ifnγ elispot pre coated plates  (Cellular Technology Ltd)

Journal: NPJ Vaccines

Article Title: An HLAII-targeted DNA vaccine against influenza H7N9 protected mice and ferrets from a virus challenge

doi: 10.1038/s41541-025-01341-4

Figure Lengend Snippet: A Study design: Ferrets were allocated to three groups ( n = 8/group) and DNA vaccinated twice at days 0 and 33 with jet injections intradermally. Blood samples were collected longitudinally, as indicated, for evaluations of antibody and T-cell responses. At day 56, ferrets were challenged with A/Anhui/1/2013 (H7N9) influenza virus. At day 60, the experiment was ended and animals necropsied. IgG in sera from vaccinated ferrets was evaluated individually at different timepoints in ELISA against HA from A/Shanghai/1/2013 (H7N9) ( B ), and HS ( C ). *p < 0.0003 for αHLAII-HS (3 mg) vs. αHLAII-HS (0.3 mg) and NaCl, Two-Way ANOVA and Tukey’s multiple comparison test. D Geometric mean (±95% CI) haemagglutination inhibition (HI) titers (reciprocal values) in sera against inactivated whole virus influenza A/Anhui/1/2013 virus. Kruskal–Wallis per timepoint followed by Wilcoxon test for pair-wise comparison, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared to NaCl. Left of “/ “ = results for αHLAII-HS (3 mg), right results of αHLAII-HS (0.3 mg). Sera after one ( E ) and two DNA vaccinations ( F ) were evaluated in a pseudotype neutralization assays against A/Shanghai/1/2013 (H7N9) and A/Shanghai/2/2013 (H7N9). * p < 0.02 as compared to NaCl, One-Way ANOVA, and Tukey’s multiple comparison test. G Number of spot-forming cells (SFC) in IFNγ ELISpot (mean ± 95% CI) after in vitro stimulation with inactivated A/Anhui/1/2013 (H7N9) influenza virus. n = 8/group, except for the 3 mg αHLAII-HS ( n = 5) and 0.3 mg αHLAII-HS ( n = 2) vaccinated ferrets on day 12. Further, on day 40, n = 6 for 0.3 mg αHLAII-HS, and n = 7 for NaCl. Kruskal Wallis per timepoint followed by Wilcoxon test for pair-wise comparison: ns: non-significant. ** p < 0.01 as compared to NaCl. Left of “/ “ = results of αHLAII-HS (3 mg), right results of αHLAII-HS (0.3 mg).

Article Snippet: On the next day, cells were transferred to V-shaped plates, re-incubated with fresh stimuli on pre-coated Ferret IFNγ-ELISpot plates (3112-4APW-2, Mabtech), and incubated for 20–24 h at 37 °C in a 5% CO 2 incubator.

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Comparison, Inhibition, Neutralization, Enzyme-linked Immunospot, In Vitro

Journal: Journal of Neuroinflammation

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1186/s12974-025-03637-7

Figure Lengend Snippet: Sez6L2 immunized mice generate Sez6L2-specific T cells. A and B Splenocyte and lymph node mixed cell cultures from Sez6L2 or sham immunized mice were left unstimulated or were stimulated with H-Sez6L2 or M-Sez6L2 protein as indicated for 24 h in an IFNγ ELISPOT assay. N = 8–10 mice (4–5 male and 4–5 female); one mouse = one culture. Cells from mice immunized with M-Sez6L2 were only stimulated with M-Sez6L2 (not H-Sez6L2) as only the reaction to M-Sez6L2 is relevant to the autoimmunity model for that group. A Representative images of ELISPOT wells. B Quantification of the number of spots per 200,000 splenocytes/lymph node cells plated. C - E Splenocyte/lymph node mixed cell cultures from Sez6L2 or sham immunized mice were stimulated with M-Sez6L2 protein for 24–72 h followed by cell surface and intracellular cytokine staining and analysis by flow cytometry. C Graphs show the percent of CD4 + cells positive for IFNγ or TNFα after stimulation in culture for 24 h. N = 8–10 (4–5 male and 4–5 female). D Graphs show the percent of CD4 + cells positive for IFNγ, TNFα, IL-4, or IL-17 A after stimulation in culture for 72 h. N = 4–5 males only. E Graphs show the percent of CD8 + cells positive for IFNγ, TNFα after stimulation in culture for 24 h. N = 8–10 (4–5 male and 4–5 female). Statistics for all graphs = Welch-ANOVAs with Dunnett’s T3 MCTs

