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Ifnγ elispot

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The IFNγ ELISPOT is a laboratory equipment designed to measure the number of cells secreting interferon-gamma (IFNγ) in a given sample. It provides a quantitative assessment of the cellular immune response.

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Lab products found in correlation

Peptide pools, by Mimotopes (2 mentions) Automated elispot reader, by Autoimmun Diagnostika (2 mentions) Extravidin alkaline phosphatase, by Merck Group (1 mentions) Multiscreen filter plate, by Merck Group (1 mentions) Biotin conjugated anti ifnγ antibody, by Mabtech (1 mentions) Brefeldin a, by Merck Group (1 mentions) Bcip nbt substrate, by Merck Group (1 mentions) Anti ifn γ, by Mabtech (1 mentions) Human cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions) Fitc cd8α, by Thermo Fisher Scientific (1 mentions) Cd3 percp, by Thermo Fisher Scientific (1 mentions) Hepes solution, by Merck Group (1 mentions) Rpmi medium, by Merck Group (1 mentions) Ketaset, by Zoetis (1 mentions) Msips4510, by Merck Group (1 mentions) Apex software 1, by Mabtech (1 mentions)

10 protocols using ifnγ elispot

1

Antigen-Specific T Cell Characterization

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T cell lines originate from anti-PDL1 (clone MIH-5) treated, MC38 immune mice as described by Sow et al. (46 (link)), and ex vivo established via coculture of splenocytes with irradiated MC38 in IL2 supplemented (5 Cetus Units) medium [as described by Hos et al., 2019 (20 (link))]. Recognition of live MC38 cells (MC38-L and MC38-K) by T cell lines was determined by cytokine production after o/n coculture in a 5:1 (effector: target) ratio and 2 µg/mL brefeldin A (Sigma-Aldrich) by IFNγ ELISPOT.
Adpgk-, Rpl18- or OTI-TCR-transduced T cells were cultured overnight at 37°C on anti-IFNγ (Mabtech, clone: AN18) pre-coated Multiscreen filter plates (Merck Millipore). 1x105 transduced T cells were stimulated with 5x104 tumor cells, i.e. B16-Ova, MC38-L or –MC38-K cells (untreated or pre-treated overnight with 20 ng/mL IFNγ), or MC38-K electroporated with Adpgk-, Rpl18- or OvaI-RNA. The spots were visualized with a biotin-conjugated anti-IFNγ antibody (Mabtech) followed by incubation with ExtrAvidin-Alkaline Phosphatase (Sigma-Aldrich) and BCIP/NBT substrate (Sigma-Aldrich). Plates were scanned using CTL’s ImmunoSpot® Series S five Versa ELISpot Analyzer (S5Versa-02-9038) and analyzed by ImmunoCapture V6.3 software. The samples were tested in duplicates and spot counts were summarized as means of technical duplicates.
Schrörs B., Hos B.J., Yildiz I.G., Löwer M., Lang F., Holtsträter C., Becker J., Vormehr M., Sahin U., Ossendorp F, & Diken M. (2023). MC38 colorectal tumor cell lines from two different sources display substantial differences in transcriptome, mutanome and neoantigen expression. Frontiers in Immunology, 14, 1102282.
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2

IFNγ ELISPOT Assay for M. tuberculosis

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IFNγ ELISPOT (MABtech, Cincinnati, OH) assays were performed as per manufacturer instructions and previously described39 (link). Briefly, PBMCs and neutrophils were obtained from macaques as previously indicated and three cell suspensions were added to duplicate wells: neutrophils only (125,000 cells/well), PMBCs (125,000 cells per well) and neutrophils with PBMCs (1:1 ratio, total 250,000 cells per well). Cells were stimulated with CFP or a cocktail of M. tuberculosis ESAT-6 and 38.1 peptides as previously described8 (link) and incubated overnight at 37°C with 5% CO2. T cell viability after this treatment was experimentally confirmed and not found to be impaired by overnight co-culture with CFP-stimulated neutrophils. Plates were read with a CTL SPOT reader (Immunospot, Cleveland, OH) and analyzed for the number of spots per well, spot size, and signal intensity per spot.
Gideon H.P., Phuah J., Junecko B.A, & Mattila J.T. (2019). Neutrophils express pro- and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques. Mucosal immunology, 12(6), 1370-1381.
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3

IFNγ ELISPOT Assay for M. tuberculosis

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IFNγ ELISPOT (MABtech, Cincinnati, OH) assays were performed as per manufacturer instructions and previously described39 (link). Briefly, PBMCs and neutrophils were obtained from macaques as previously indicated and three cell suspensions were added to duplicate wells: neutrophils only (125,000 cells/well), PMBCs (125,000 cells per well) and neutrophils with PBMCs (1:1 ratio, total 250,000 cells per well). Cells were stimulated with CFP or a cocktail of M. tuberculosis ESAT-6 and 38.1 peptides as previously described8 (link) and incubated overnight at 37°C with 5% CO2. T cell viability after this treatment was experimentally confirmed and not found to be impaired by overnight co-culture with CFP-stimulated neutrophils. Plates were read with a CTL SPOT reader (Immunospot, Cleveland, OH) and analyzed for the number of spots per well, spot size, and signal intensity per spot.
Gideon H.P., Phuah J., Junecko B.A, & Mattila J.T. (2019). Neutrophils express pro- and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques. Mucosal immunology, 12(6), 1370-1381.
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4

