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Guidelines for the automated evaluation of Elispot assays


The presented protocol for Elispot plate evaluation summarizes how to implement the recommendations developed following the establishment of a large-scale international Elispot plate-reading panel and subsequent multistep consensus-finding process. The panel involved >100 scientists from various immunological backgrounds. The protocol includes the description and justification of steps for setting reading parameters to obtain accurate, reliable and precise automated analysis results of Elispot plates. Further, necessary adjustments for out-of-specification situations are described and examples are provided. The plate analysis, including parameter adjustments, auditing of results and necessary annotations, should be achievable within a time range of 10–30 min per plate. Adoption of these guidelines should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot. These guidelines conclude the ongoing harmonization efforts for the enzymatic Elispot assay.

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Figure 1: Examples of Elispot plate evaluation issues observed at the 2011 Cancer Immunotherapy Consortium Elispot panel.
Figure 2: Summary of evaluation results of three plates by panelists.
Figure 3: Stages of an Elispot experiment.
Figure 4: Differences in well appearance owing to the biological system character of samples.
Figure 5: Overall plate reading approaches documented in the Cancer Immunotherapy Consortium Elispot panel.
Figure 6: Plate reading steps and decision.
Figure 7: Elispot well examples with artifacts.
Figure 8: Examples of background spots.
Figure 9: Examples for spot diffuseness and disintegration.
Figure 10: Examples of spot crowdedness.
Figure 11: Example of differences in PBMC sample reactivity levels from the same donor at two different time points.
Figure 12: The influence of reader settings on reported spot counts.


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C.M.B. and H.S. were supported by a grant from the Wallace Coulter Foundation (Florida, USA). We thank all participants in the Elispot plate reading panel and consensus process. The full list of participants, including several authors of this paper, is given in the Supplementary Note. We also thank A. Szterenfeld for helpful comments on the manuscript.

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Authors and Affiliations



S.J., L.P., C.M.B. and M.J.P.W. designed the study. S.J., L.P., H.S., C.M.B., M.J.P.W. and A.H. organized and led the panel. All panelists listed under acknowledgement provided data and feedback leading to the final protocol. S.J. and L.P. compiled and analyzed the data. S.J. wrote the manuscript.

Corresponding author

Correspondence to Sylvia Janetzki.

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Competing interests

S.J. is founder and president of ZellNet Consulting, Inc. L.P. is the founder and director of LBPrice Statistical Consulting, Ltd. All other authors declare no competing financial interests.

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Supplementary Data, Supplementary Table 1 and Supplementary Note (PDF 2985 kb)

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Janetzki, S., Price, L., Schroeder, H. et al. Guidelines for the automated evaluation of Elispot assays. Nat Protoc 10, 1098–1115 (2015).

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