Previously we demonstrated that multiple
cytokines could be simultaneously detected using an antibody-based
protein array system with high sensitivity and specificity from
conditioned medium and serum. Here, we created a higher density array system to simultaneously detect 35
cytokines from cell lysates and tissue lysates. This assay combines the advantages of the specificity of
enzyme-linked
immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, capture
antibodies dissolved in
methanol were spotted onto
polyvinylidene difluoride (
PVDF) membranes. The membranes were then incubated with tissue lysates or cell lysates. After removing unbound
proteins by extensive washing, the membranes were exposed to
horseradish peroxidase (HRP)-conjugated antibody(ies). The signals were visualized with an ECL system. High specificity, sensitivity, and accuracy of this approach were demonstrated. This approach can be used in any general laboratory setting without any sophisticated equipment. It should be feasible to extend this concept to develop a high-throughput
protein array system. Combining
nitrocellulose membrane-based and
PVDF membrane-based approaches, the human
cytokine array system can be applied to detect multiple
cytokine expression from cell lysate, tissue lysate, serum, plasma, and
conditioned medium. Future applications of this new approach include direct
protein expression profiling,
immunological disease diagnostics, and discovery of new
biomarkers.