25 mol/l sulfuric acid after 15 minutes. The optical density of the samples was read using a microplate reader (SLT, Crailsheim, Germany) at a wavelength of 450 nm/620 nm. Statistical Analysis Assay characteristics were compared by performing ROC curve analysis according to Hanley & McNeil. 14 Correlation of data was calculated by determination of Spearmen s coefficient of rank correlation. A difference of P < 0.05 was considered to be statistically significant. The software program MedCalc TM (MedCalc, Mariakerke, Belgium) wasemployedforstatisticalanalysis. Results Assay Characteristics of the Anti-PR3plus In order to determine the assay characteristics of the novel third-generation anti-pr3 ELISA, patient and healthy blood donor samples were run in the Anti-PR3plus. Remarkably, 33 out of 34 patients suffering from WG demonstrated a positive reactivity in the assay. None of the 11 MPO-positive patients suffering from other AASV and none of 137 blood donor sera showed elevated anti-pr3 reactivity. Only three out of 65 patients with SLE demonstrated anti-pr3 autoantibodies above the recommended cut-off of 10 U/mL. Receiver operating characteristics curve analysis revealed a sensitivity of 97.1 (95% confidence interval [CI]: ) and a specificity of 98.4 (CI: ) with an area under the ROC curve (AUC) of (CI: , P = 0001 for AUC = 0.5) (Fig. 1A). For these patient cohorts the optimal cutoff was calculated at 8.4 U/mL. Comparison of Different Anti-PR3 Assay Generations To compare different assay generations, 34 WG sera and 43 control sera comprising 11 MPO-positive AASV and 32 blood donor sera were run in the anchor ELISA Anti-PR3plus, in addition to second- and first-generation anti-pr3 ELISA (Fig. 1B). Remarkably, the third-generation ELISA Anti-PR3plus demonstrated the highest AUC compared with the second- and first-generation ELISA (P = 0.001, P = 0.011, respectively). In fact, only 16 and 14 out of 34 WG patients displayed a positive reactivity to PR3 in the second- and

4 44 Annals of the New York Academy of Sciences Figure 1. (A) Receiver operating characteristics curve (solid line) for the detection of reactivity to PR3 by the Anti-PR3plus in 34 patients with WG, 11 patients with MPO-positive AASV, 65 patients with SLE, and 137 blood donors. The 95% CI curves are given by dashed lines. The calculated AUC for the Anti-PR3plus was (CI: , P = for AUC = 0.5). (B) Receiver operating characteristics curve analysis of third-generation ELISA (Anti-PR3plus; solid line), second- (dashed line) and first-generation ELISA (dotted line) for the detection of anti-pr3 autoantibodies in 34 WG patients sera and 43 control sera. The third-generation ELISA displayed a significantly higher AUC of (CI: ) in comparison with the second-generation ELISA (0.904, CI: , P = 0.011) and the first-generation ELISA (0.804, CI: , P = 0.001). first-generation assays, respectively. None of the control sera displayed elevated anti-pr3 autoantibody, whereas one blood donor serum scored positive in both the second and the first generation ELISA. Comparison of Third-Generation Anti-PR3 Assays To compare the novel Anti-PR3plus with another commercial third-generation anti-pr3 (Anti-PR3 hs) assay, sera of 31 patients with WG were run in both assays. In fact, 30 WG sera demonstrated a positive reactivity to PR3 in both assays (Fig. 2). However, statistical analysis did not reveal a significant relationship between both assays with a Spearman s coefficient of rank correlation of (CI: ; P = 0.054). Discussion The novel Anti-PR3plus ELISA presented in this study is a highly sensitive but very Figure 2. Anti-PR3 reactivity of 31 patients with WG in Anti-PR3plus compared with another commercial third-generation (anchor) ELISA Spearman s coefficient of rank correlation: (CI: ; P = 0.054). specific diagnostic tool in the serological diagnosis of WG. In samples collected from patients with WG demonstrating C-ANCA reactivity in IIF, the novel anchor ELISA Anti- PR3plus detected autoantibodies to PR3 in 97.1% of patients with a high diagnostic specificity for WG of 98.4%. In accordance with these data Hellmich et al. reported elevated

