Abstract
BackgroundPemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids.MethodsWe expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis.ResultsThe optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n = 105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n = 31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value.ConclusionsWe developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients with pemphigoids. This immunoassay should prove a useful tool for clinical and translational research and should essentially improve the diagnosis and disease monitoring of patients with IgA pemphigoid. Moreover, our findings strongly suggest that IgA pemphigoid and IgG bullous pemphigoid represent two ends of the clinical spectrum of an immunological loss of tolerance against components of hemidesmosomes, which is mediated by both IgG and IgA autoantibodies.
Highlights
Pemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/ bullous pemphigoid antigen of 180 kDa (BP180)
A major target of pemphigoid autoantibodies is the bullous pemphigoid antigen of 180 kDa (BP180), referred to as collagen XVII, a hemidesmosomal transmembrane protein with a type II orientation whose extracellular domain consists of 15 collagenous regions interrupted by non-collagenous portions (Figure 1A) [1,4,6]
The pathogenic relevance of IgG autoantibodies against BP180 is supported by several lines of evidence: 1) the transplacental transfer of pemphigoid IgG autoantibodies from mothers to the fetus induces transient skin blistering in the newborn [14,15,16]; 2) serum levels of IgG autoantibodies against BP180 correlate with disease activity in patients with bullous pemphigoid and pemphigoid gestationis [17,18,19,20]; 3) patients autoantibodies against BP180 recruit leukocytes to the dermal-epidermal junction and induce dermal-epidermal separation of human skin [21,22]; 4) IgG antibodies against BP180 induce subepidermal blistering when passively transferred into neonatal autoantigen humanized, wild type mice and hamsters [23,24,25,26]; 5) grafting of human BP180 transgenic mouse skin induces an autoimmune response resulting in subepidermal blistering in wild-type animals [27,28]
Summary
Pemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/ BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. The aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids. IgE autoantibodies against BP180 correlate with disease activity in pemphigoid patients and induce eosinophil infiltration and dermal-epidermal separation when injected into human skin grafted on immunodeficient mice [29,30,31]
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