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2 M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Enzyme-linked Immunospot, Staining, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1186/s12974-025-03637-7

Figure Lengend Snippet: Immunodominant T cell epitopes and antibody peptide epitopes from Sez6L2-immunized mice. A Splenocytes from H-Sez6L2 or sham-immunized mice were stimulated with 15-mer/10aa overlapping peptide library containing peptides from the extracellular domain of mouse and human Sez6L2 and assayed in an IFNγ ELISPOT assay. The 15-mer immunodominant peptide sequences are shown with the MHC-II core binding region predicted by IEDB Resource highlighted in red. For the peptide ELISPOT, H-Sez6L2-immunized N=5 (3 male, 2 female). B ELISA absorbance from serum of 6-week post-immunization mice tested for binding to the Sez6L2 15-mer peptide library. 12 H-Sez6L2-immunized mice were tested. For both A and B) Circle data points represent results from peptides that are identical in human and mouse Sez6L2, diamond data points are from peptides unique to humans, and triangle data points are from peptides unique to mouse. The colors represent individual mice. A schematic of Sez6L2’s domain structure is aligned to the A and B graphs. The red lines highlight the location of immunodominant T cell epitopes and the green lines highlight the antibody epitopes. C Summary table from 15-mer experiments showing peptide sequences that had positive hits in the ELISPOT and/or ELISA experiments. MHC-II core binding region predicted by IEDB Resource are highlighted in red. Overlapping peptides sharing 10 amino acids in common are underlined.

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2 M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Enzyme-linked Immunospot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: Initial immune characterization of HA DNA-LNP formulations and immunogenicity (A) Schematic of DNA-LNP N/P ratios and immunization regimen. (B–D) Biophysical characterization of DNA-LNPs at different N/P ratios. (B) Particle size; (C) polydispersity index (PDI); (D) zeta potential. (E) Representative fluorescence-activated cell sorting (FACS) plots of GC B cells. (F) Bar plots quantifying frequency of GC B cells. (G) Frequency of CA09 HA-specific GC B cells. (H) Frequency of activated Tfh cells. (I) IFNγ ELISpot of splenocytes. (J) Representative TEM images of HA DNA-LNP (top) and HA mRNA-LNP (bottom). Scale bar 100 nm (K and L) Fold change cytokine induction in DLNs at 4 h (K) and 24 h (L) after immunization quantified using Luminex. (M and N) ELISpot assay measuring IFNα (M) and IFNγ (N) 20 h after stimulation of splenocytes ex vivo with DNA-LNP, plasmid DNA, or DNA-LNP in the presence of chemical inhibitors to the indicated DNA sensors. (O) Schematic of relevant pathways implicated in DNA-LNP sensing. Dots represent individual animals; n = 8–9 (E–H), n = 5 (I, K, and L), or n = 3–4 animals per group (L and M); data pooled or representative from two independent experiments (E–H, M, and N) or from one independent experiment (I–L). Plots show mean with SD (B–D and I) or geometric mean with geometric SD (F–H and K–N). Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (F–H, K, and L) or compared to DNA-LNP control (M and N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells and stimuli were incubated for 20 h and either mouse IFNγ pre-coated ELISpot plates (Mabtech) or mouse IFNα (Mabtech 3326-2A) developed according to manufacturer’s protocol.

Techniques: Immunopeptidomics, Zeta Potential Analyzer, Fluorescence, FACS, Enzyme-linked Immunospot, Luminex, Ex Vivo, Plasmid Preparation, Control

