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Biotinylated anti ifnγ antibody

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Biotinylated anti-IFNγ antibody is a laboratory reagent used to detect and quantify the presence of interferon-gamma (IFNγ) in biological samples. It functions by binding to IFNγ, allowing for its identification and measurement through various analytical techniques.

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Lab products found in correlation

Anti ifn γ antibody, by BD (4 mentions) Hts ip plate, by Merck Group (3 mentions) Avidin horseradish peroxidase, by BD (3 mentions) Multiscreen ha plates, by Merck Group (1 mentions) Streptavidin horseradish peroxidase, by Merck Group (1 mentions) 3 amino 9 ethylcarbazole, by Merck Group (1 mentions) Elispot plate, by Merck Group (1 mentions) Streptavidin alkaline phosphatase, by BD (1 mentions) Gilgfvftl, by ProImmune (1 mentions) Golgiplug, by BD (1 mentions) Nitroblue tetrazolium, by Thermo Fisher Scientific (1 mentions) Ly6g fitc, by BD (1 mentions) Mhcii fitc, by BioLegend (1 mentions) Anti mouse ifn γ antibody, by BD (1 mentions) Golgistop, by BD (1 mentions) Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions) Cd4 buv737, by BD (1 mentions)

Spelling variants of this product

Biotinylated anti-IFNγ detection antibody (5 citations) Biotinylated anti-human IFNγ detection antibody (3 citations) Biotinylated anti-mouse IFNγ antibody (2 citations) Biotinylated mouse anti-porcine IFNγ antibody (clone P2C11, (2 citations) Biotinylated rat anti-mouse IFNγ antibody (2 citations)

6 protocols using biotinylated anti ifnγ antibody

1

Quantifying HPV16 E7 Antigen-Specific T Cells

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Functional IFNγ producing tumor antigen–specific cells specific for HPV16 E7(49–57) were detected 10 days after final vaccination. A total of 5 × 106 freshly isolated splenocytes (n = 5 per group, 3 independent experiments) were stimulated without or with E7 peptide (final concentration 1 µg/mL) and with 5 IU interleukin (IL)-2/mL in culture medium for 24 h. Multiscreen HA plates (EMD Millipore, Burlington, MA, USA.) were coated with 10 μg/mL anti-IFNγ antibody (BD Biosciences, San Jose, CA, USA). Plates were washed and blocked with culture medium. Splenocytes were added to the coated plates in 2-fold serial dilutions ranging from 5 × 105 to 6.25 × 104 cells per well. After 20 h, plates were washed and incubated with 1 μg/mL of biotinylated anti-IFNγ antibody (BD Biosciences). Washed plates were incubated with 100 μL of 1:4000 diluted streptavidin–horseradish peroxidase (Sigma Aldrich, St. Louis, MO, USA) per well. Spots were developed using 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO, USA) for 5 min, and the reaction was stopped with water. Spots were counted using the Zeiss KS enzyme-linked immunospot system. Assays were performed in triplicate, and results were calculated as spot-forming cells (SFC) per 106 splenocytes after subtracting medium background.
Da Silva D.M., Martinez E.A., Bogaert L, & Kast W.M. (2022). Investigation of the Optimal Prime Boost Spacing Regimen for a Cancer Therapeutic Vaccine Targeting Human Papillomavirus. Cancers, 14(17), 4339.
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2

Tumor-specific CD8+ T Cell Functional Assay

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For tumor-specific CD8+ T cell functional assay in the MC38 model, 10 days after anti-CD47 Ab treatment, CD8+ T cells were purified from spleen. 2.5×105 CD8+ T cells were re-stimulated with MC38 cells at the ratio of 20:1 for 48 hours. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFN-γ antibody (BD Bioscience) with a 1:250 dilution overnight at 4° C. After co-culture, cells were removed. 2µg/ml biotinylated anti-IFN-γ antibody (BD Bioscience) with a 1:250 dilution was added and incubated for 2h at room temperature or overnight at 4°C. Avidin-horseradish peroxidase (BD Bioscience) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (BD Bioscience).
Xu M.M., Pu Y., Han D., Shi Y., Cao X., Liang H., Chen X., Li X.D., Deng L., Chen Z.J., Weichselbaum R.R, & Fu Y.X. (2017). Dendritic cells but not macrophages sense tumor mitochondrial DNA for cross-priming through signal regulatory protein α signaling. Immunity, 47(2), 363-373.e5.
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3

