FC-08   APPLICATION OF A cDNA LIBRARY TO DETECT TARGET AUTOANTIGENS IN AN ALOPECIA AREATA MOUSE MODEL (C3H/HeJ)

L. Mooney, NJ Lindsey, JP Sundberg*, DJ Tobin. Dept. Biomedical Sciences, University of Bradford, Bradford, UK. *The Jackson Laboratory2, Maine, USA.

Alopecia areata (AA) is a common cause of hair loss afflicting approximately 2% of the general human population. Whilst the aetiology of AA is still unresolved, an immune-mediated pathogenesis is suspected. We have previously shown that high titre antibodies in mammals with AA can be specifically directed to the hair follicle (HF) Moreover, it has been shown that an abnormal antibody response to HF is present in symptomatic, and less so, in clinically unaffected C3H/HeJ AAlittermates, indicating that anti-HF antibodies may appear before onset of clinically- detectable hair loss. However, the exact HF antigens targeted by these autoantibodies are yet to be identified. We, therefore, aim to identify possible antigens that may be of relevance in the pathogenesis of AA. Proteins expressed by a mouse skin (full anagen) cDNA library, using the Uni-Zap XR vector system, were screened with sera collected monthly from C3H/HeJ mice with hair loss. Briefly, proteins expressed by bacteriophage were transferred onto nitrocellulose membranes and probed with pooled sera from 10 selected mice comprising three months pre- and post-symptoms. Reactivity was detected with anti-mouse IgG-HRP and chemiluminescence. Bacteriophages of interest were excised and plasmids containing the cDNA insert subcloned into Escherichia coli SOLR and XL1-MRF’ strains. Cultures of each bacterial clone were grown on nitrocellulose membranes, blocked and re-screened to confirm the expression and detection of proteins. Ongoing, screening of the cDNA library with pooled sera has identified 23 reactive proteins to date. Immunoscreening of each bacterial clone with pooled sera has demonstrated that subsequent expression and detection of each protein was successful, indicating that this method is both reliable and reproducible. These preliminary data suggest that this is an appropriate method with which to identify putative antigens targeted by autoantibodies in AA mice. We are currently screening each protein with separate monthly (12) aliquots of sera from 10 AA mice to determine at what point in the pathogenesis of the disease these proteins are targeted by autoantibodies. We intend to sequence all DNA inserts encoding proteins of interest to elucidate their putative identity and function, contributing to the understanding of the pathogenesis of AA.