Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients

Abstract

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are intraepidermal blistering skin diseases. PV is characterised by autoantibodies directed against desmoglein (Dsg) 3 and in patients with the mucocutaneous variant also against Dsg 1, whereas in PF, only Dsg 1 is targeted. Here, ectodomains of Dsg 3 and Dsg 1 were recombinantly expressed in a human cell line (HEK293) and applied as authentic solid phases in ELISA test systems. Autoantibodies against Dsg 3 and ⁄ or Dsg 1 could be detected in 71 (100%) of 71 PV sera and against Dsg 1 in 48 (96%) of 50 PF sera. Control sera showed reactivity with Dsg 3 and Dsg 1 in 0.2% and 0.7%, respectively, of 401 healthy blood donors and in 2.1% of 48 randomly selected patients with bullous pemphigoid. No reactivity with Dsg 1 and 3 was detected in 21 patients with linear IgA disease. For both pemphigus variants, a statistically significant correlation between clinical severity and autoantibody levels was observed as demonstrated for 10 PV and 5 PF patients. In conclusion, the use of the ectodomains of Dsg 3 and 1 as target antigens expressed in a human cell line resulted in sensitive and specific ELISA systems for both diagnosis and monitoring of PV and PF.Key words: autoimmunity -detection -disease activity -ELISA Please cite this paper as: Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients.

Anti-Skin Specific Autoantibodies Detected by a New Immunofluorescence Multiplex Biochip Method in Patients with Autoimmune Bullous Diseases

“…Some authors showed that serum anti-Dsg antibody levels reflect clinical activity during the disease course and indicate that monitoring of serum autoantibodies in PV may be helpful [10]; others observed that there was no correlation between variation in disease extent and the level of autoantibodies against Dsg3 and Dsg1 [34]. Further studies showed that anti-Dsg autoantibody levels were significantly reduced after treatment and remission of disease.…”

“…In detail, each field of the slide is a mosaic of 6 substrates: (1) frozen primate esophagus section; (2) EU90 recombinant cells transfected with Dsg1 protein ectodomain (aa 1–569) [10]; (3) EU90 recombinant cells transfected with Dsg3 protein ectodomain (aa 2–640) [10]; (4) EU90 recombinant cells transfected with full-length BP230 protein (aa 1–2649) [24]; (5) EU90 recombinant cells transfected with C-terminal globular domain of the BP230 protein (aa 1875–2649) [24]; (6) microdrops of NC16A-4X BP180 free antigen (tetramer of the immunogenic NC16A noncollagenosus domain of the BP180 protein, aa 490–562, expressed in Escherichia coli ) [18]. …”

“…The four Euroimmun ELISA microplates were coated with: (1) the extracellular domain of Dsg1 (aa 1–548) [10] and (2) Dsg3 (aa 2–616) [10] – both proteins based on human cDNA were produced recombinantly in mammalian cells (HEK293); (3) biochemically purified C-terminal sequence (aa 2326–2649) [24] of the human 230-kDa BP antigen expressed in E. coli; (4) recombinant BP180-NC16A-4X, a tetramer (four copies fused to a carboxyterminal polyhistidine tag) of the immunogenic NC16A domain (aa 490–562) [18] of BP180, based on human cDNA, expressed in E. coli. …”

“…In recent years, human recombinant Dsg1 and Dsg3 proteins have been applied to the development of sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of circulating autoantibodies [6,7] and it has been shown that ELISA reactivities correlate with the disease activity of pemphigus patients [8,9]. A new ELISA method using as antigen ectodomains of Dsg1 and Dsg3 generated in human HEK293 cells for the detection of autoantibodies to Dsg1, anti-Dsg3 has also been recently marketed [10]. It would seem that the mature Dsg1 and Dsg3 isoforms preferentially recognize pathogenic autoantibodies compared to the proprotein isoform [11,12].…”

The Value of Anti-Desmoglein Enzyme-Linked Immunosorbent Assay in the Immunological Follow-Up of Pemphigus

“…However, the study population was Indian, having a higher frequency of Dsg1 antibodies than European patients [13]. Other studies showed a correlation between Dsg ELISA findings and clinical status during follow-up [10,11,14,15], but still others did not find a correlation between Dsg1 ELISA results and cutaneous severity [16,17] or Dsg3 ELISA results and mucosal severity [18,19], including disease activity scoring with arbitrary and non-validated clinical activity scores [9,16,17]. In contrast, other studies did not find a correlation during follow-up [9,16,20].…”