Current Pharmaceutical Design, 2006, 12, 3735-3742 3735 1381-6128/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. New Analytical Tools for Studying Autoimmune Diseases Matthias Kalbas * , Angelika Lueking, Axel Kowald # and Stefan Muellner Protagen AG, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany and # MPI für Molekulare Genetik, Ihnestr. 73, D-14195 Berlin, Germany Abstract: Protein microarrays with immobilised proteins on their surface are new analytical tools to overcome the current limits with respect to sample volume and throughput. They have a great potential as well with respect to multiplexing of complex samples, as a research tool and in diagnostics. Based on recent advances in this technology, new applications for protein microarrays in studying autoimmune diseases were described. Required tools for bioinformatical analysis of pro- tein microarrays concerning normalisation, clustering and classification methods are discussed. The huge potential of this technology as well as future requirements such as protein microarray based diagnostics are presented. Key Words: autoimmune disease, protein microarray, biomarker discovery, autoantibody profiling. INTRODUCTION Protein biochips are emerging analytical tools to follow DNA microarrays as a new possible screening technology to identify any protein-ligand interactions [for review see 1]. They have a great potential as well in parallel processing of complex samples, as a research tool and in diagnostics. Traditional methods for the analysis of proteomes include two dimensional gel electrophoresis or chromatography which, when combined with mass spectrometry, enables large-scale separation and identification of proteins, includ- ing many of their postranslational modifications. These pro- teome studies have been already applied to the comparative study of expression patterns, e.g. of differentially developed states or diseased versus normal tissues [2, 3] and even for the comparison of related pathogenic versus non-pathogenic organisms [4]. While such experiments can be referred to an “unbiased” or “discovery-orientated” approach towards pro- teomics, they generate large data collections that need to be further processed by bioinformatical analysis. However, the experimental design of such experiments is crucial, since the pattern of proteins detected is very sensitive to even slight changes in culture growth, extract preparation, running con- ditions and gel composition. Additionally, the key bottle- necks of these methods are the requirement of large sample volumes and highly sophisticated automation, whereby the aspect of multiplexing still remains a challenge. Therefore, microarray based technologies in the field of proteomics are growing due to the versatility of applications, time and cost effectiveness, feasible automation, high sensi- tivity and the high degree of potential parallel analysis. Con- sequently, for the generation of protein biochips, highly automated technologies have been developed for the produc- tion of cDNA expression libraries, high-throughput protein expression, large-scale analysis of proteins and protein mi- croarray generation [reviewed in 5]. Based on these advances *Address correspondence to this author at the Protagen AG, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany; Tel: +49 231 9742-6300; Fax: +49 231 9742-6301; E-mail: matthias.kalbas@protagen.de in technology, several new applications for protein microar- rays in studying autoimmune diseases have been described. Autoimmune diseases affect 5% of the world population and our understanding and treatment of human autoimmune diseases has to be improved [6]. While the incidence of autoimmune diseases has increased over the last decades, reliable methods for the detection of individual riks of devel- oping autoimmune diseases are not available yet. Often, the current diagnostic tools are limited, as there is no assay to detect the quality of the patients’ response to drugs. Thus, in addition to improved diagnostics, the discrimination between drug-responder and drug non-responder before the onset of therapy might be a goal for future strategies in the treatment of autoimmune diseases. A characteristic feature of autoimmune diseases is the presence of high-affinity autoantibodies [7]. Although the pathogenic role for most of the autoantibodies in various autoimmune diseases needs to be elucidated, the identifica- tion of those autoantigens that are targeted by the autoanti- bodies during the immune response may present an impor- tant tool for diagnosis, classification and prognosis [8, 9]. Additionally, profiling the autoantibody repertoire may help to elucidate the pathophysiology of autoimmunity, enabling novel treatments such as antigen-tolerating therapy [10, 11]. In first attempts, protein microarrays have been used for the detection and validation of autoantibodies in biological fluids [10, 12-14]. APPLICATIONS OF PROTEIN MICROARRAYS In recent years, different formats of biochips such as protein arrays, peptide arrays, antibody arrays, living cell arrays, tissue arrays, and carbohydrates arrays have been developed (Fig. 1). The different formats are associated with a range of applications and show specific advantages as well as definite bottlenecks. Protein arrays comprised of immobilised proteins are an emerging biochip format. One bottleneck in manufacturing such high-throughput, high-content protein arrays is the effi-