Skip to main content

Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring

  • Protocol
  • First Online:
Handbook of ELISPOT

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1808))

Abstract

The ELISpot assay, as a sensitive and specific method, enables the detection of cytokines for immunological purposes and in vaccine development. Here we describe the successful transfer of the manual procedure to a commercially available automated liquid handling platform, based on the work described in Neubauer et al. (Cytotechnology 69:57–73, 2017). Different kinds of technical issues (dead volume reduction, instrumental handling limitations, liquid class improvement) have been solved and biological effects (reagents concentration, selectivity tests, dispensing way, etc.) have been controlled during the implementation process. At the end a maximum of 6% mean delta difference and a lower mean dispersion than the manual assay were reached as well as a turnaround time of four to six times higher than the manual process.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cox JH, Ferrari G, Janetzki S (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods 38(4):274–282. https://doi.org/10.1016/j.ymeth.2005.11.006

    Article  PubMed  CAS  Google Scholar 

  2. Janetzki S, Price L, Schroeder H et al (2015) Guidelines for the automated evaluation of Elispot assays. Nat Protoc 10(7):1098–1115. https://doi.org/10.1038/nprot.2015.068

    Article  PubMed  CAS  Google Scholar 

  3. Janetzki S, Schaed S, Blachere NEB et al (2004) Evaluation of Elispot assays. Influence of method and operator on variability of results. J Immunol Methods 291(1–2):175–183. https://doi.org/10.1016/j.jim.2004.06.008

    Article  PubMed  CAS  Google Scholar 

  4. Maecker HT, Moon J, Bhatia S et al (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6:17. https://doi.org/10.1186/1471-2172-6-17

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Moodie Z, Price L, Janetzki S et al (2012) Response determination criteria for ELISPOT. Toward a standard that can be applied across laboratories. Methods Mol Biol 792:185–196. https://doi.org/10.1007/978-1-61779-325-7_15

    Article  PubMed  CAS  Google Scholar 

  6. Maecker HT, Hassler J, Payne JK et al (2008) Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:9. https://doi.org/10.1186/1471-2172-9-9

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Hawkins N, Self S, Wakefield J (2006) The automated counting of spots for the ELISpot assay. J Immunol Methods 316(1–2):52–58. https://doi.org/10.1016/j.jim.2006.08.005

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Zadorozhny SS, Martynov NN (2012) Mathematical algorithms for automatic search, recognition, and detection of spots in ELISPOT assay. Methods Mol Biol 792:145–153. https://doi.org/10.1007/978-1-61779-325-7_12

    Article  PubMed  CAS  Google Scholar 

  9. Janetzki S, Panageas KS, Ben-Porat L et al (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57(3):303–315. https://doi.org/10.1007/s00262-007-0380-6

    Article  PubMed  Google Scholar 

  10. Almeida C-AM, Roberts SG, Laird R et al (2009) Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses. J Immunol Methods 344(1):1–5. https://doi.org/10.1016/j.jim.2009.02.007

    Article  PubMed  CAS  Google Scholar 

  11. Franscini N, Wuertz K, Patocchi-Tenzer I et al (2011) Development of a novel automated cell isolation, expansion, and characterization platform. J Lab Autom 16(3):204–213. https://doi.org/10.1016/j.jala.2011.01.002

    Article  PubMed  CAS  Google Scholar 

  12. Neubauer JC, Sébastien I, Germann A et al (2017) Towards standardized automated immunomonitoring. An automated ELISpot assay for safe and parallelized functionality analysis of immune cells. Cytotechnology 69(1):57–73. https://doi.org/10.1007/s10616-016-0037-4

    Article  PubMed  CAS  Google Scholar 

  13. Tecan Schweiz AG (2009) Freedom EVOware Software Manual. 393172, en, version 2.3

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H. Zimmermann .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Neubauer, J.C., Sébastien, I., Germann, A., von Briesen, H., Zimmermann, H. (2018). Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring. In: Kalyuzhny, A. (eds) Handbook of ELISPOT . Methods in Molecular Biology, vol 1808. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8567-8_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8567-8_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8566-1

  • Online ISBN: 978-1-4939-8567-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics