Journal: Microbial biotechnology

Article Title: Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

doi: 10.1111/j.1751-7915.2011.00316.x

Figure Lengend Snippet: Cellular immune responses stimulated in mice after the immunizations with EHEC O157:H7 (N°CIP 105282) BGs. Mice were immunized as described in Fig. 1 . Spleen cells (1 × 10 5 per well) were incubated for 16 h in the presence of EHEC O157:H7 BGs (specific Ag); measles inactivated Ag (strain Edmonston, non‐specific Ag) or as a negative control spleen cells were left without stimulation. The numbers of IFN‐γ‐producing cells were determined by ELISPOT assays. Rectal immunization with EHEC O157:H7 BGs effectively increased the number of IFN‐γ‐producing spleen cells after recognition of specific Ag (A). The stimulatory effect of the boost immunization was detected on day 42 (14 days after the second immunization) and remained significant until the end of experiment (B). Data represent the mean of three independent experiments ± SD (three mice per time point). P ‐values < 0.05 were considered significant and are indicated with asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001). a, day of the first immunization in all groups; b, day of the second immunization in groups B1 and D1; c, day of the challenge.

Article Snippet: The number of IFN‐γ‐producing cells was determined by Mouse IFN‐γ ELISpot Kit (R&D Systems, Inc, Minneapolis, USA) following the manufacturer's instructions.

Techniques: Incubation, Negative Control, Enzyme-linked Immunospot

Journal: Gene Therapy

Article Title: Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells

doi: 10.1038/sj.gt.3303085

Figure Lengend Snippet: Determination of interferon-γ (IFN-γ) and interleukin-2 (IL-2) in mice receiving either Ad-NP or tAd-NP. The mice were administrated with either Ad-NP (open bar) or tAd-N (filled bar) and boosted 4 weeks later. The splenocytes were then harvested on days 0, 15 and 30 for the analyses of Ag-specific IFN-γ or IL-2 using ELISPOT as described in the ‘Materials and methods’ section. Data represent spots per 10 5 splenocytes stimulated by 10 μg ml −1 of NP. ( a ) IFN-γ levels in mice injected with either Ad-NP or tAd-NP (i.d. route); ( b ) IL-2 levels in mice injected with either Ad-NP or tAd-NP (i.d. route); ( c ) IFN-γ levels in mice injected with either Ad-NP or tAd-NP through the i.n. route); ( d ) IL-2 levels in mice injected with either Ad-NP or tAd-NP (i.n. route). All data represent the means±s.d. from four mice. Asterisks ( * ) indicate significant difference between Ad-NP and tAd-NP (same below). Statistical comparisons were conducted with the use of a two-tailed t -test, with P <0.05 being considered significant. Open bar depicts Ad-NP, while filled bar designates tAd-NP.

Article Snippet: Anti-CD40 mAb, rh-IL-4, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), the ELISPOT kits with various controls, including IFN-γ (EL485) and IL-2 (EL402), were purchased from R&D systems, Minneapolis, MN, USA.

Techniques: Enzyme-linked Immunospot, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

doi: 10.1101/2022.11.03.515007

Figure Lengend Snippet: Mice were immunized (priming) with chimeric proteins either alone (pO, pNO), or bound to T5 CLP (pO-CLP, pNO-CLP) or supplemented with CFA. Mice were subsequently boosted under the same conditions (except that CFA was replaced by incomplete Freund adjuvant, ICF). (a-b) IFNγ-producing splenocytes were quantified by ELISPOT and ( c ) IFNγ production by splenocytes was measured by ELISA after in vitro restimulation with Ova 257-264 peptide 10 days after boosting. The interval between priming and boosting was of two ( a ) or six months ( b ). The bars correspond to the mean of each group (n = 5-6) and the circles to results of individual mice. ns , non-significant; **, p < 0 . 01 ; ***, p < 0 . 001 .

