Protein microarrays for diagnosis and research of allergic diseases

Abstract

Summary: For both the systematic as well as for the system-wide analysis of protein function, interaction and abundance, protein microarrays represent one of the pillars underlying modern high-throughput Proteomics. The prospect of analysing samples in a highly parallel while miniaturized fashion has fuelled efforts to develop novel diagnostic applications in the cancer, autoimmune or allergy field. Here, I discuss recent advancements in the development of protein microarrays for the profiling of IgE antibodies in the diagnosis of Type 1-related allergic diseases. Protein microarrays, i.e. multiplex solid-phase immunoassays in a miniaturized form, represent just one of the emerging application in Proteomics (13,14). In principle, protein biochips are the counterparts of DNA biochip technology, using spatially separated and individually addressable microspots of antibo dies (15), proteins (16), small molecules (17) or cell extracts (18) contained in a microarray to monitor the function, interaction or expression of (protein) analytes of interest. Apart from the power of analyzing protein function on the proteome level, the prospect of deve loping diagnostic applications has become an attractive goal for researchers in the protein microarray field. For the development of novel diagnostic applications, protein microarray technology has been employed to immobilize purified (natural or recombinant) antigens or antibodies as capturing agents for the screening of analytes (e.g. IgEs or IgGs) in the serum of diseased patients. Examples include the monitoring of autoantibody responses in autoimmune diseases (19,20), alopecia areata (21), diabetes (22), systemic lupus erythematosus (23), allergy (16, 24Ê26), cancer (27), rheumatoid arthritis (28), and the profiling of linear allergen epitopes (29,30).Like their DNA biochip counterparts, protein microarrays are usually built on planar substrates, such as high-quality glass microscopy slides, silicon wafers or plastic devices. For the stable and functional immobilisation of proteins, the surface of the substrate is usually modified in order to bind protein compounds in a stable and biologically active manner. The latter is achieved by adding chemical modifications to the surface, or by the application of 3D-like functional layers, such as nitrocellulose or hydrogels. Different types and numbers of capture molecules (e.g. native or recombinant proteins, antibodies, peptides or aptamer molecules) are subsequently microarrayed on these substrates by robotic equipment in order to create individually addressable reaction sites (spots, features). Each of these (usually) mm-sized spots is later employed in a miniaturised ligand binding assay where each biological interaction can subsequently be monitored by applying specific detection antibodies (e.g. labelled with a fluorescence dye) combined with sensitive detection methods (e.g. fluorescence laser scanning microscopes). As a result fluorescence images are produced, and the signal intensities of each spot can be used for analyte...