Development of an ELISA for sensitive and specific detection of IgA autoantibodies against BP180 in pemphigoid diseases.

Abstract

BACKGROUND:

Pemphigoids 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.

METHODS:

We 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.

RESULTS:

The 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.

CONCLUSIONS:

We 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.

• Authors: Kinga Csorba, Sabine Schmidt, Florina Florea, Norito Ishii, Takashi Hashimoto, Michael Hertl, Sarolta Kárpáti, Leena Bruckner-Tuderman, Wataru Nishie, Cassian Sitaru
• Journal: Orphanet journal of rare diseases (Orphanet J Rare Dis) Vol. 6 Pg. 31 (May 28 2011) ISSN: 1750-1172 [Electronic] England
• PMID: 21619684 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2011 Csorba et al; licensee BioMed Central Ltd.

Chemical References

• Autoantibodies
• Autoantigens
• Immunoglobulin A
• Non-Fibrillar Collagens

Topics

• Autoantibodies (blood)
• Autoantigens (blood, immunology, metabolism)
• Cell Line
• Enzyme-Linked Immunosorbent Assay (methods, standards)
• Humans
• Immunoglobulin A (blood)
• Non-Fibrillar Collagens (blood, immunology, metabolism)
• Pemphigoid, Bullous (blood, diagnosis, immunology)
• Sensitivity and Specificity
• Collagen Type XVII