Abstract
The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.
| Original language | English |
|---|---|
| Pages (from-to) | 18-29 |
| Number of pages | 12 |
| Journal | Journal of Immunological Methods |
| Volume | 320 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - Mar 30 2007 |
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Keywords
- CD4+
- CD8+
- Cellular immune responses
- ELISPOT
- HIV
- IL-2
- Interferon-gamma
- T cells
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology
Cite this
A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-γ HIV-specific immune responses. / Boulet, Salix; Ndongala, Michel L.; Peretz, Yoav; Boisvert, Marie Pierre; Boulassel, Mohamed Rachid; Tremblay, Cecile; Routy, Jean Pierre; Sekaly, Rafick P.; Bernard, Nicole F.
In: Journal of Immunological Methods, Vol. 320, No. 1-2, 30.03.2007, p. 18-29.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-γ HIV-specific immune responses
AU - Boulet, Salix
AU - Ndongala, Michel L.
AU - Peretz, Yoav
AU - Boisvert, Marie Pierre
AU - Boulassel, Mohamed Rachid
AU - Tremblay, Cecile
AU - Routy, Jean Pierre
AU - Sekaly, Rafick P.
AU - Bernard, Nicole F.
PY - 2007/3/30
Y1 - 2007/3/30
N2 - The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.
AB - The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.
KW - CD4+
KW - CD8+
KW - Cellular immune responses
KW - ELISPOT
KW - HIV
KW - IL-2
KW - Interferon-gamma
KW - T cells
UR - http://www.scopus.com/inward/record.url?scp=33847380680&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33847380680&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2006.11.010
DO - 10.1016/j.jim.2006.11.010
M3 - Article
VL - 320
SP - 18
EP - 29
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -