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

  • Barabas, Sascha1
  • Spindler, Theresa1
  • Kiener, Richard2
  • Tonar, Charlotte1
  • Lugner, Tamara1
  • Batzilla, Julia1
  • Bendfeldt, Hanna1
  • Rascle, Anne1
  • Asbach, Benedikt2
  • Wagner, Ralf2
  • Deml, Ludwig1, 2
  • 1 Lophius Biosciences GmbH, Am BioPark 13, Regensburg, 93053, Germany , Regensburg (Germany)
  • 2 University Regensburg, Institute of Medical Microbiology and Hygiene, Franz-Josef-Strauss-Allee 11, Regensburg, 93053, Germany , Regensburg (Germany)
Published Article
BMC Immunology
Springer (Biomed Central Ltd.)
Publication Date
Mar 07, 2017
DOI: 10.1186/s12865-017-0195-y
Springer Nature


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.

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