Article Snippet: For ELISPOT, plates were revealed with supplied reagents (murine IFNγ ELISPOT kit, Diaclone) and spots were counted with the ImmunoSpot® S6 FluoroSpot Line Plate Reader (C.T.L.).

Techniques: Adjuvant, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Frontiers in Microbiology

Article Title: Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell immune responses

doi: 10.3389/fmicb.2024.1404558

Figure Lengend Snippet: Detection of IFN-γ secretion from PBMCs induced with the predicted epitope peptide groups and the individual peptide in selected groups with an ELISpot assay. PBMCs isolated from PRRSV HuN4-F112-vaccinated Hp-4.0 or Hp-26.0 haplotype piglets and unvaccinated piglets were seeded in 96-well plates pre-coated with anti-IFN-γ antibody. After stimulation with six groups of peptides, the spot forming cells (SFC) were counted. PHA was used as the positive control and ddH 2 O or DMSO was used as the negative control. The results are expressed as the average number of SFC per one million PBMCs (SFC/10 6 PBMCs). The predicted epitope peptide groups (A) and 12 predicted peptides in groups 4, 5, and 6 (B) were used for the IFN-γ ELISpot assay, respectively. * p < 0.05; ** p < 0.01.

Article Snippet: Peptide-specific IFN-γ-secreting cells were analyzed with the porcine IFN-γ ELISpot Kit (R&D Systems, United States), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Isolation, Positive Control, Negative Control

Journal: Journal of Virology

Article Title: Human Papillomavirus Type 16 and 18 E7-Pulsed Dendritic Cell Vaccination of Stage IB or IIA Cervical Cancer Patients: a Phase I Escalating-Dose Trial

doi: 10.1128/jvi.02343-07

Figure Lengend Snippet: FIG. 5. CD4 ELISpot counts versus E7 and KLH, ratios relative to time zero. ELISpot analysis for enumeration of IFN--secreting CD4

Article Snippet: Gamma interferon (IFN- ) ELISpot kits (R&D Systems) were used to determine the frequency of cytokineexpressing in vitro-stimulated CD8 and CD4 T cells after overnight activation with KLH and E7-antigen-loaded and unloaded stimulator DC.

Techniques: Enzyme-linked Immunospot

Journal: Journal of Virology

Article Title: Human Papillomavirus Type 16 and 18 E7-Pulsed Dendritic Cell Vaccination of Stage IB or IIA Cervical Cancer Patients: a Phase I Escalating-Dose Trial

doi: 10.1128/jvi.02343-07

Figure Lengend Snippet: FIG. 6. CD8 ELISpot counts versus E7 and KLH, ratios relative to time zero. ELISpot analysis for the enumeration of IFN--secreting CD8

Article Snippet: Gamma interferon (IFN- ) ELISpot kits (R&D Systems) were used to determine the frequency of cytokineexpressing in vitro-stimulated CD8 and CD4 T cells after overnight activation with KLH and E7-antigen-loaded and unloaded stimulator DC.

Techniques: Enzyme-linked Immunospot

Journal: Veterinary Research

Article Title: Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

doi: 10.1186/1297-9716-45-6

Figure Lengend Snippet: Comparison of xenodiagnosis , initial conventional serology , multiplex serology , and IFNγ ELISpot assays

Article Snippet: Cells were assayed for IFNγ production using the Canine IFN-γ ELISpot kit (cat no. EL781, R& D Systems, Inc, Minneapolis, MN, USA) following standard kit protocol.

