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Desmoglein 3-ELISA: a Pemphigus Vulgaris Specific Diagnostic Tool

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Desmoglein 3-ELISA: a Pemphigus Vulgaris Specific Diagnostic Tool STUDY Desmoglein 3-ELISA A Pemphigus Vulgaris–Specific Diagnostic Tool Petra Lenz, MD; Masayuki Amagai, MD; Beatrix Volc-Platzer, MD; Georg Stingl, MD; Reinhard Kirnbauer, MD Background: Pemphigus vulgaris (PV) is an autoim- patients with various nonbullous skin disorders, and 10 mune-blistering disease of the skin and mucous mem- healthy individuals were tested. branes caused by autoantibodies against desmoglein 3 (Dsg3), an epidermal desmosomal adhesion protein of Results: Forty-seven (98%) of 48 serum samples from pa- the cadherin family. Cloning of the Dsg3 gene and ex- tients with PV that were positive by IIF on monkey esopha- pression of the protein in a native conformation en- gus were also reactive by Dsg3-ELISA, whereas 4 of 4 IIF- abled the recent development of a specific and sensitive negative PV serum samples showed no reactivity by ELISA. enzyme-linked immunosorbent assay (ELISA) for the de- In addition, negative ELISA results were obtained from 11 tection of PV autoantibodies. of 11 serum samples from patients with bullous pemphi- goid, 10 of 12 serum samples from patients with other bul- Objectives: To evaluate serum samples from patients lous skin disorders, 7 of 9 serum samples from patients with with PV and other dermatologic diseases for anti-Dsg3 autoimmune-connective tissue diseases, and 13 of 13 se- antibodies. To compare ELISA values with autoanti- rum samples from patients with other nonbullous skin dis- body titers obtained by classic indirect immunofluo- eases. Interestingly, 1 patient with paraneoplastic pemphi- rescence (IIF). gus had positive ELISA results. There was a positive correlation (r = 0.654) between ELISA values and IIF ti- Design: Serum samples from patients with PV and vari- ters within the whole population with PV. In addition, when ous other bullous and nonbullous skin diseases were tested multiple serum samples from 1 patient with PV sampled for anti-Dsg3 reactivity by ELISA. over a 2-year period were tested, ELISA reactivity paral- leled both the IIF titers and the clinical course. Setting: Ambulatory and hospitalized patients from a university hospital. Conclusion: The Dsg3-ELISA is a sensitive, objective, and PV-specific test that should be considered as an ad- Patients: Fifty-two serum samples from 11 patients with junct test for the management of patients with PV. PV, and serum samples from 11 patients with bullous pemphigoid, 12 patients with other bullous diseases, 22 Arch Dermatol. 1999;135:143-148 EMPHIGUS VULGARIS (PV) is tinocytes.5,6 In animal studies7 and in an autoimmune-blistering vitro8,9 experiments, anti-Dsg3 antibodies disease of the skin and have been shown to induce loss of cell mucous membranes. Histo- adhesion, indicating an important role in logically, PV is character- disease pathognesis. Pized by suprabasal acantholysis and From the Laboratory of Viral intraepidermal blister formation, and For editorial comment Oncology, the Division of clinically by flaccid blisters and erosions see page 195 Immunology, Allergy, and that usually arise on normal-appearing Infectious Diseases, Department skin and/or mucosa.1 Immunologically, Pemphigus antibodies are routinely of Dermatology, University of antibodies directed against the keratino- detected by direct or indirect immuno- Vienna Medical School, Vienna, cyte cell surface are detected in the skin fluorescence (IIF) testing using monkey Austria (Drs Lenz, and in the circulation; they recognize the esophagus as a substrate to establish the Volc-Platzer, Stingl, and 1-4 Kirnbauer); and the extracellular domain of Dsg3. Desmo- diagnosis, and for monitoring disease Department of Dermatology, glein 3, a 130-kd transmembrane glyco- activity that frequently follows antibody 10-13 Keio University School of protein of the cadherin family, mediates titers. However, these methods are Medicine, Tokyo, Japan weak calcium-dependent homophilic subjective, require an experienced exam- (Dr Amagai). cell-to-cell adhesion of epidermal kera- iner, and often fail to clearly distinguish ARCH DERMATOL / VOL 135, FEB 1999 143 ©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 tion and ELISA buffers to maintain the conformational epi- PATIENTS, MATERIALS, 18 AND METHODS topes of Dsg1 and Dsg3. WESTERN BLOT PATIENTS AND CONTROLS Fractions obtained during rDsg1 and rDsg3 purification were A total of 52 serum samples (stored at −20°C until assayed) sized by gel electrophoresis, transferred to an Immobi- from 11 patients with PV (6 women, 5 men; mean age, 56.8 lon-P membrane (Millipore Corporation, Bedfort, Mass), years; age range, 34-85 years) treated in the Department of and incubated overnight at 4°C with 5000-fold diluted an- Dermatology at the University of Vienna, Vienna, Austria, over tibody against the E-tag (Pharmacia Biotech, Uppsala, Swe- 2 years (September 1995-November 1997) were tested for anti- den), followed by incubation with a 40 000-fold diluted per- Dsg3 reactivity. The diagnosis of PV was