A Highly Sensitive and Simple Assay for the Detection of Circulating Autoantibodies against Full-Length Bullous Pemphigoid Antigen 180

Schmidt, Enno; Kromminga, Arno; Mimietz, S.; Leinfelder, Ulrich; Sitaru, Cassian; Bröcker, Eva‐Bettina; Zillikens, Detlef; Zimmermann, U.

doi:10.1006/jaut.2002.0589

Cited by 17 publications

(11 citation statements)

References 38 publications

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“…Dsg3 Elisa (97.5-100% and 95.8-97.9%), Dsg1 Elisa (96.0-97.9% and 95.8-97.9%) BP180NC16A Elisa (84.4-89.9% and 97.8-98.9%), and BP230 Elisa (63.0-72.4% and 93–99.5%) [17,19,20,22,30,51,52] (Tables 2 and 3). Previously, indirect IF microscopy of Sf21 insect cells expressing full-length BP180 has shown a sensitivity and specificity of 89% and 100%, respectively, when probed with BP and control sera [53], however, this test is not widely available. The use of HEK293 cells results in the exclusive expression of the mature Dsg1 and Dsg3 forms, which have previously been shown to harbor major pathogenic epitopes on these two ectodomains [54,55].…”

Section: Discussionmentioning

confidence: 99%

Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

Beek

Rentzsch

Probst

et al. 2012

Orphanet J Rare Dis

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116

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BackgroundVarious antigen-specific immunoassays are available for the serological diagnosis of autoimmune bullous diseases. However, a spectrum of different tissue-based and monovalent antigen-specific assays is required to establish the diagnosis. BIOCHIP mosaics consisting of different antigen substrates allow polyvalent immunofluorescence (IF) tests and provide antibody profiles in a single incubation.MethodsSlides for indirect IF were prepared, containing BIOCHIPS with the following test substrates in each reaction field: monkey esophagus, primate salt-split skin, antigen dots of tetrameric BP180-NC16A as well as desmoglein 1-, desmoglein 3-, and BP230gC-expressing human HEK293 cells. This BIOCHIP mosaic was probed using a large panel of sera from patients with pemphigus vulgaris (PV, n = 65), pemphigus foliaceus (PF, n = 50), bullous pemphigoid (BP, n = 42), and non-inflammatory skin diseases (n = 97) as well as from healthy blood donors (n = 100). Furthermore, to evaluate the usability in routine diagnostics, 454 consecutive sera from patients with suspected immunobullous disorders were prospectively analyzed in parallel using a) the IF BIOCHIP mosaic and b) a panel of single antibody assays as commonly used by specialized centers.ResultsUsing the BIOCHIP mosaic, sensitivities of the desmoglein 1-, desmoglein 3-, and NC16A-specific substrates were 90%, 98.5% and 100%, respectively. BP230 was recognized by 54% of the BP sera. Specificities ranged from 98.2% to 100% for all substrates. In the prospective study, a high agreement was found between the results obtained by the BIOCHIP mosaic and the single test panel for the diagnosis of BP, PV, PF, and sera without serum autoantibodies (Cohen’s κ between 0.88 and 0.97).ConclusionsThe BIOCHIP mosaic contains sensitive and specific substrates for the indirect IF diagnosis of BP, PF, and PV. Its diagnostic accuracy is comparable with the conventional multi-step approach. The highly standardized and practical BIOCHIP mosaic will facilitate the serological diagnosis of autoimmune blistering diseases.