Techniques: Comparison, Multiplex Assay, Enzyme-linked Immunospot

Journal: Veterinary Research

Article Title: Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

doi: 10.1186/1297-9716-45-6

Figure Lengend Snippet: Seropositive dogs have significantly higher frequencies of T. cruzi -specific PBMCs and reactive T cell compartment. A , Interferon (IFN)-γ enzyme-linked immmunosorbent spot (ELISpot) well images of 4 × 10 5 PBMCs cultured with media, PMA/ionomycin (PMA/ion), or T. cruzi lysate (TcLysate/Tc) and 1 × 10 4 PBMCs stimulated with PMA/ionomycin (PMA/ion (10 000)). Individual dog identification (ID) and results of multiplex serology are denoted above wells. B-C, Numbers of IFNγ-producing PBMCs in the ELISpot assay responding to T. cruzi parasite lysate (B) or PMA (C) adjusted to 1 × 10 6 or per 1 × 10 4 PBMCs, respectively. Each square and dot represents an individual dog, while each diamond represents one of three US dog controls to ensure plate reproducibility. Dogs are grouped by multiplex serology negative (Sero -) or serology positive (Sero +) from Argentina or seronegative dogs from United States. Statistical significance was determined by one-way ANOVA using Prism v4.0c (GraphPad). Horizontal line, threshold for positive/negative response.

Article Snippet: Cells were assayed for IFNγ production using the Canine IFN-γ ELISpot kit (cat no. EL781, R& D Systems, Inc, Minneapolis, MN, USA) following standard kit protocol.

Techniques: Enzyme-linked Immunospot, Cell Culture, Multiplex Assay

Journal: Veterinary Research

Article Title: Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

doi: 10.1186/1297-9716-45-6

Figure Lengend Snippet: Intracellular IFNγ staining identifies T. cruzi -specific CD4 + and CD8 + T cells. A , Representative flow plots of intracellular cytokine staining of T cells from ELISpot positive (71, 69, 43, 67) and negative canine (1,2) PBMC cultures incubated with media, PMA/ionomycin, or T. cruzi lysate (TcLysate). Numbers indicate percentage of IFNγ-producing CD4 + or CD8 + T cells. B-C, Percentage of IFNγ-producing CD4 + (B) and CD8 + (C) T cells from individual dogs. Each bar represents a dog from Argentina (red) or United States (gray). Results of reactivity in IFNγ ELISpot assay are denoted below each bar.

Article Snippet: Cells were assayed for IFNγ production using the Canine IFN-γ ELISpot kit (cat no. EL781, R& D Systems, Inc, Minneapolis, MN, USA) following standard kit protocol.

Techniques: Staining, Enzyme-linked Immunospot, Incubation

Journal: mAbs

Article Title: Development of a nonhuman primate challenge model to evaluate CD8 + T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors

doi: 10.1080/19420862.2021.1979447

Figure Lengend Snippet: Study design. MHC I-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent adenovirus serotype 5 (Ad5) encoding Gag, Nef or Pol SIV (Simian immunodeficiency virus) proteins. One group received the anti-PD-L1 atezolizumab and the second group received an immune checkpoint-targeting bi-specific antibody mAbX in early development. Blood samples, from all animals, were taken on a weekly basis to characterize the T cell phenotype (Immunophenotyping) and function (IFNγ ELIspot and cytolytic T cell assay)

Article Snippet: ELIspot assays were conducted according to the Primate IFN-gamma ELISpot Kit manufacturer’s protocol (R&D Systems).

Techniques: Virus, Enzyme-linked Immunospot

Journal: mAbs

Article Title: Development of a nonhuman primate challenge model to evaluate CD8 + T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors

doi: 10.1080/19420862.2021.1979447

Figure Lengend Snippet: Detection of cellular IFNγ response by ELISPOT assay following ex-vivo stimulation of freshly isolated PBMCs (sixteen immunized MCMs) with a peptide pool covering the complete sequence of the Adenovirus hexon, at 0.6 µM (total peptide concentration, A and C) or with a pool of three Nef peptides (RM9, HW8 and LT9 at 10 µM each, B and D) or Conditions for which spots were too numerous to count were arbitrary set at 3000 SFCs per 10 6 PBMCs. *, p < .05 indicates statistical significance compared to the untreated group

Article Snippet: ELIspot assays were conducted according to the Primate IFN-gamma ELISpot Kit manufacturer’s protocol (R&D Systems).

Techniques: Enzyme-linked Immunospot, Ex Vivo, Isolation, Sequencing, Concentration Assay