Adapting cDNA Microarray Format to Cytokine Detection Protein Arrays

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• 作者：Yiwen Li and W. Monty Reichert
• 刊名：Langmuir
• 出版年：2003
• 出版时间：March 4, 2003
• 年：2003
• 卷：19
• 期：5
• 页码：1557 - 1566
• 全文大小：471K
• 年卷期：v.19,no.5(March 4, 2003)
• ISSN：1520-5827

文摘

A cytokine detection protein array was developed that combines the advantages of the cDNA microarraytechnology and sandwich fluoroimmunoassay. The protein array was printed by robotically spotting fivehuman cytokine and growth factor capture antibodies onto planar substrates. The printed arrays wereincubated with cytokine samples, bound by biotin-conjugated detection antibodies, and then detected bystreptavidin-conjugated Cy5. This assay protocol was prepared specifically for the special requirementsof 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 thelowest background. The dynamic ranges of the parallel assay for IL-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">, TNF-mages/gifchars/alpha.gif" BORDER=0>, VEGF, MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">, and TGF-mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">1 were 4 orders of magnitude with a detection limit (2× background) of 10 pg/mL. The system was testedagainst patient sera and samples from an in vitro VEGF release study, measuring very low cytokine levelswithout any detectable nonspecific cross reactivity. This cytokine detection protein array can be extendedto a larger menu of cytokines and growth factors for applications such as profiling the molecular signalingin wound healing.