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

Sascha Barabas, Theresa Spindler, Richard Kiener, Charlotte Tonar, Tamara Lugner, Julia Batzilla, Hanna Bendfeldt, Anne Rascle, Benedikt Asbach, Ralf Wagner, Ludwig Deml

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. 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. 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 × 10(4) and 2 × 10(5) PBMC per well upon stimulation with T-activated® IE-1 (R(2) = 0.97) and pp65 (R(2) = 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. 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.