Protein Array-Based Multiplexed Cytokine Assays

  • Cheng C. Wang
Part of the Methods in Molecular Biology™ book series (MIMB, volume 385)


We have demonstrated a microarray format immunoassay using HydroGel-coated slides. HydroGel is a porous substrate based on a polymer matrix that provides a three-dimensional hydrophilic environment similar to free solution suitable for biomolecular interactions. This substrate has been used to develop fluorescence-based multiplexed cytokine immunoassays. Forty-three monoclonal antibodies (mAbs) of cytokines and chemokines are printed at a volume of 350 pL per spot using a PerkinElmer BioChip Arrayer®. Cytokines that are captured by the arrayed mAb are detected using another biotinylated mAb, following by the addition of a Texas red conjugated streptavidin. The fluorescent images of arrays are recorded using a PerkinElmer ScanArray™ 5000 confocal slide scanner and quantitated using PerkinElmer QuantArray™ software. Experiments demonstrate that 43 cytokines can be simultaneously screened and quantitated in conditioned culture media, cell lysate, and human plasma. Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.

Key Words

Microarray protein chip chemokine cytokine immunoassay multiplex assays HydroGel 


  1. 1.
    Zhu, H. and Snyder, M. (2003) Protein chip technology. Curr. Opin. Chem. Biol. 7(1), 55–63.CrossRefGoogle Scholar
  2. 2.
    Wiese, R., Belosludtsev, Y., Powdrill, T., Thompson, P., and Hogan, M. (2001) Simultaneous multianalyte ELISA performed on a microarray platform. Clin. Chem. 47(8), 1451–1457.Google Scholar
  3. 3.
    Mendoza, L. G., McQuary, P., Mongan, A., Gangadharan, R., Brigna, S., and Eggers, M. (1999) High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). Biotechniques 27(4), 778–780, 782–786, 788.Google Scholar
  4. 4.
    Moody, M. D., Van Arsdell, S. W., Murphy, K. P., Orencole, S. F., and Burns, C. (2001) Array-based ELISAs for high-throughput analysis of human cytokines. Biotechniques 31(1), 186–190, 192–194.Google Scholar
  5. 5.
    Schweitzer, B., Wiltshire, S., Lambert, J., et al. (2000) Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. USA 97(18), 10,113–10,119.CrossRefGoogle Scholar
  6. 6.
    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(2), RESEARCH0004 Epub.Google Scholar
  7. 7.
    Huang, R. P. (2004) Cytokine protein arrays. Methods Mol. Biol. 264, 215–231.Google Scholar
  8. 8.
    Lin, Y., Huang, R., Chen, L. P., et al. (2003) Profiling of cytokine expression by biotin-labeled-based protein arrays. Proteomics 3(9), 1750–1757.CrossRefGoogle Scholar
  9. 9.
    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(10), 2806–2812.Google Scholar
  10. 10.
    Carson, R. T. and Vignali, D. A. (1999) Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J. Immunol. Methods 227(1–2), 41–52.CrossRefGoogle Scholar
  11. 11.
    Mirzabekov, A. and Zlatanova, J. (2001) Gel-immobilized microarrays of nucleic acids and proteins. Production and application for macromolecular research. Methods Mol. Biol. 170, 17–38.Google Scholar
  12. 12.
    Carding, S. R., Hayday, A. C., and Bottomly, K. (1991) Cytokines in T-cell development. Immunol. Today 12(7), 239–245.CrossRefGoogle Scholar
  13. 13.
    Murphy, P. M. (2001) Chemokines and the molecular basis of cancer metastasis. N. Engl. J. Med. 345(11), 833–835.CrossRefGoogle Scholar
  14. 14.
    Bienvenu, J., Monneret, G., Fabien, N, and Revillard, J. P. (2000) The clinical usefulness of the measurement of cytokines. Clin. Chem. Lab. Med. 38(4), 267–285.CrossRefGoogle Scholar
  15. 15.
    Haverty, A. A., Harmey, J. H., Redmond, H. P., and Bouchier-Hayes, D. J. (1997) Interleukin-6 upregulates GP96 expression in breast cancer. Surg. Res. 69(1), 145–149.CrossRefGoogle Scholar
  16. 16.
    Kleine-Lowinski, K., Gillitzer, R., Kuhne-Heid, R., and Rosl, F. (1999) Monocyte-chemo-attractant-protein-1 (MCP-1)-gene expression in cervical intra-epithelial neoplasias and cervical carcinomas. Int. J. Cancer 82(1), 6–11.CrossRefGoogle Scholar
  17. 17.
    Riethdorf, S., Riethdorf, L., Richter, N., and Loning, T. (1998) Expression of the MCP-1 gene and the HPV 16 E6/E7 oncogenes in squamous cell carcinomas of the cervix uteri and metastases. Pathobiology 66(6), 260–267.CrossRefGoogle Scholar
  18. 18.
    Huang, R. P., Fan, Y. de Belle, I., et al. (1997) Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation. Int. J. Cancer 72(1), 102–109.CrossRefGoogle Scholar
  19. 19.
    MacBeath, G. and Schreiber, S. L. (2000) Printing proteins as microarrays for high-throughput function determination. Science 289(5485), 1760–1763.Google Scholar
  20. 20.
    Stillman, B. A. and Tonkinson, J. L. (2000) FAST slides: a novel surface for microarrays. Biotechniques 29(3), 630–635.Google Scholar
  21. 21.
    Kodadek, T. (2001) Protein microarrays: prospects and problems. Chem. Biol. 8, 105–115.CrossRefGoogle Scholar
  22. 22.
    Ekins, R. P. (1989) Multi-analyte immunoassay. J. Pharm. Biomed. Anal. 7(2), 155–168.CrossRefGoogle Scholar
  23. 23.
    Service, R. F. (2001) Proteomics. Searching for recipes for protein chips. Science 294, 2080–2082.CrossRefGoogle Scholar
  24. 24.
    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(4), 337–343.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Cheng C. Wang
    • 1
  1. 1.QiagenShanghaiChina

Personalised recommendations