title: A sensitive and restricted enzyme-linked immunosorbent assay for detecting a heterogeneous antibody population in serum from people suffering from a new variant of endemic pemphigus.
abstract: We recently described a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, that resembles Senear-Usher syndrome and identified autoantibodies to desmoglein 1 (Dsg1), as well as to multiple known and unknown antigens including plectins, in the serum of these patients. Here, we developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these EPF patients, and useful for serum epidemiological studies. A protein extract obtained from trypsin-digested fresh bovine skin and further purified on a concanavalin A matrix was used as antigen. This extract contains an important conformational epitope (a 45 kDa tryptic fragment of the Dsg1 ectodomain), which is recognized by antibodies in serum from patients with all varieties of pemphigus foliaceus (PF), and from half of those with pemphigus vulgaris with active clinical disease. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for PF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients diagnosed with EPF, non-endemic PF and other bullous diseases. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibilities of 98% (intraassay) and 95% (interassay). Comparing the ELISA with other tests to detect EPF autoantibodies, this ELISA was the most sensitive, followed by direct immunofluorescence (DIF), indirect immunofluorescence using anti-IgG4 monoclonal antibodies and immunoprecipitation (IP), respectively. The most specific assay was IP, followed by DIF. Immunoblotting to Dsg1 exhibited both poor sensitivity and poor specificity, although plectins were well visualized. We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples simultaneously and for detecting, with appropriate restriction and sensitivity, the heterogeneous antibody population seen in patients with this variant of EPF. Finally, autoantibody serum levels obtained with this ELISA correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.
authors.affiliation: Institute for Molecular Medicine and Genetics, Medical College of Georgia, CB 2803, 1120 15th Street, GA 30912-2630, Augusta, USA, aavelez@mail.mcg.edu
authors.forename: Ana María
authors.initials: AM
authors.lastname: Abréu-Vélez
authors.forename: Maria Mercedes
authors.initials: MM
authors.lastname: Yepes
authors.forename: Pablo Javier
authors.initials: PJ
authors.lastname: Patiño
authors.forename: Wendy B
authors.initials: WB
authors.lastname: Bollag
authors.forename: Fernando
authors.initials: F
authors.lastname: Montoya
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology / Q000302:isolation & purification; D015820:Cadherins / Q000276:immunology; D002417:Cattle; D003362:Cost-Benefit Analysis; D051183:Desmoglein 1; D019353:Endemic Diseases; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods* / Q000592:standards; D000939:Epitopes; D019085:Fluorescent Antibody Technique, Direct / Q000592:standards; D019084:Fluorescent Antibody Technique, Indirect / Q000592:standards; D006801:Humans; D010392:Pemphigus / Q000175:diagnosis / Q000453:epidemiology* / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry; D014020:Tissue Extracts / Q000276:immunology
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D015820:Cadherins; D051183:Desmoglein 1; D000939:Epitopes; D010446:Peptide Fragments; D014020:Tissue Extracts