Flu-specific CD8+ T Cell Activation

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PBMCs derived from buffy coat of healthy donors were co-cultured with K562 cells, pulsed with HLAI-A2 restricted Flu peptide (GILGFVFTL) (10 μg/mL) (Proimmune, Oxford, UK) for 12 days in RPMI+10% FCS in presence of IL-2 (10 U/ml) and IL-15 (10ng/ml). Flu-enriched CD8+ T cells were isolated employing the human microbeads CD8 (Miltenyi Biotech) and co-cultured with autologous Tregs (+/- rhMGL-Fc) and/or mDCs pulsed with Flu peptide in the anti-IFN-γ precoated (1:200; Pharmingen San Diego, CA, USA) ELISpot plates (Millipore, Bedford, MA, USA) overnight at 4°C. Cytokine release was detected with biotinylated anti-IFN-γ antibody (Pharmingen; 1:250, 2 hrs at RT) revealed with streptavidin-alkaline phosphatase (Pharmingen) (1:1000, 100 mL/well, 1 h at RT) and chromogen substrate (5-bromo-4- chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium alkaline phosphatase substrate, Sigma) (50 μL/well). Spots were counted using the ImmunoSpot Image Analyzer (Aelvis).
Zizzari I.G., Martufi P., Battisti F., Rahimi H., Caponnetto S., Bellati F., Nuti M., Rughetti A, & Napoletano C. (2015). The Macrophage Galactose-Type C-Type Lectin (MGL) Modulates Regulatory T Cell Functions. PLoS ONE, 10(7), e0132617.
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4

CD8+ T Cell IFN-γ ELISPOT Assay in B16-OVA Tumor Model

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For antigen-specific CD8+ T cell functional assay in the B16-OVA model, 12 days after tumor inoculation, 3×105 lymphocytes were re-stimulated with 1μg/ml SIINFEKEL or MC38 tumor cells (lymphocyte:MC38 = 50:1) for 48 hours. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution overnight at 4 °C. After co-culture, cells were removed. 2 mg/ml biotinylated anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution was added and incubated for 2 h at room temperature or overnight at 4°C. Avidin-horseradish peroxidase (BD Bioscience) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (BD Bioscience).
Han D., Liu J., Chen C., Dong L., Liu Y., Chang R., Huang X., Liu Y., Wang J., Dougherty U., Bissonnette M., Shen B., Weichselbaum R., Xu M.M, & He C. (2019). Anti-tumor immunity controlled through mRNA m6A and YTHDF1 in dendritic cells. Nature, 566(7743), 270-274.
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5

CD8+ T Cell IFN-γ ELISPOT Assay in B16-OVA Tumor Model

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For antigen-specific CD8+ T cell functional assay in the B16-OVA model, 12 days after tumor inoculation, 3×105 lymphocytes were re-stimulated with 1μg/ml SIINFEKEL or MC38 tumor cells (lymphocyte:MC38 = 50:1) for 48 hours. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution overnight at 4 °C. After co-culture, cells were removed. 2 mg/ml biotinylated anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution was added and incubated for 2 h at room temperature or overnight at 4°C. Avidin-horseradish peroxidase (BD Bioscience) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (BD Bioscience).
Han D., Liu J., Chen C., Dong L., Liu Y., Chang R., Huang X., Liu Y., Wang J., Dougherty U., Bissonnette M., Shen B., Weichselbaum R., Xu M.M, & He C. (2019). Anti-tumor immunity controlled through mRNA m6A and YTHDF1 in dendritic cells. Nature, 566(7743), 270-274.
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6

Evaluating T-cell Responses in Mice

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T-cell responses in mouse spleen were evaluated 1 week after the second immunization. For ELISpot assays, plate wells were coated with anti-mouse IFN-γ antibody (BD Biosciences) and incubated overnight at 4°C. Plates were then blocked with tissue culture media (RMPI-1640, 10% fetal calf serum and 5% Penicillin-Streptomycin), and single-cell suspensions (2 × 105 cells/well) were incubated with 1 μg/mL NS1 protein, 1 μM OVA CD4 peptide (265TEWTSSNVMEERKIKV280), or 1 μM OVA CD8 peptide (257SIINFEKL264) for a minimum of 18 hours at 37°C. Biotinylated anti-IFN-γ antibody (BD Biosciences) was then added, followed by a washing step and addition of streptavidin–conjugated alkaline phosphatase. The assay was developed with nitro-blue tetrazolium and 5-bromo-4-chloro-3’-indolyphosphate substrate (ThermoFisher Scientific). For intracellular cytokine staining (ICS), 2 × 106 cells were incubated at 37°C for 1 hour with 1 μg/mL DENV NS1 protein, followed by 4 hours in the presence of 1 μg/ml brefeldin A (GolgiPlug, BD Biosciences) and 2 μM monensin (GolgiStop, BD Biosciences). Cells were stained with the viability dye Zombie Green (BioLegend), CD8a-BUV395, CD4-BUV737, Ly6G-FITC, CD90.2 V500 (BD Biosciences), and MHCII-FITC (BioLegend). Cells were then treated with Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) and stained with IL-2 PE, TNF-α-PEcy7, IFN-γ APC/647 (BioLegend).
Espinosa D.A., Beatty P.R., Reiner G.L., Sivick K.E., Glickman L.H., Dubensky TW J.r, & Harris E. (2019). Cyclic dinucleotide-adjuvanted dengue virus nonstructural 1 protein induces protective antibody and T-cell responses. Journal of immunology (Baltimore, Md. : 1950), 202(4), 1153-1162.
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