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Correction published on 21 April 2015, see Cells 2015, 4(2), 133-134.

Open AccessArticle
Cells 2015, 4(1), 1-18;

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway Port St. Lucie, FL 34987, USA
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA
Cedars-Sinai, 8700 Beverly Blvd., Los Angeles, CA 90048, USA
ImmunoCellular Therapeutics Ltd., 23622 Calabasas Road, Calabasas, CA 91302, USA
ZellNet Consulting, 555 North Ave., Fort Lee, NJ 07024, USA
Present address: Lion biotechnologies, 21900 Burbank Blvd., Woodland Hills, CA 91367, USA.
Authors to whom correspondence should be addressed.
Academic Editor: Alexander E. Kalyuzhny
Received: 9 October 2014 / Accepted: 16 December 2014 / Published: 24 December 2014
(This article belongs to the Special Issue ELISPOT Research)
Full-Text   |   PDF [2628 KB, uploaded 24 December 2014]   |  


Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning. View Full-Text
Keywords: overnight resting; PBMC; IFNg ELISPOT; immune monitoring overnight resting; PBMC; IFNg ELISPOT; immune monitoring

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Santos, R.; Buying, A.; Sabri, N.; Yu, J.; Gringeri, A.; Bender, J.; Janetzki, S.; Pinilla, C.; Judkowski, V.A. Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells 2015, 4, 1-18.

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