established clini- oxide–conjugated antimouse IgG antibody (Biorad cally and by the typical histologic and immunopathologic find- Laboratories, Hercules, Calif). Specific binding was visu- ings. Forty-eight of these PV serum samples obtained from alized using a chemiluminescence Western blotting detec- 9 patients were positive by IIF on monkey esophagus and 4 tion kit (ECL; Amersham, Buckinghamshire, England). serum samples from 2 patients with PV were negative by IIF at the time of serum sampling (Table 1). In addition, se- PV SERUM ABSORPTION WITH rDSG rum samples from 45 patients with a variety of other bul- lous and nonbullous skin diseases were included in the study. To determine the antigenicity of the rDsg3 protein, immu- Of these patients, 11 were diagnosed as having bullous pem- noabsorption studies were performed using a PV serum phigoid; 12 had other bullous diseases including pemphi- sample with an IIF titer of 320. The serum sample was se- gus foliaceus (1 patient), PNP (2 patients), epidermolysis bul- rially diluted and incubated overnight with column elu- losa acquisita (1 patient), porphyria cutanea tarda (1 patient), ates containing native rDsg3, rDsg1, or denatured (at 96°C) and unclassified bullous diseases (4 patients); 9 had autoim- rDsg3 protein and reevaluated by IIF analysis. mune–connective tissue diseases and high-titer antibodies against nuclear antigens, and 13 had other nonbullous skin ELISA diseases including eczema (2 patients), lichen ruber planus (1 patient), Behc¸et disease (2 patients), erythema migrans (3 Immunomicrotiter plates (Nunc; Roskilde, Denmark) were patients), thermal burn (1 patient), and sexually transmit- coated with 50 µL of eluate and incubated overnight at 40°C. ted diseases (4 patients). To establish a cutoff value for the After extensive washing in ice-cold TBS-Ca containing 0.05% ELISA we tested serum samples from 10 healthy individuals. Tween (TBS-Ca/T), the wells were blocked with 1% bovine serum albumin (Sigma, St Louis, Mo) in TBS-Ca/T. Tested IIF ANALYSIS serum samples were coded to allow blinded testing, serially diluted from 1:100 to 1:10 000 in bovine serum albumin and Serum samples were tested at serial dilutions of 1:10 to 1:320 TBS-Ca/T and added for 1 hour at room temperature. In ini- for the presence of antibodies against keratinocyte cell sur- tial experiments the most specific optical density (OD) value face proteins by IIF analysis with cryosections of monkey with the least background was achieved at a serum dilution esophagus as a substrate using 1:50 dilution of fluorescein of 1:10 000 and subsequently serum samples were tested at isothiocyanate–conjugatedanti–humanIgGantibodies(INOVA this dilution only. Plates were then incubated with 2000-fold Diagnostics, San Diego, Calif) as a second antibody. diluted peroxidase–conjugated rabbit anti–human IgG an- tibodies (monoclonal antibody against the k-light chain of PRODUCTION AND PURIFICATION all immunoglobulin classes) for 1 hour at room temperature. OF rDSG BACULOVIRUS PROTEIN Color development was achieved by adding 100 µL of ABTS (Boehinger Mannheim, Mannheim, Germany) for 20 min- Desmoglein 1 and Dsg3 were expressed and purified as de- utes, and OD at 405 nm was determined using an ELISA reader scribed previously.16,17 Recombinant baculovirus encoding the (Dynatech, Guerney, Channel Island). All assays were per- entire extracellular domain of Dsg1 or Dsg3 fused with formed in duplicate and background values determined for complementary DNA encoding for an E-tag and a histidine- each test serum sample in uncoated wells were subtracted. tag was used to infect “high-five” insect cells (Invitrogen, San Each plate contained at least 1 negative control serum sample Diego) cultured under serum-free conditions (Sf 900 II SFM; and a positive PV reference serum sample (IIF titer, 160). Gibco BRL, Grand Island, NY) for 3 days at 27°C, resulting The OD of the tested serum samples obtained for each plate in the secretion of rDsg into the culture supernatant. Cell de- were normalized relative to the mean OD obtained for the bris was removed by centrifugation, and supernatants were PV reference serum sample (dilution, 10 000) tested on the immediately affinity purified or stored at −70°C. For purifi- same 96-well plate. The mean of the normalized OD was used cation of rDsg, culture supernatants were applied to nickel in the analysis. A test serum sample was considered posi- nitrilotriacetic acid–agarose (Qiagen Inc, Chatsworth, Calif) tive by ELISA when the mean OD was 3 SDs above the mean columns allowing binding of the protein via the histidine- OD of serum samples from 10 healthy volunteers (OD, 0.09). tag. The protein was eluted with sequential 1-mL volumes of 200 mmol/L of imidazole in a 20-mmol/L sodium phos- STATISTICS phate and 500-mmol/L sodium chloride solution (pH 6.3) containing 1 mmol of calcium chloride per liter, dialyzed To determine the correlation between ELISA OD and IIF ti- against 50-mmol/L Tris-buffered saline (TBS)–calcium, ana- ter, we applied the Spearman correlation for nonparametric lyzed by Western blot and used for coating of the ELISA plates.
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