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 ? 2.5 pg/ml for all 50 cytokines studied. Studies are continuing to improve this level of sensitivity as well as further optimize the chip reading mechanism. The chip exhibits very good selectivity and a high degree of specificity when using both model mixtures of cytokines and human clinical samples. The chip is now in the evaluation stage prior to its use in such applications as the analysis of important regulatory pathways including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing in the clinical arena. ? ? Work has continued to develop and improve a static antibody array for measuring cytokines and growth factors in clinical samples. The acquisition of an automated chip reader greatly improved both sensitivity and selectivity of the antibody arrays. Additionally the use of dual laser system has expanded the usefulness of the array. Collaborative studies recently established with the Critical Care Center of the NIH Clinical Center have opened up new clinical applications for the antibody chip. At present, a panel of 50-100 specific antibodies is being immunochemically evalauated for inclusion in a patient-screening chip. This chip will be used to screen pediatric patients with inflammatory lung disease for regulatory cytokines, pain-associated neuropeptides, and other modulators associated with immune function in respiratory disease.