5 Roggenbuck et al.: High-sensitivity Proteinase-3 Autoantibody Detection 45 PR3 autoantibodies in 96.0% of WG patient samples collected, prospectively employing a commercial third-generation assay. 11 The specificity of 98.5% in their patient cohort compares nicely with the specificity reported in this study. In accordance with a previous report, our data also support the superior sensitivity of third (anchor)-generation ELISA in comparison with first- and second-generation assays. 11 The higher sensitivity of the investigated anchor technique for immobilizing human PR3 compared with direct and capture methods may be explained by creating a higher number of autoantigenic epitopes on the surface of ELISA plates or by stabilizing the epitope structure of autoantibody binding sites. Direct immobilization in first-generation assays is mainly achieved by interaction of hydrophobic regions of the antigen with the plastic surface of microtiter plates leading to conformational changes of the antigen and blocking of epitopes adjacent to the interaction region. Both effects can impair the binding of anti-pr3 autoantibody, ultimately causing falsely negative results. The employment of capture molecules like PR3-specific monoclonal antibodies may preserve the epitope structure of PR3 during the coating process in second-generation assays. Therefore, sensitivity of capture ELISA is reportedly often higher than that of firstgeneration assays. 9 However, PR3-specific autoantibodies binding to the epitope of the capture antibody or directed to the vicinity of this epitope may be hindered to interact with the captured PR3 molecule. Consequently, this may also lead to false negative results in capture ELISA. Third-generation assays with the anchor technique employ a bridging molecule that probably prevents direct adhesion to the plastic surface, preserves the conformation of the PR3 molecule and renders the antigen more autoantigenic. 11 That may explain the higher sensitivity of anchor ELISA in this study and reported elsewhere. Surprisingly, data presented in this study revealed no significant correlation between the Anti-PR3plus and another commercial thirdgeneration assay described by Hellmich et al.. 11 This fact points to a different PR3 epitope structure after immobilization in both assays and needs further investigation. In summary, this study confirms the superior sensitivity of anchor ELISA for the detection of PR3 antibodies in WG patients. However, further studies confirming the high sensitivity and specificity of third (anchor)-generation assays are warranted to support the suggestion by Hellmich et al. thatanchorelisa mayreplace the need to perform IIF and ELISA in routine clinical testing. 11,15,16 Acknowledgments We thank Simone Berndt for excellent technical assistance. Competing Interests Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH, a diagnostic manufacturer. The remaining authors declare that they have no competing financial interests. References 1. Bosch, X., A. Guilabert, & J. Font Antineutrophil cytoplasmic antibodies. Lancet 368: Van der Woude, F.J., et al Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener s granulomatosis. Lancet 1: Cohen Tervaert, J.W., et al Association between active Wegener s granulomatosis and anticytoplasmic antibodies. Arch. Intern. Med. 149: Savige, J., W. Pollock & M. Trevisin What do antineutrophil cytoplasmic antibodies tell us? Best Pract. Res. Clin. Rheumatol. 19: Savige, J., et al Addendum to the International Consensus Statement on testing and reporting of anti-neutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune disease. Am. J. Clin. Path. 120:

6 46 Annals of the New York Academy of Sciences 6. Savige, J., et al International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am. J. Clin. Pathol. 111: Wang, G., et al Comparison of eight commercial kits for quantification of antineutrophil cytoplasmic antibodies (ANCA). J. Immunol. Meth. 208: Holle, J.U., et al Validations in performance characteristics of commercial enzyme immunoassay kits for the detection of antineutrophil cytoplasmatic antibodies: What is the optimal cut-off? Ann. Rheum. Dis. 64: Csernok, E., et al Evaluation of capture ELISA for detection of antineutrophil cytoplasmic antibodies against proteinas-3 in Wegener s granulomatosis: First results from a multicenter study. Rheumatology 43: Lee, A.S., et al A novel capture-elisa for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxyterminally tagged recombinant neutrophil serine protease. J. Immunol. Meth. 307: Epub 2005 Oct Hellmich, B., et al A novel high sensitive ELISA for detection of antineutrophil cytoplasm antibodies against proteinase-3. Clin. Exp. Rheumatol. 25(Suppl. 44): S1 S Jenette, J., et al Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum. 37: Hochberg, M.C Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis Rheum. 40: Hanley, J. & B. McNeil The meaning and use of the area under a receiver operating characteristics (ROC) curve. Radiology 143: Miller, A., et al Assesment of systemic vasculitis. Autoimmun. Rev. 8: Müller, A., et al Human proteinase 3 (PR3) and its binding molecules: implications for inflammatory and PR3-related autoimmune responses. Ann. N. Y. Acad. Sci. 1109: