Adapting cDNA microarray format to cytokine detection protein arrays

A cytokine detection protein array was developed that combines the advantages of the cDNA microarray technology and sandwich fluoroimmunoassay. The protein array was printed by robotically spotting five human cytokine and growth factor capture antibodies onto planar substrates. The printed arrays were incubated with cytokine samples, bound by biotin-conjugated detection antibodies, and then detected by streptavidin-conjugated Cy5. This assay protocol was prepared specifically for the special requirements of the cytokine detection, with special attention paid to identifying the substrate, array printing buffer, blocking buffer, and the fluorescent dyes that yielded the highest sensitivity and selectivity against the lowest background. The dynamic ranges of the parallel assay for IL-1beta, TNF-alpha, VEGF, MIP-1beta, and TGF-beta1 1 were 4 orders of magnitude with a detection limit (2 x background) of 10 pg/mL. The system was tested against patient sera and samples from an in vitro VEGF release study, measuring very low cytokine levels without any detectable nonspecific cross reactivity. This cytokine detection protein array can be extended to a larger menu of cytokines and growth factors for applications such as profiling the molecular signaling in wound healing.