An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

BMC Immunol. 2017 Mar 7;18(1):14. doi: 10.1186/s12865-017-0195-y.


Background: In 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.

Methods: Objective 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.

Results: Optimized 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.

Conclusion: The 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.

Keywords: CD4 +; CD8 +; CMV; Cell-mediated immunity; Cytomegalovirus; Cytotoxic T lymphocyte (CTL); ELISpot; IE-1; NKT-like; Natural killer (NK); T helper (Th); pp65.

Publication types

  • Validation Study

MeSH terms

  • Adult
  • CD4-Positive T-Lymphocytes / immunology*
  • CD4-Positive T-Lymphocytes / virology
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / virology
  • Cells, Cultured
  • Cytomegalovirus / physiology*
  • Cytomegalovirus Infections / diagnosis*
  • Cytomegalovirus Infections / immunology
  • Cytotoxicity, Immunologic
  • Enzyme-Linked Immunospot Assay / methods*
  • Female
  • Humans
  • Immediate-Early Proteins / immunology
  • Immunity, Cellular
  • Interferon-gamma / metabolism
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / virology
  • Male
  • Middle Aged
  • Monitoring, Immunologic
  • Natural Killer T-Cells / immunology*
  • Natural Killer T-Cells / virology
  • Observer Variation
  • Phosphoproteins / immunology
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Viral Matrix Proteins / immunology
  • Young Adult


  • IE1 protein, cytomegalovirus
  • Immediate-Early Proteins
  • Phosphoproteins
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa
  • Interferon-gamma