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Section: Discussionmentioning

confidence: 99%

Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

Beek

Rentzsch

Probst

et al. 2012

Orphanet J Rare Dis

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116

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“…In BP, autoantibodies may react with epitopes outside the NC16A domain of BP180 (38). Therefore, recombinant forms of full‐length and of the entire extracellular domain BP180 have been generated and used to develop immunoassays (31,39,40). However, the expression of collagenous proteins of high molecular mass requires eukaryotic expression systems and the yield of these recombinant forms is relatively low.…”

Section: Discussionmentioning

confidence: 99%

Enzyme‐linked immunosorbent assay using multimers of the 16th non‐collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies

Sitaru

Dähnrich

Probst

et al. 2007

Experimental Dermatology

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137

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Bullous pemphigoid (BP) and pemphigoid gestationis (PG) are acquired autoimmune subepidermal blistering diseases characterized by autoantibodies against the hemidesmosomal proteins BP180 ⁄ type XVII collagen and BP230. In the vast majority of BP and PG patients, these autoantibodies bind to epitopes clustered within the 16th non-collagenous domain of BP180. An ELISA system for the detection of these autoantibodies was developed and evaluated using 16th non-collagenous domain (NC16A) tetramers instead of monomers. In contrast to antigens fused to large proteins used in the past for the detection of autoantibodies against type XVII collagen, tetrameric antigen fragments bearing a small hexahistidine tag allow for high expression levels without the need to cleave off the fusion partner. Using tetrameric BP180 NC16A, positive reactions were found in 106 (89.8%) of 118 randomly selected BP sera and in all of 20 (100%) randomly selected PG sera, whereas only 2.2% of a large cohort of control subjects were positive in this assay, including patients with rheumatoid arthritis (two of 107), progressive systemic sclerosis (two of 50), systemic lupus erythematosus (one of 72), and healthy blood donors (10 of 494). Thus, the sensitivity and specificity of the new anti-tetrameric NC16A ELISA were 89.9% and 97.8% respectively. Levels of circulating autoantibodies against BP180 paralleled disease activity in the pemphigoid patients. In conclusion, the use of tetrameric NC16A in ELISA results in a sensitive and specific tool for diagnosis and monitoring of BP and PG.Key words: autoantigen -autoimmunity -BPAG2 -collagen -ELISA -tetramer Please cite this paper as: Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies. Experimental Dermatology 2007; 16: 770-777.

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“…First, many BP patients react with other epitopes outside of the NC16A domain. This could be shown by Western blot analysis using the soluble BP180 ectodomain (LAD‐1) derived from cultured human keratinocytes or by indirect IF microscopy of BP sera without BP180 NC16A reactivity on Sf21 insect cells where the entire BP180 ectodomain was expressed in a transmembrane fashion [8, 9].…”

Section: The Development Of New Diagnostic Testsmentioning

confidence: 99%

“…Circulating autoantibodies against BP230 can be found in 60–70% of BP patients [9, 13]. The great majority of sera recognize epitopes on the C‐terminal end which mediates interaction with keratin filaments of the cytoskeleton [13].…”

Section: The Development Of New Diagnostic Testsmentioning

confidence: 99%

Research in practice: diagnosis of subepidermal autoimmune bullous disorders

Schmidt

Zillikens

2009

J Deutsche Derma Gesell

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Subepidermal autoimmune bullous disorders are a heterogeneous group of diseases that are associated with autoantibodies to hemidesmosomal proteins (pemphigoid group, epidermolysis bullosa acquisita) or epidermal/tissue transglutaminase (dermatitis herpetiformis). Characterization of the target antigens has led to the description of novel entities such as anti-p200 pemphigoid and has greatly facilitated the diagnosis of these diseases. Precise identification of target antigens is of therapeutic relevance since treatment options may greatly differ between these disorders. By the use of various in vitro and in vivo systems, the pathogenic relevance of autoantibodies to different hemidesmosomal constituents, including BP180 (type XVII collagen), laminin 332 (laminin 5), alpha6beta4 integrin, and type VII collagen has been demonstrated. The translation of this basic scientific work has subsequently led to the development of specific autoantibody detection systems that are not only of diagnostic use but also allow the monitoring of circulating autoantibody levels during the course of the disease and thus are helpful in guiding treatment decisions in these patients.

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A Highly Sensitive and Simple Assay for the Detection of Circulating Autoantibodies against Full-Length Bullous Pemphigoid Antigen 180

Cited by 17 publications

References 38 publications

Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

Enzyme‐linked immunosorbent assay using multimers of the 16th non‐collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies

Research in practice: diagnosis of subepidermal autoimmune bullous disorders

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