Detection of antigen-specific T cell interferon γ expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques

Bapi Pahar, Jun Li, Tracy Rourke, Chris J Miller, Michael B. McChesney

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) methods have been developed for the detection of low-frequency, antigen-specific, cytokine-producing T cells following short-term in vitro stimulation. Peptide-based ELISPOT and CFC assays were compared for the quantitative detection of interferon γ-positive (IFN-γ+) antigen-specific T cells in rhesus macaques. Ten normal and nine simian immunodeficiency virus (SIV)-infected monkeys were tested for the detection of IFN-γ+ memory T cells specific for p27gag peptides of SIV with both assays. The CFC assay detected more IFN-γ+ cells than the ELISPOT assay and this assay was more informative in identifying the phenotype of responding cells. Cryopreserved cells were as functional as fresh cells in heparinized blood samples and compared to EDTA, heparin was the better anticoagulant for yielding IFN-γ+ cells. Using overlapping peptide pools, 20-mer peptides were more efficient in stimulating CD4+ T cells than 15-mer peptides in the ELISPOT assay, but there was no significant difference between 20- and 15-mer peptides in detecting CD4 or CD8+, IFN-γ+ T cells in the CFC assay.

Original languageEnglish (US)
Pages (from-to)103-115
Number of pages13
JournalJournal of Immunological Methods
Volume282
Issue number1-2
DOIs
StatePublished - Nov 2003

Fingerprint

Macaca mulatta
Interferons
Flow Cytometry
Cytokines
T-Lymphocytes
Antigens
Peptides
Enzymes
Enzyme-Linked Immunospot Assay
Simian Immunodeficiency Virus
Edetic Acid
Anticoagulants
Haplorhini
Heparin
Phenotype

Keywords

  • Interferon γ
  • Rhesus monkey
  • T cell

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Detection of antigen-specific T cell interferon γ expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques. / Pahar, Bapi; Li, Jun; Rourke, Tracy; Miller, Chris J; McChesney, Michael B.

In: Journal of Immunological Methods, Vol. 282, No. 1-2, 11.2003, p. 103-115.

Research output: Contribution to journalArticle

@article{bdc6cf32c2144bb5ad94ea5188a08577,
title = "Detection of antigen-specific T cell interferon γ expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques",
abstract = "Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) methods have been developed for the detection of low-frequency, antigen-specific, cytokine-producing T cells following short-term in vitro stimulation. Peptide-based ELISPOT and CFC assays were compared for the quantitative detection of interferon γ-positive (IFN-γ+) antigen-specific T cells in rhesus macaques. Ten normal and nine simian immunodeficiency virus (SIV)-infected monkeys were tested for the detection of IFN-γ+ memory T cells specific for p27gag peptides of SIV with both assays. The CFC assay detected more IFN-γ+ cells than the ELISPOT assay and this assay was more informative in identifying the phenotype of responding cells. Cryopreserved cells were as functional as fresh cells in heparinized blood samples and compared to EDTA, heparin was the better anticoagulant for yielding IFN-γ+ cells. Using overlapping peptide pools, 20-mer peptides were more efficient in stimulating CD4+ T cells than 15-mer peptides in the ELISPOT assay, but there was no significant difference between 20- and 15-mer peptides in detecting CD4 or CD8+, IFN-γ+ T cells in the CFC assay.",
keywords = "Interferon γ, Rhesus monkey, T cell",
author = "Bapi Pahar and Jun Li and Tracy Rourke and Miller, {Chris J} and McChesney, {Michael B.}",
year = "2003",
month = "11",
doi = "10.1016/j.jim.2003.08.003",
language = "English (US)",
volume = "282",
pages = "103--115",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Detection of antigen-specific T cell interferon γ expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques

AU - Pahar, Bapi

AU - Li, Jun

AU - Rourke, Tracy

AU - Miller, Chris J

AU - McChesney, Michael B.

PY - 2003/11

Y1 - 2003/11

N2 - Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) methods have been developed for the detection of low-frequency, antigen-specific, cytokine-producing T cells following short-term in vitro stimulation. Peptide-based ELISPOT and CFC assays were compared for the quantitative detection of interferon γ-positive (IFN-γ+) antigen-specific T cells in rhesus macaques. Ten normal and nine simian immunodeficiency virus (SIV)-infected monkeys were tested for the detection of IFN-γ+ memory T cells specific for p27gag peptides of SIV with both assays. The CFC assay detected more IFN-γ+ cells than the ELISPOT assay and this assay was more informative in identifying the phenotype of responding cells. Cryopreserved cells were as functional as fresh cells in heparinized blood samples and compared to EDTA, heparin was the better anticoagulant for yielding IFN-γ+ cells. Using overlapping peptide pools, 20-mer peptides were more efficient in stimulating CD4+ T cells than 15-mer peptides in the ELISPOT assay, but there was no significant difference between 20- and 15-mer peptides in detecting CD4 or CD8+, IFN-γ+ T cells in the CFC assay.

AB - Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) methods have been developed for the detection of low-frequency, antigen-specific, cytokine-producing T cells following short-term in vitro stimulation. Peptide-based ELISPOT and CFC assays were compared for the quantitative detection of interferon γ-positive (IFN-γ+) antigen-specific T cells in rhesus macaques. Ten normal and nine simian immunodeficiency virus (SIV)-infected monkeys were tested for the detection of IFN-γ+ memory T cells specific for p27gag peptides of SIV with both assays. The CFC assay detected more IFN-γ+ cells than the ELISPOT assay and this assay was more informative in identifying the phenotype of responding cells. Cryopreserved cells were as functional as fresh cells in heparinized blood samples and compared to EDTA, heparin was the better anticoagulant for yielding IFN-γ+ cells. Using overlapping peptide pools, 20-mer peptides were more efficient in stimulating CD4+ T cells than 15-mer peptides in the ELISPOT assay, but there was no significant difference between 20- and 15-mer peptides in detecting CD4 or CD8+, IFN-γ+ T cells in the CFC assay.

KW - Interferon γ

KW - Rhesus monkey

KW - T cell

UR - http://www.scopus.com/inward/record.url?scp=0242384226&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0242384226&partnerID=8YFLogxK

U2 - 10.1016/j.jim.2003.08.003

DO - 10.1016/j.jim.2003.08.003

M3 - Article

C2 - 14604545

AN - SCOPUS:0242384226

VL - 282

SP - 103

EP - 115

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -