A Gel-Based Dual Antibody Capture and Detection Method for Assaying of Extracellular Cytokine Secretion

  • Lisa A. Spencer
  • Rossana C. N. Melo
  • Sandra A. C. Perez
  • Peter F. Weller
Part of the Methods in Molecular Biology™ book series (MIMB, volume 302)


A distinguishing feature of eosinophils is their ability to rapidly release preformed cytokines from intracellular pools. Cytokines are delivered to the cell surface from granule stores by transport vesicles and are released in small packets at discrete locations along the cell surface through a process termed “piecemeal” degranulation. The study of this process has been hindered by lack of an assay sensitive enough to register minute protein concentrations and the inability to visualize morphology of cytokine secreting cells. These hindrances have necessitated our development of the EliCell assay, an agarose-based dual cytokine capture and detection system through which cytokine secretion and cellular morphology may be analyzed in concert. Cells are embedded within capture antibody-containing agarose and stimulated under conditions of interest. Extracellularly released cytokine is captured within the matrix at the point of release from the cell and can be labeled with a fluorochrome-conjugated antibody. Cytokine release and cellular morphology are visualized in parallel by phase contrast and fluorescence microscopy, respectively.

Key Words

EliCell agarose matrix eosinophil cytokine piecemeal degranulation vesicular transport secretion 


  1. 1.
    Gleich GJ, Adolphson C. R., and Leiferman K. M. (1992) Eosinophils, in Inflammation: Basic Principles and Clinical Correlates (Gallin J. I., Goldstein I. M., and Synderman R., eds.), Raven Press, New York. pp. 663–700.Google Scholar
  2. 2.
    Kita H. A. (1998) Biology of eosinophils, in Allergy: Principles and Practice (Adkinson N. F., Busse W. W., Ellis E. F., Middleton E., Jr., Reed C. E., and Yunginger J. W., eds.) Mosby St. Louis. pp. 242–260.Google Scholar
  3. 3.
    Lacy P. and Moqbel R. (1997) Eokines: synthesis, storage and release from human eosinophils. Mem Inst Oswaldo Cruz. 92Suppl 2, 125-33.Google Scholar
  4. 4.
    Weller P. F., and Dvorak A. M. (1994) Human eosinophils—development, maturation and functional morphology, in Asthma and Rhinitis (Busse W. M., and Holgate S., eds.), Blackwell Scientific Publications, Boston. pp. 225–274.Google Scholar
  5. 5.
    Gleich G. J., Adolphson C. R., and Leiferman K. M. (1993) The biology of the eosinophilic leukocyte. Annu Rev Med. 44, 85–101.PubMedCrossRefGoogle Scholar
  6. 6.
    Beil W. J., Weller P. F., Tzizik D. M., Galli S. J., and Dvorak A. M.. (1993) Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome. J. Histochem. Cytochem. 41, 1611–1615.PubMedGoogle Scholar
  7. 7.
    Moller G. M., de Jong T. A., van der Kwast T. H., et al. (1996) Immunolocalization of interleukin-4 in eosinophils in the bronchial mucosa of atopic asthmatics. Am. J. Respir. Cell Mol. Biol. 14, 439–443.PubMedGoogle Scholar
  8. 8.
    Moqbel R. (1996) Synthesis and storage of regulatory cytokines in human eosinophils. Adv Exp Med Biol. 409, 287–294.PubMedGoogle Scholar
  9. 9.
    Ying S., Meng Q., Taborda-Barata L., et al. (1996) Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein. Eur J Immunol. 26, 70–76.PubMedCrossRefGoogle Scholar
  10. 10.
    Dvorak A. M., Estrella P., and Ishizaka T. (1994) Vesicular transport of peroxidase in human eosinophilic myelocytes. Clin. Exp. Allergy. 24, 10–18.PubMedCrossRefGoogle Scholar
  11. 11.
    Dvorak A. M., and Ishizaka T. (1994) Human eosinophils in vitro. An ultrastructural morphology primer. Histol Histopathol. 9, 339–374.PubMedGoogle Scholar
  12. 12.
    Dvorak A. M., Ackerman S. J., and Weller P. F. (1990) Subcellular morphology and biochemistry of eosinophils, in Blood Cell Biochemistry: Megakaryocytes, Platelets, Macrophages and Eosinophils (H.J. R., ed), Plenum Publishing, London. pp. 237–344.Google Scholar
  13. 13.
    Dunzendorfer S., Feistritzer C., Enrich B., and Wiedermann C. J. (2002) Neuropeptide-induced inhibition of IL-16 release from eosinophils. Neuroimmunomodulation. 10, 217–223.PubMedCrossRefGoogle Scholar
  14. 14.
    Schmid-Grendelmeier P., Altznauer F., Fischer B., et al. (2002) Eosinophils express functional IL-13 in eosinophilic inflammatory diseases. J Immunol. 169, 1021–1027.PubMedGoogle Scholar
  15. 15.
    Bandeira-Melo C., Sugiyama K., Woods L. J., and Weller P. F. (2001) Cutting edge: eotaxin elicits rapid vesicular transport-mediated release of preformed IL-4 from human eosinophils. J Immunol. 166, 4813–4817.PubMedGoogle Scholar
  16. 16.
    Sabin E. A., Kopf M. A., and Pearce E. J. (1996) Schistosoma mansoni egg induced early IL-4 production is dependent upon IL-5 and eosinophils. J Exp Med. 184, 1871–1878.PubMedCrossRefGoogle Scholar
  17. 17.
    Bandeira-Melo C., Gillard G., Ghiran I., and Weller P. F. (2000) EliCell: a gelphase dual antibody capture and detection assay to measure cytokine release from eosinophils. J. Immunol. Methods. 244, 105–115.PubMedCrossRefGoogle Scholar
  18. 18.
    Bandeira-Melo C., Perez S. A., Melo R. C., Ghiran I., and Weller P. F.(2003) EliCell assay for the detection of released cytokines from eosinophils. J. Immunol. Methods. 276, 227–237.PubMedCrossRefGoogle Scholar
  19. 19.
    Alon R., Bayer E. A., and Wilchek M. (1992) Cell-adhesive properties of streptavidin are mediated by the exposure of an RGD-like RYD site. Eur. J. Cell Biol. 58, 271–279.PubMedGoogle Scholar
  20. 20.
    Alon R., Bayer E. A., and Wilchek M. (1993) Cell adhesion to streptavidin via RGD-dependent integrins. Eur. J. Cell Biol. 60, 1–11.PubMedGoogle Scholar
  21. 21.
    Alon R., Hershkoviz R., Bayer E. A., Wilchek M., and Lider O. (1993) Streptavidin blocks immune reactions mediated by fibronectin-VLA-5 recognition through an Arg-Gly-Asp mimicking site. Eur. J. Immunol. 23, 893–898.PubMedCrossRefGoogle Scholar
  22. 22.
    Ferguson T. A., Mizutani H., and Kupper T. S. (1991) Two integrin-binding peptides abrogate T-cell-mediated immune responses in vivo. Proc. Natl. Acad. Sci. USA. 88, 8072–8076.PubMedCrossRefGoogle Scholar
  23. 23.
    Bandeira-Melo C., Phoofolo M., and Weller P. F. (2001) Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils. J. Biol. Chem. 276, 22779–22787.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Lisa A. Spencer
    • 1
  • Rossana C. N. Melo
    • 1
    • 2
  • Sandra A. C. Perez
    • 1
  • Peter F. Weller
    • 1
  1. 1.Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical CenterHarvard Medical SchoolBoston
  2. 2.Department of BiologyFederal University of Juiz de ForaJuiz de ForaBrazil

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