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A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses
Salix Boulet1, Michel L Ndongala, Yoav Peretz
1Research Institute of the McGill University Health Center, Montreal, Canada.
Insights
A new dual color ELISPOT assay simultaneously detects Interleukin-2 (IL-2) and Interferon-gamma (IFN-gamma) for improved HIV immune response assessment. This method offers a more comprehensive view of virus-specific immunity in HIV-infected individuals.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- The Interferon-gamma (IFN-gamma) ELISPOT assay is standard for HIV immunity assessment.
- Single cytokine detection may be insufficient for comprehensive HIV immune response evaluation.
Purpose of the Study:
- To design and validate a dual color ELISPOT assay for simultaneous detection of IL-2 and IFN-gamma secreting cells in response to HIV antigens.
- To assess the assay's performance, including cell input requirements and variability, for HIV immune monitoring.
Main Methods:
- Development and validation of a dual color ELISPOT assay.
- Simultaneous detection of IL-2 and IFN-gamma secreting cells using HIV antigens.
- Evaluation of cell input numbers (200,000 cells/well) and assay variability.
Main Results:
- The dual color assay effectively detects both IL-2 and IFN-gamma secreting cells.
- A cell input of 200,000 cells/well balances cell availability and detection sensitivity.
- Intra- and inter-assay variability were comparable to single-cytokine assays (CV < 20%).
- Both CD4+ and CD8+ T cells were found to mediate HIV-specific responses.
Conclusions:
- The dual color ELISPOT assay provides a more comprehensive assessment of HIV-specific immune responses.
- This assay is a valuable tool for screening HIV immunity in clinical settings due to its high throughput and specific cytokine detection capabilities.
Abstract:
The single color IFN-gamma 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-gamma 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-gamma resulted in a decreased magnitude of IFN-gamma 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-gamma 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.