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP elicits potent antigen-specific CD8 + and CD4 + T cell responses (A) Schematic of immunization regimen. (B and C) Representative FACS plots (B) and frequency (C) of IFNγ + effector CD8 + T cells. (D) CD107a + effector CD8 + T cells. (E) TNF-α + effector CD8 + T cells. (F) IFNγ + effector CD4 + T cells. (G) TNF-α + effector CD4 + T cells. (H) IL-2 + effector CD4 + T cells. (I–K) Frequency of effector CD8 + T cells expressing IFNγ (I), CD107a (J), or TNF-α (K) after dose de-escalation. Dots represent individual animals; for (C–H), n = 10 animals per group; for (I–K), n = 5 animals per group. Data pooled from two (C–H) or one (I–K) independent experiment(s). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells and stimuli were incubated for 20 h and either mouse IFNγ pre-coated ELISpot plates (Mabtech) or mouse IFNα (Mabtech 3326-2A) developed according to manufacturer’s protocol.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP induces potent memory responses in mice and rabbits (A) Schematic of mouse immunization regimen. (B) IFNγ-secreting cells in splenocytes by ELISpot. (C) IFNγ-secreting effector CD8 + T cells by flow cytometry. (D) CA09 HA-specific ASC responses in bone marrow by ELISpot. (E) Representative FACS plot of CA09 HA-specific MBCs. (F and G) Bar plots show frequency (F) and numbers (G) of CA09 HA-specific MBCs. (H) Schematic of rabbit immunization regimen. (I–K) IFNγ ELISpot on peripheral blood mononuclear cells (PBMCs) at day 42 (I), day 105 (J), and day 202 (K). (L) AUC of total A/California/04/2009 HA-specific serum IgG ELISA data. (M and N) HAI titers to A/Netherlands/602/2009 (M) and A/New York City/PV63249/2022 (N). Dots represent individual animals (C, D, F, and G); n = 9–10 animals per group (B–D, F, and G), n = 5 animals per group (I–N); data pooled from two independent experiments. Plots show mean with SD (B and I–K) or geometric mean with geometric SD (C, D, F, G, and L–N). Unpaired one-way or two-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ANOVA was performed at the final time point for (L–N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells and stimuli were incubated for 20 h and either mouse IFNγ pre-coated ELISpot plates (Mabtech) or mouse IFNα (Mabtech 3326-2A) developed according to manufacturer’s protocol.

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet:

Article Snippet: Cells and stimuli were incubated for 20 h and either mouse IFNγ pre-coated ELISpot plates (Mabtech) or mouse IFNα (Mabtech 3326-2A) developed according to manufacturer’s protocol.

Techniques: Virus, Recombinant, Lysis, Reporter Gene Assay, Cell Stimulation, Electron Microscopy, Luminex, Enzyme-linked Immunospot, Luciferase, Plasmid Preparation, Software, Synthesized

Journal: Clinical Cancer Research

Article Title: Integrating Multisector Molecular Characterization into Personalized Peptide Vaccine Design for Patients with Newly Diagnosed Glioblastoma

doi: 10.1158/1078-0432.CCR-23-3077

Figure Lengend Snippet: T-cell characterization. A–C, immunoSEQ Analyzer differential abundance analysis displayed in pairwise scatter plots are shown for subjects 1, 2, and 3, respectively. The frequency of productive TCRβ rearrangements (clones) from the peripheral blood before and after NeoVax is compared. No data are available for subject 4 because they did not receive NeoVax. D–M, Bar graphs displaying IFNγ spots from IFNγ ELISPOT assays. Experiments were performed with duplicate or triplicate wells, and biological replicates were performed at least twice. *, P < 0.05 (Student t test) between pre- and post-NeoVax responses. There were 84, 77, and 35 days between pre- and post-NeoVax peripheral blood collections for subjects 1, 2, and 3, respectively. D, Direct ex vivo stimulation on select CD8 + T cells with minimal epitope peptide pools (m) or DMSO control for subject 1 post-NeoVax. E, Direct ex vivo stimulation on select CD4 + T cells with SLPs or DMSO control for subject 1 post-NeoVax. F, Direct ex vivo stimulation on select CD8 + T cells with minimal epitope peptide pools (m) or DMSO control for subject 1 pre-NeoVax. G, Direct ex vivo stimulation on select CD4 + T cells with SLPs or DMSO control for subject 1 pre-NeoVax. H–M, In vitro expanded PBMCs from pre- and postvaccination time points stimulated with IL2 and corresponding SLPs (green bars), minimal epitope peptide pools (dark orange bars), or DMSO control (purple bars) for 12 days prior to restimulation and analysis for subjects 1, 2, and 3.

Article Snippet: Approximately 100,000 double-negative autologous PBMCs were incubated on pre-coated human IFN-γ ELISPOT plates (Cellular Technology, Ltd.) with 10 μmol/L of indicated peptide(s) (GenScript) plus ∼400,000 CD8 + or CD4 + PBMCs for 18 to 20 hours at 37°C.

Techniques: Clone Assay, Enzyme-linked Immunospot, Ex Vivo, Control, In Vitro