Abstract
Cytokines play important roles in many aspects of cell physiology and pathology. The simultaneous determination of multiple cytokine-expression levels is receiving much attention in the research community. Multiple cytokine-expression levels can be simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology. In this approach, target proteins are captured by the arrayed capture antibody and then detected in a sandwich ELISA format using a cocktail of biotinylated detection antibodies. The signals are visualized by either horseradish peroxidase (HRP)-conjugated streptavidin and enhanced chemiluminescence, or cy3-conjugated streptavidin and laser scanner. Several key factors and steps are described, including selection of solid supports, selection of suitable antibodies, determination of specificity and sensitivity of cytokine protein arrays, array design, sample preparation, and detailed experimental procedures for macroarray and microarray formats. An account of the successful development and application of cytokine protein arrays is presented.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
MacEwan, D. J. (2002) TNF receptor subtype signalling: differences and cellular consequences. Cell Signal. 14, 477–492.
Vitkovic, L., Maeda, S., and Sternberg, E. (2001) Anti-inflammatory cytokines: expression and action in the brain. Neuroimmunomodulation 9, 295–312.
Lotem, J. and Sachs, L. (2002) Cytokine control of developmental programs in normal hematopoiesis and leukemia. Oncogene 21, 3284–3294.
Slifka, M. K. and Whitton, J. L. (2000) Clinical implications of dysregulated cytokine production. J. Mol. Med. 78, 74–80.
Baraldi-Junkins, C. A., Beck, A. C., and Rothstein, G. (2000) Hematopoiesis and cytokines: relevance to cancer and aging. Hematol. Oncol. Clin. North Am. 14, 45–61, viii.
Talmadge, J. E., Singh, R., Ageitos, A., et al. (2001) Potential for cytokine and product manipulation to improve the results of autologous stem cell transplantation for rheumatoid arthritis. J. Rheumatol. Suppl. 64, 32–38.
Kurzrock, R. (2001) Cytokine deregulation in cancer. Biomed. Pharmacother. 55, 543–547.
Moody, M. D., Van Arsdell, S. W., Murphy, K. P., et al. (2001) Array-based ELISAs for high-throughput analysis of human cytokines. Biotechniques 31, 186–190, 192–194.
Schweitzer, B., Roberts, S., Grimwade, B., et al. (2002) Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365.
Huang, R., Lin, Y., Wang, C. C., et al. (2002) Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology. Cancer Res. 62, 2806–2812.
Huang, R. P. (2001) Detection of multiple proteins in an antibody-based protein microarray system. J. Immunol. Methods 255, 1–13.
Huang, R. P., Huang, R., Fan, Y., et al. (2001) Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem. 294, 55–62.
Wang, C. C., Huang, R-P., Sommer, M., et al. (2002) Array-based multiplexed screening and quantitation of human cytokines and chemokines. J. Proteome Res. 1, 337–343.
Lin, Y., Huang, R., Santanam, N., et al. (2002) Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array. Cancer Lett. 187, 17–24.
MacBeath, G. and Schreiber, S. L. (2000) Printing proteins as microarrays for high-throughput function determination [see comments]. Science 289, 1760–1763.
Huang, R. P. (2001) Simultaneous detection of multiple proteins with an array-based ELISA and enhanced chemiluminescence (ECL). Clin. Chem. Lab Med. 39, 209–214.
Mendoza, L. G., McQuary, P., Mongan, A., et al. (1999) High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). Biotechniques 27, 778–780, 782–786, 788.
Wiese, R., Belosludtsev, Y., Powdrill, T., et al. (2001) Simultaneous multianalyte ELISA performed on a microarray platform. Clin. Chem. 47, 1451–1457.
Bussow, K., Cahill, D., Nietfeld, W., et al. (1998) A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Res. 26, 5007, 5008.
Joos, T. O., Schrenk, M., Hopfl, P., et al. (2000) A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics. Electrophoresis 21, 2641–2650.
Arenkov, P., Kukhtin, A., Gemmell, A., et al. (2000) Protein microchips: use for immunoassay and enzymatic reactions. Anal. Biochem. 278, 123–131.
Haab, B. B., Dunham, M. J., and Brown, P. O. (2001) Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2, RESEARCH0004.
Wang, Y., Wu, T. R., Cai, S., et al. (2000) Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation. Mol. Cell Biol. 20, 4505–4512.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Huang, RP. (2004). Cytokine Protein Arrays. In: Fung, E.T. (eds) Protein Arrays. Methods in Molecular Biology, vol 264. Humana Press. https://doi.org/10.1385/1-59259-759-9:215
Download citation
DOI: https://doi.org/10.1385/1-59259-759-9:215
Publisher Name: Humana Press
Print ISBN: 978-1-58829-255-1
Online ISBN: 978-1-59259-759-8
eBook Packages: Springer Protocols