Leucocytes pp 15-30

Part of the Methods in Molecular Biology book series (MIMB, volume 844)

Detection and Quantification of Cytokines and Other Biomarkers

  • Evan L. Chiswick
  • Elizabeth Duffy
  • Brian Japp
  • Daniel Remick


Accurate measurement of cytokine concentrations is a powerful and essential approach to the study of inflammation. The enzyme-linked immunosorbent assay (ELISA) is a simple, low-cost analytical tool that provides both the specificity and sensitivity required for the study of cytokines in vitro or in vivo. This communication describes a systematic approach to develop an indirect sandwich ELISA to detect and quantify cytokines, or other biomarkers, with accuracy and precision. Also detailed is the use of sequential ELISA assays to analyze multiple cytokines from samples with limited volumes. Finally, the concept of a multiplex ELISA is discussed with considerations given to cost and additional time required for development.

Key words

Cytokines Sandwich ELISA Sequential ELISA Multiplex ELISA Antibodies 


  1. 1.
    Feghali, C.A., Wright, T.M. (1997) Cytokines in acute and chronic inflammation, Front Biosci 2, d12–26.PubMedGoogle Scholar
  2. 2.
    Sokol, H., Pigneur, B., Watterlot, L. et al. (2008) Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients, Proc Natl Acad Sci USA 105, 16731–16736.PubMedCrossRefGoogle Scholar
  3. 3.
    Polpitiya, A. D., McDunn, J. E., Burykin, A. et al. (2009) Using systems biology to simplify complex disease: immune cartography, Crit Care Med 37, S16–21.PubMedCrossRefGoogle Scholar
  4. 4.
    De Santo, C., Arscott, R., Booth, S. et al. (2010) Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A, Nat Immunol 11, 1039–1046.PubMedCrossRefGoogle Scholar
  5. 5.
    DeForge, L. E., Remick, D. G. (1991) Sandwich ELISA for detection of picogram quantities of interleukin-8, Immunol Invest 20, 89–97.PubMedCrossRefGoogle Scholar
  6. 6.
    Osuchowski, M. F., Remick, D. G. (2006) The repetitive use of samples to measure multiple cytokines: the sequential ELISA, Methods (San Diego, Calif) 38, 304–311.Google Scholar
  7. 7.
    Findlay, J.W.A., Dillard, R. F. (2007) Appropriate calibration curve fitting in ligand binding assays, AAPS Journal 9, 2 E260-E267. doi: 10.1208/aapsj0902029.PubMedCrossRefGoogle Scholar
  8. 8.
    Morgan, E., Varro, R., Sepulveda, H. et al. (2004) Cytometric bead array: a multiplexed assay platform with applications in various areas of biology, Clin Immunol 110, 252–266.PubMedCrossRefGoogle Scholar
  9. 9.
    Nemzek, J. A., Siddiqui, J., Remick, D. G. (2001) Development and optimization of cytokine ELISAs using commercial antibody pairs, J Immunol Methods 255, 149–157.PubMedCrossRefGoogle Scholar
  10. 10.
    Natarajan, S., Remick, D.G. (2008) The ELISA Standard Save: Calculation of sample concentrations in assays with a failed standard curve, J Immunol Methods 336, 242–245.PubMedCrossRefGoogle Scholar
  11. 11.
    Crowther, J. R. (2000) The ELISA guidebook, Methods in molecular biology (Clifton, NJ) 149, III-IV, 1–413.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Evan L. Chiswick
    • 1
  • Elizabeth Duffy
    • 1
  • Brian Japp
    • 1
  • Daniel Remick
    • 1
  1. 1.Department of Pathology and Laboratory MedicineBoston University School of Medicine and Boston Medical CenterBostonUSA

Personalised recommendations