title: A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses.
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.
authors.affiliation: Research Institute of the McGill University Health Center, Montreal, Canada.
authors.forename: Salix
authors.initials: S
authors.lastname: Boulet
authors.forename: Michel L
authors.initials: ML
authors.lastname: Ndongala
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peretz
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Boisvert
authors.forename: Mohamed-Rachid
authors.initials: MR
authors.lastname: Boulassel
authors.forename: Cecile
authors.initials: C
authors.lastname: Tremblay
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Routy
authors.forename: Rafick-P
authors.initials: RP
authors.lastname: Sekaly
authors.forename: Nicole F
authors.initials: NF
authors.lastname: Bernard
mesh_terms: D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015658:HIV Infections / Q000097:blood / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000097:blood* / Q000378:metabolism; D007376:Interleukin-2 / Q000097:blood* / Q000378:metabolism; D010455:Peptides / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D007376:Interleukin-2; D010455:Peptides; D007371:Interferon-gamma