Journal: BMC Immunology

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

doi: 10.1186/s12865-017-0195-y

Figure Lengend Snippet: Determination of optimal concentrations of T-activated® pp65 and IE-1 for the stimulation of PBMC. PBMC were isolated from one CMV-seropositive healthy donor blood sample and stimulated for 19 h with increasing concentrations of T-activated® pp65 (31.6 fg/ml-31.6 μg/ml; black circles) or IE-1 (0.01-31.6 μg/ml; black squares). IFN-γ ELISpot results of four replicates are expressed as mean SFC/200,000 PBMC. Error bars represent standard deviations. The Y-axis scale was adjusted to pp65- (left) and IE-1- (right) specific values to optimize data resolution. Curve fitting was made using the four parameter logistic function of GraphPad Prism 5.04

Article Snippet: Statistics applied to assay development was performed using SigmaPlot Version 11.0 and GraphPad Prism 5.04.

Techniques: Isolation, Enzyme-linked Immunospot

Journal: BMC Immunology

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

doi: 10.1186/s12865-017-0195-y

Figure Lengend Snippet: IFN-γ ELISpot assay linearity. a Working range of PBMC per ELISpot assay . Increasing number of PBMC from one CMV-seropositive healthy donor were seeded per well and ELISpot assays were performed as described, following stimulation with T-activated® IE-1 or pp65. Mean SFC values and standard deviation obtained for 60,000-200,000 PBMC per well are depicted. Scale of the Y-axes was adjusted for pp65 (left) and IE-1 (right) for a better data resolution. b Linearity between the number of CMV-reactive PBMC and enumerated SFC. The indicated numbers of PBMC from one CMV-seropositive healthy donor were mixed with PBMC from one CMV-seronegative donor (up to 200,000 total PBMC) and stimulated with T-activated® pp65 antigen according to the optimized protocol. Mean SFC values and standard deviation of quadruplicate measurements are shown for two donor pairs (d034 + d219, d204 + d067). In both panels, regression lines and corresponding coefficient of determination R 2 were generated using the regression line tool of GraphPad Prism 5.04

Article Snippet: Statistics applied to assay development was performed using SigmaPlot Version 11.0 and GraphPad Prism 5.04.

Techniques: Enzyme-linked Immunospot, Standard Deviation, Generated