Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology

Abstract

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

“…This had lead to the profiling of the antibody repertoire of patients with various diseases. In a series of pioneering studies a number of putative novel autoantigens have been identified from one of the largest collections of recombinant human protein expression clones (Uniclone ᮋ technology, www.protagen.de) using pools of patient sera (91)(92)(93). The UNIclone ᮋ collection consists of ; 11,000 different, sequence-characterized human recombinant proteins.…”

Probing the mRNA processing body using protein macroarrays and "autoantigenomics"

“…The bacteria are robotically transferred, at high density, to nitrocellulose membranes and are induced to express the cDNA in situ. This technique was used by other investigators to identify the targets of autoantibodies in patients with cardiac and dermatologic disorders (Lueking et al 2005;Horn et al 2006). In the present study, we used serum from patients with primary biliary cirrhosis and protein macroarray technology to identify potential P-body autoantigens and show that Y-box protein 1 (YB-1) is a novel component of these structures.…”

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

“…Our laboratory has developed protein array based technologies and methodologies since first developing the hEx1 protein expression library [18] with which we have performed studies on the binding of autoantibodies to arrayed proteins in Alopecia areata and Dilated Cardiomyopathy [19,20] , binding of antibodies to proteins identified from tumour neovasculature in humans [21],context independent motif identification in the human proteome [22] and of identification of novel proteinprotein interaction networks [23,24]. We have employed this library screening method as part of an ovarian cancer research consortium, Discovary, performing a pilot study on autoantibody identification screening the hEx1 protein library with ovarian cancer serum samples from a well characterised patient cohort with stage I ovarian cancer of mixed histology, stage III serous papillary adenocarcinoma, primary peritoneal carcinoma and normal/healthy individuals.…”