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 the 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. Sensitivity of the chip was found to be 100 - 150 pg/ml for all five cytokines studied. Further studies require optimization of the chip sensor and reading mechanism to further increase sensitivity. Selectivity studies indicated that the chip was capable of a high degree of specificity when using mixed cytokine samples. Development of new, increased selectivity chips open the door to studying a number of important regulatory pathways such as hematopoesis, inflammation neurogenic regulation, and wound healing simulataneously on the same chip. Future models are expected to contain 30-100 ligands making multiple analyses possible.
