Abstract
The recent renaissance of enzyme-linked immunospot (ELISPOT) assays largely is the result of advances in image analysis. Information on the frequency of antigen-specific T-cells and also on the secretion rate of the individual cells is captured in spots generated using this technique. Although the overall assessment of ELISPOT results can be conducted visually, this is inevitably subjective, inaccurate, and cumbersome. In contrast, objective, and accurate measurements are fundamental to good science. Validated image analysis algorithms and procedures, therefore, have become critical for elevating the quality of ELISPOT assays results. As cytokine and granzyme B ELISPOT assays become the gold standard for monitoring antigen-specific T-cell immunity in clinical trials, the pressure increases to make ELISPOT analysis transparent, reproducible and tamperproof, complying with Good Laboratory Practice and Code for Federal Regulations Part 11 guidelines. In addition, ELISPOT assays in clinical and basic science settings frequently require high degrees of throughput, thus further raising the need for advanced data management and statistical analysis. The ImmunoSpot software port-folio has been specifically designed to meet all these needs, using the techniques described in this chapter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guerkov R. E., Targoni O. S., Kreher C. S., Boehm B. O., Herrera M. T., Tary-Lehmann M., et al. (2003) Detection of low-frequency antigen-specific IL-10-producing CD4+ T-cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine.J. Immunol. Methods 279, 111–121.
Hesse M. D., Karulin A. Y., Boehm B. O., Lehmann P. V., and Tary-Lehmann M. (2001) A T-cell clone’s avidity is a function of its activation state. J. Immunol. 167, 1353–1361.
Ott P. A. Berner B. R., Herzog B. A., Guerkov R., Yonkers N. L., Boehm B. O., et al. (2004) CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4+ and CD8+cell populations in human diseases. J. Immunol. Methods 285, 223–235.
Helms T., Boehm B. O., Assad R. J., Trezza R. T., Lehmann P. V., and Tary-Lehmann M. (2000) Direct visualization of cytokine-producing, recall antigen specific CD4 memory T-cells in healthy individuals and HIV patients. J. Immunol. 164, 3723–3732.
Kreher C. R., Dittrich M. T., Guerkov R., Boehm B. O., and Tary-Lehmann M M. (2003). CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J. Immunol. Methods. 278, 79–93.
Karulin A. Y., Hesse M. D., Tary-Lehmann M., and Lehmann P. V. (2000) Single-cytokine-producing CD4 memory cells prevail in vivo, in type 1/type 2 immunity.J. Immunol. 164, 1862–1872.
Rininsland F. H., Helms T., Asaad R. J., Boehm B. O., and Tary-Lehmann M. (2000) Granzyme B ELISPOT assay for ex vivo measurement of T-cell immunity. J. Immunol. Methods 240, 143–155.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Lehmann, P.V. (2005). Image Analysis and Data Management of ELISPOT Assay Results. In: Kalyuzhny, A.E. (eds) Handbook of ELISPOT. Methods in Molecular Biology™, vol 302. Humana Press. https://doi.org/10.1385/1-59259-903-6:117
Download citation
DOI: https://doi.org/10.1385/1-59259-903-6:117
Publisher Name: Humana Press
Print ISBN: 978-1-58829-469-2
Online ISBN: 978-1-59259-903-5
eBook Packages: Springer Protocols