TY - JOUR
T1 - 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
AU - Abréu-Vélez, Ana María
AU - Yepes, Maria Mercedes
AU - Patiño, Pablo Javier
AU - Bollag, Wendy B
AU - Montoya, Fernando
N1 - Funding Information:
Acknowledgements Initial studies were funded by the Immuno-dermatology Laboratory of the Medical College of Wisconsin, Milwaukee, Wis., under the guidance of Dr. Luis A. Diaz. Later, this project was funded by the University of Antioquia, Basic Bio- medical Science Corporation, Medellin, Colombia (Abreu and Montoya) and by the Institute for Molecular Medicine, Medical College of Georgia, Augusta, Ga. (Abreu) and NIH # AR45212 (Bollag). We want to thank Drs. Monica Olague and Jose M. Mas-caro Jr. and Ms. Argelia Lopez for their team work. We also thank Dr. Detleff Zillikens (University of Wuerzburg, Germany) for providing serum from BP patients and Dr. George Guidice of the Department of Dermatology at the Medical College of Wisconsin for the NC16 peptide for the immunoadsorption studies. Dr. Abreu was the recipient of a scholarship from Colciencias, Colombia.
PY - 2004/3
Y1 - 2004/3
N2 - 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 45kDa 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.
AB - 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 45kDa 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.
KW - Autoimmunity
KW - ELISA
KW - Pemphigus
UR - http://www.scopus.com/inward/record.url?scp=1842781097&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1842781097&partnerID=8YFLogxK
U2 - 10.1007/s00403-003-0441-4
DO - 10.1007/s00403-003-0441-4
M3 - Article
C2 - 14730452
AN - SCOPUS:1842781097
SN - 0340-3696
VL - 295
SP - 434
EP - 441
JO - Archives of Dermatological Research
JF - Archives of Dermatological Research
IS - 10
ER -