IFNγ ELISPOT Assay for CD8+ T Cell Responses

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Purified CD8+ T cells from patient blood were analyzed by IFNγ ELISPOT (MABTECH), according to the manufacturer’s instructions. Briefly, plates were coated with human IFNγ capture antibody and blocked with RPMI media containing 7% FBS. CD8+ T cells were magnetically sorted from PBMCs (Miltenyi Biotec Inc, human CD8+ T cell Isolation Kit) and added to wells at 3×105 cells/100ul/well. HLA-A2.1 transduced target cells (K562A2.1, a gift from Alan Houghton, MSKCC) were pulsed with MHC-I restricted peptides (MART-126–35 ELAGIGILTV, GP100209–217 ITDQVPFSV, TYR368–376 YMDGTMSQV; or no peptide for control), at 37oC for 30 minutes, irradiated, and added to each well at 3×104 cells/50ul/well. Cells were incubated for 20h at 37°C, and ELISPOT was then developed with aminoethylcarbazole chromogen. Spots per well were counted on an automated reader system with KS 4.3 software (Carl Zeiss).
Han J., Zhao Y., Shirai K., Molodtsov A., Kolling F.W., Fisher J.L., Zhang P., Yan S., Searles T.G., Bader J.M., Gui J., Cheng C., Ernstoff M.S., Turk M.J, & Angeles C.V. (2021). Resident and circulating memory T cells persist for years in melanoma patients with durable responses to immunotherapy. Nature cancer, 2(3), 300-311.
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5

Quantifying Antigen-Specific CD8+ T cells

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Splenocytes were treated with anti-CD16/CD32 and labelled with PE-conjugated DCT180-188 peptide-loaded MHC-I tetramer (Baylor College), PerCP-CD3, and FITC-CD8α (eBioscience) for 30 minutes in FACS buffer (1% PBS, 5% fetal calf serum, 2.5% NaZ). For ELISpot analyses, the exact number of DCT-MHC-I tetramer+/CD8+ T cells were calculated from flow cytometry proportion results and added to an IFNγ ELISpot following manufacturer’s instructions (Mabtech).
Ananth A.A., Tai L.H., Lansdell C., Alkayyal A.A., Baxter K.E., Angka L., Zhang J., Tanese de Souza C., Stephenson K.B., Parato K., Bramson J.L., Bell J.C., Lichty B.D, & Auer R.C. (2016). Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence. PLoS ONE, 11(5), e0155947.
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6

IFN-γ ELISpot Assay for CCHF Virus Antigen

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Splenocytes were assessed for antigen recall response via IFN-γ ELISpot (Mabtech, Sweden), performed as per the manufacturer's instructions. Cells were seeded in PVDF microtitre plates at 2 × 105 splenocytes per well and re-stimulated with peptide pools (Mimotopes, Australia). Overlapping peptides spanning the length of the CCHF virus nucleoprotein consisting of 20mers, offset by 8 residues, were applied at a final concentration of 25 μg/ml per peptide in pools of 28–32 peptides. Plates were developed after 18 hours at 37°C in a humidified incubator supplemented with 5% CO2. Spots were counted visually on an automated ELISpot reader (Autoimmun Diagnostika GmbH, Germany). Background values from wells containing medium but no peptides were subtracted and pools were summed across the target protein. Results were expressed as spot forming units (SFU) per 106 cells.
Dowall S., Buttigieg K., Findlay-Wilson S., Rayner E., Pearson G., Miloszewska A., Graham V., Carroll M, & Hewson R. (2015). A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease. Human Vaccines & Immunotherapeutics, 12(2), 519-527.
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7

Splenocyte IFN-γ ELISpot for Antigen Recall

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Spleens from vaccinated animals were collected aseptically, homogenised, and red blood cells were lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2 mM L-Glutamine, 100 U penicillin & 0.1 mg/ml streptomycin, 50 μM 2-mercaptoethanol and 25 mM HEPES solution (Sigma-Aldrich). Splenocytes were assessed for antigen recall response via IFN-γ ELISpot (Mabtech, Sweden), performed as per the manufacturer's instructions. Cells were seeded in PVDF microtitre plates at 2×105 per well and re-stimulated with peptide pools (Mimotopes, Australia). Peptides spanning the tPA-GP-V5 fusion protein sequence were 20 residues long, with an overlap of 12 residues between peptides. They were applied to cells at a final concentration of 2.5 μg/ml per peptide, with 28–32 peptides per pool. Plates were developed after 18 hours at 37°C, 5% CO2 in a humidified incubator. Spots were counted visually on an automated ELISpot reader (Autoimmun Diagnostika, Germany). Background values from wells containing cells and medium but no peptides, were subtracted and pools summed across the target protein. Results were expressed as spot forming units (SFU) per 106 cells.
Buttigieg K.R., Dowall S.D., Findlay-Wilson S., Miloszewska A., Rayner E., Hewson R, & Carroll M.W. (2014). A Novel Vaccine against Crimean-Congo Haemorrhagic Fever Protects 100% of Animals against Lethal Challenge in a Mouse Model. PLoS ONE, 9(3), e91516.
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8

