A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-γ HIV-specific immune responses
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- 作者：Salix Boulet ; Michel L. Ndongala ; Yoav Peretz ; Marie-Pierre Boisvert ; Mohamed-Rachid Boulassel ; Cecile Tremblay ; Jean-Pierre Routy ; Rafick-P Sekaly ; Nicole F. Bernard
- 关键词：ELISPOT ; HIV ; Interferon-gamma ; IL-2 ; Cellular immune responses ; CD4+ ; CD8+ ; T cells
- 刊名：Journal of Immunological Methods
- 出版年：2007
- 出版时间：30 March 2007
- 年：2007
- 卷：320
- 期：1-2
- 页码：18-29
- 全文大小：7135 K
文摘
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.