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Allergy affects more than 25% of Western populations (1) and is estimated to be the sixth leading cause of chronic disease in the United States and Western Europe. The complexity of the condition is such that hundreds of common allergens have been described, and in order to maximize diagnostic efficiency there is an urgent clinical requirement for assays to provide multiple-allergen determination in a timely and cost-effective manner. Miniaturized immunoassays that utilize protein microarray technology now offer the possibility of circumventing most of the current limitations in the serodiagnosis of allergic disease. The heterogeneous nature of allergens presents many challenges in all aspects of developing such arrays, from immobilization of the capture molecule to detection of the bound ligand. In addition, there is no simple method of protein amplification (such as PCR for nucleic acids), and stabilization is yet a further major consideration. Notwithstanding these challenges, protein microarrays have been developed for the serodiagnosis of allergies and other complex clinical conditions. These assays exhibit good analytical and clinical performance and deliver significant advantages in convenience and cost compared with traditional ELISA test formats. This chapter details the techniques employed in the construction and processing of an allergen array specific for the serodiagnosis of allergic disease. An overview of protein microarray technology is provided and the principles that underpin the suitability for use of this technology in the identification and measurement of particular proteins in patient sera (serum profiling) are discussed. [ABSTRACT FROM AUTHOR]

Protein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats. [ABSTRACT FROM AUTHOR]