Mathematics Faculty Articles
Document Type
Article
Publication Date
12-24-2014
Publication Title
Cells
Keywords
overnight resting, PBMC, IFNg ELISPOT, immune monitoring
ISSN
2073-4409
Volume
4
Issue/No.
1
First Page
1
Last Page
18
Abstract
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.
NSUWorks Citation
Santos, Radleigh; Bunying, Alcinette; Sabri, Nazila; Yu, John; Gringeri, Anthony; Bender, James; Janetzki, Sylvia; Pinilla, Clemencia; and Judkowski, Valeria A., "Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis" (2014). Mathematics Faculty Articles. 234.
https://nsuworks.nova.edu/math_facarticles/234
Supplementary File
DOI
10.3390/cells4010001
Comments
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).