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

Cells

Radleigh Santos¹  Alcinette Buying³  Nazila Sabri³  John Yu⁴  Anthony Gringeri⁵  James Bender⁵  Sylvia Janetzki²  Clemencia Pinilla³  Valeria A. Judkowski³ 

[1] Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway Port St. Lucie, FL 34987, USA; E-Mail:;ZellNet Consulting, 555 North Ave., Fort Lee, NJ 07024, USA; E-Mail:;Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA; E-Mails:;Cedars-Sinai, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; E-Mail:;ImmunoCellular Therapeutics Ltd., 23622 Calabasas Road, Calabasas, CA 91302, USA; E-Mails:

关键词: overnight resting;    PBMC;    IFNγ ELISPOT;    immune monitoring;   

DOI  :  10.3390/cells4010001

来源: mdpi

【 摘 要 】

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 IFNγ ELISPOT assay is a well-standardized and validated method for the determination of functional IFNγ-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 IFNγ 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 IFNγ 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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.