A sensitive ELISPOT assay to detect low-frequency human T lymphocytes

M. McCutcheon, N. Wehner, A. Wensky, M. Kushner, S. Doan, L. Hsiao, Peter Calabresi, T. Ha, T. V. Tran, K. M. Tate, J. Winkelhake, E. G. Spack

Research output: Contribution to journalArticle

Abstract

We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 105 PBMC with the standard assay compared to 19 SFC per 2 x 105 PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocytes cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.

Original languageEnglish (US)
Pages (from-to)149-166
Number of pages18
JournalJournal of Immunological Methods
Volume210
Issue number2
DOIs
StatePublished - Dec 29 1997
Externally publishedYes

Fingerprint

Enzyme-Linked Immunospot Assay
Blood Cells
T-Lymphocytes
Antigens
Cytokines
Biomarkers
Myelin Basic Protein
Signal-To-Noise Ratio
Antigen-Presenting Cells
Plastics
Multiple Sclerosis
In Situ Hybridization
Organism Cloning
Software
Vaccines
Enzyme-Linked Immunosorbent Assay
Tissue Donors
Therapeutics
Population

Keywords

  • Autoantigen
  • Cytokine
  • ELISPOT
  • Human

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

McCutcheon, M., Wehner, N., Wensky, A., Kushner, M., Doan, S., Hsiao, L., ... Spack, E. G. (1997). A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. Journal of Immunological Methods, 210(2), 149-166. https://doi.org/10.1016/S0022-1759(97)00182-8

A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. / McCutcheon, M.; Wehner, N.; Wensky, A.; Kushner, M.; Doan, S.; Hsiao, L.; Calabresi, Peter; Ha, T.; Tran, T. V.; Tate, K. M.; Winkelhake, J.; Spack, E. G.

In: Journal of Immunological Methods, Vol. 210, No. 2, 29.12.1997, p. 149-166.

Research output: Contribution to journalArticle

McCutcheon, M, Wehner, N, Wensky, A, Kushner, M, Doan, S, Hsiao, L, Calabresi, P, Ha, T, Tran, TV, Tate, KM, Winkelhake, J & Spack, EG 1997, 'A sensitive ELISPOT assay to detect low-frequency human T lymphocytes', Journal of Immunological Methods, vol. 210, no. 2, pp. 149-166. https://doi.org/10.1016/S0022-1759(97)00182-8
McCutcheon M, Wehner N, Wensky A, Kushner M, Doan S, Hsiao L et al. A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. Journal of Immunological Methods. 1997 Dec 29;210(2):149-166. https://doi.org/10.1016/S0022-1759(97)00182-8
McCutcheon, M. ; Wehner, N. ; Wensky, A. ; Kushner, M. ; Doan, S. ; Hsiao, L. ; Calabresi, Peter ; Ha, T. ; Tran, T. V. ; Tate, K. M. ; Winkelhake, J. ; Spack, E. G. / A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. In: Journal of Immunological Methods. 1997 ; Vol. 210, No. 2. pp. 149-166.
@article{be283ecb0fad49ceb8c7e4df9975c418,
title = "A sensitive ELISPOT assay to detect low-frequency human T lymphocytes",
abstract = "We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 105 PBMC with the standard assay compared to 19 SFC per 2 x 105 PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocytes cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.",
keywords = "Autoantigen, Cytokine, ELISPOT, Human",
author = "M. McCutcheon and N. Wehner and A. Wensky and M. Kushner and S. Doan and L. Hsiao and Peter Calabresi and T. Ha and Tran, {T. V.} and Tate, {K. M.} and J. Winkelhake and Spack, {E. G.}",
year = "1997",
month = "12",
day = "29",
doi = "10.1016/S0022-1759(97)00182-8",
language = "English (US)",
volume = "210",
pages = "149--166",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - A sensitive ELISPOT assay to detect low-frequency human T lymphocytes

AU - McCutcheon, M.

AU - Wehner, N.

AU - Wensky, A.

AU - Kushner, M.

AU - Doan, S.

AU - Hsiao, L.

AU - Calabresi, Peter

AU - Ha, T.

AU - Tran, T. V.

AU - Tate, K. M.

AU - Winkelhake, J.

AU - Spack, E. G.

PY - 1997/12/29

Y1 - 1997/12/29

N2 - We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 105 PBMC with the standard assay compared to 19 SFC per 2 x 105 PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocytes cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.

AB - We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 105 PBMC with the standard assay compared to 19 SFC per 2 x 105 PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocytes cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.

KW - Autoantigen

KW - Cytokine

KW - ELISPOT

KW - Human

UR - http://www.scopus.com/inward/record.url?scp=0031590668&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031590668&partnerID=8YFLogxK

U2 - 10.1016/S0022-1759(97)00182-8

DO - 10.1016/S0022-1759(97)00182-8

M3 - Article

C2 - 9520298

AN - SCOPUS:0031590668

VL - 210

SP - 149

EP - 166

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 2

ER -