Cytokines serve a variety of regulatory functions, one of the most important being the regulation of T-cell differentiation. Secreted cytokine profiles have been demonstrated to be predictive of different T-cell differentiation, especially cytokines regulating cell-mediated and humoral immunity. In order to measure different cytokine profiles simultaneously in individual samples, a basic cytokine chip has been developed based on solid-phase immunoaffinity extraction of the analytes of interest. Arrays of recombinant streptavidin spots were derivatized to the surfaces of silanized glass microscope slides via a carbonyldiimidazole bridge. Anti-cytokine antibodies were biotinylated via their carbohydrate moieties and bound to the steptavidin. Samples were labeled with a laser dye prior to analysis and incubated with the chip. Following removal of non-reactive materials by washing, the bound analytes were read in a scanning laser densitometer. Improvements in both the immobilization chemistry and techniques for keeping the antibody molecules active have increased the sensitivity of the chip to 1.0 - 1.8 pg/ml for all five cytokines studied. Further studies are underway to improve this sensitivity as well as optimize the chip reading mechanism. The chip now available exhibits very good selectivity and a high degree of specificity when using model mixtures of cytokine. However, further studies are required to determine the efficiency of this chip against human samples and to increase the chip selectivity to 50 cytokines, chemokines, and growth factors. This refined chip will have applications in the analysis of a number of important regulatory pathways including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing.