Rhesus Macaque TB Vaccine Trial

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Twenty-four, three-year-old, male rhesus macaques of Indian genotype were obtained from an established UK breeding colony. Absence of previous exposure to mycobacterial antigens was confirmed by a tuberculin skin test and screening using an ex-vivo IFN-γ ELISPOT (MabTech, Nacka. Sweden) to measure responses to mycobacterial antigens: PPD (SSI, Copenhagen, Denmark), and pooled 15-mer peptides of ESAT6 and CFP10 (Peptide Protein Research LTD, Fareham, U.K.).
Animals were housed in compatible social groups, in accordance with the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989), and the National Committee for Refinement, Reduction and Replacement (NC3Rs), Guidelines on Primate Accommodation, Care and Use, August 2006 (NC3Rs, 2006). Animals were sedated by intramuscular (IM) injection of ketamine hydrochloride (Ketaset, 100 mg/ml, Fort Dodge Animal Health Ltd, Southampton, UK; 10 mg/kg) for procedures requiring removal from their housing. None of the animals had been used previously for experimental procedures and each socially compatible group was randomly assigned to a particular study treatment. All animal procedures and study design were approved by the Public Health England, Porton Down Ethical Review Committee, and authorized under an appropriate UK Home Office project license.
Sharpe S., White A., Sarfas C., Sibley L., Gleeson F., McIntyre A., Basaraba R., Clark S., Hall G., Rayner E., Williams A., Marsh P.D, & Dennis M. (2016). Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations. Tuberculosis (Edinburgh, Scotland), 101, 174-190.
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9

SARS-CoV-2-Specific T Cell Responses

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Peripheral blood mononuclear cells (PBMCs) were thawed 1 day before seeding to the ELISpot plate and rested overnight. The IFN-γ ELISpot (Mabtech, Nacka Strand, Sweden) was performed in filterplates (MSIPS4510, Merck Millipore, Burlington, USA) according to the manufacturer’s instructions. PBMCs were seeded with a concentration of 3 × 105 cells/100 µL/well in X-Vivo 10 medium supplemented with 2% human AB serum and co-cultured with the following stimuli for 24 h: 25 ng/mL purified anti-CD3 (clone OKT-3; positive control) and a mix of 1.25 µg/mL S-Protein, M-Protein, and N-Protein Peptivator (Miltenyi, Bergisch-Gladbach, Germany; SARS-CoV-2 specific response)), respectively, of medium alone (negative control). Measurements were performed in triplicates. Quantification of spot forming units (SFU)/3 × 105 PBMCs was performed with the ELI-Analyze ELISpot Image Analysis Software from A.EL.VIS. and normalized to the unspecific response (SFU/3 × 105 PBMCs without stimulus).
von Metzler I., Campe J., Huenecke S., Raab M.S., Goldschmidt H., Schubert R., Rabenau H.F., Ciesek S., Serve H, & Ullrich E. (2021). COVID-19 in multiple-myeloma patients: cellular and humoral immunity against SARS-CoV-2 in a short- and long-term view. Journal of Molecular Medicine (Berlin, Germany), 100(3), 463-470.
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10

Cellular Immunity Assessment via IFN-γ ELISpot

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Cellular immunity was determined by IFN-γ ELISpot (Mabtech, Nacka Strand, Sweden) to ancestral strains on a subset of participants for each group (n = 20). Peripheral blood mononuclear cells (PBMCs) were counted and stimulated with S-peptides that consisted of 100 peptides from spike proteins, and nucleoprotein-membrane protein-open reading frame protein (NMO)-peptide pools, consisting of 101 peptides from nucleocapsid (n), membrane (M), open reading frame (ORF) 1, non-structural protein (nsp) 3, ORF-3a, ORF-7a, and ORF8 proteins. Negative controls contained only cell culture media, while positive controls contained an anti-cluster of differentiation 3 (CD3) at a dilution of 1:1000. ELISpot plates were then incubated for 20 h at 37 °C and 5% CO2, washed and developed using a conjugated secondary antibody that was bound to membrane-captured IFN-γ. The plates were read using IRIS (Mabtech) and spots were analyzed using Apex software 1.1 (Mabtech) and converted to spot-forming units (SFU) per million cells.
Niyomnaitham S., Chatsiricharoenkul S., Toh Z.Q., Senawong S., Pheerapanyawaranun C., Phumiamorn S., Licciardi P.V, & Chokephaibulkit K. (2022). Evaluation of the Safety and Immunogenicity of Fractional Intradermal COVID-19 Vaccines as a Booster: A Pilot Study. Vaccines, 10(9), 1497.
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