Abstract
BackgroundIn healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.MethodsObjective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.ResultsOptimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3−CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.ConclusionThe combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.
Highlights
In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cellmediated immunity (CMI)
Interferon gamma (IFN-γ) enzyme-linked immunospot assay (ELISpot) assay optimization following peripheral blood mononuclear cells (PBMC) stimulation with T-activated® Immediate early-1 protein (IE-1) and pp65 CMV antigens Freshly isolated PBMC were used for the ELISpot assay
A total number of 2 × 105 PBMC per well was chosen for the development of the ELISpot assay protocol as this cell count is below confluency and can usually be obtained from samples of less than 15 ml whole blood
Summary
Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cellmediated immunity (CMI). Reliable monitoring of CMV-specific CMI in immunocompromized individuals, such as solid-organ or allogeneic stem cell transplant recipients, requires a specific, standardized and highly sensitive assay capable of detecting low numbers of CMV-reactive cells. Such sensitivity might be achieved by using highly immunogenic stimulants and via the reactivation of a broad spectrum of physiological effector cells involved in the protection against CMV replication in vivo, notably CD4+ (T helper or Th), CD8+ (cytotoxic T lymphocytes or CTL), and natural killer (NK) and natural killer T (NKT) cells [5,6,7,8,9,10,11,12]
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