Human ifnγ elisa kit

Manufactured by BioLegend

Sourced in United States

The Human IFNγ ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of interferon-gamma (IFNγ) levels in human serum, plasma, and cell culture supernatants.

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12 protocols using human ifnγ elisa kit

Isolation and Activation of CD8+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy peripheral blood by Ficoll-Paque Plus (Sigma-Aldrich) density-gradient centrifugation. CD8⁺ T cells were then purified by positive selection using immunomagnetic beads according to the manufacturer’s protocol (anti-CD8α (OKT8), MicroBeads, Miltenyi Biotec, Auburn, CA) and passed through a magnetic cell sorting column (Miltenyi Biotec, Auburn, CA). CD8⁺ T cells were stimulated with plate-bound anti-human CD3 (OKT3) antibodies for 96h in presence or absence of human IL-36γ protein (S18-D169, 6835-IL-010/CF, R&D Systems), the level of IFN-γ in the supernatant was measured by human IFN-γ ELISA Kit (Biolegend).

Wang X., Zhao X., Feng C., Weinstein A., Xia R., Wen W., Lv Q., Zuo S., Tang P., Yang X., Chen X., Wang H., Zang S., Stollings L., Denning T.L., Jiang J., Fan J., Zhang G., Zhang X., Zhu Y., Storkus W, & Lu B. (2015). IL-36γ transforms the tumor microenvironment and promotes type 1 lymphocyte-mediated antitumor immune responses. Cancer cell, 28(3), 296-306.

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Flow Cytometry and Microscopy Antibody Staining

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Antibodies used for flow cytometry and immunofluorescent microscopy included those against human CD1b (SN13, Biolegend), Siglec-7 (QA79, eBioscience), CD14 (HCD14, Biolegend), CD56 (HCD56, Biolegend), CD34 (HM34, Biolegend), CD3 (UCHT1, BD Biosciences), CD19 (HIB19, Biolegend), HLA-DR (L243, Biolegend), CD11c (3.9, Biolegend), CD123 (6H6, Biolegend), EEA-1 (ab2900, Abcam) and LAMP-1 (ab24170, Abcam). For the blocking of LDN5 cell activation and liposome binding, anti-CD1b (BCD1b.3) (36 (link)) and anti-Siglec-7 (S7.7, Biolegend) were used, respectively. C80 GMM was isolated as described (36 (link)). Ficoll Plaque plus (GE Healthcare) was used for the density gradient centrifugation to separate peripheral blood mononuclear cells. Human IFNγ ELISA kit (Biolegend) was used to measure human IFNγ.

Kawasaki N., Rillahan C.D., Cheng T.Y., Van Rhijn I., Macauley M.S., Moody D.B, & Paulson J.C. (2014). Targeted delivery of mycobacterial antigens to human dendritic cells via Siglec-7 induces robust T cell activation. Journal of immunology (Baltimore, Md. : 1950), 193(4), 1560-1566.

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CAR T Cell Cytotoxicity and Activation Assay

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FR positive cancer cell lines (e.g., KB or MDA-MB-231 cells) were seeded at density of 10⁴ cells/100 μl media into 96 well plates and grown overnight. Anti-fluorescein CAR T cells were added to each well in the absence or presence of bispecific adapters. After co-incubation for 6–24 h, plates were centrifuged at 350xg for 10 min to remove debris and supernatants were analyzed for lactate dehydrogenase release (cell death analysis) using Pierce^TM LDH cytotoxicity assay kit (Thermo Fisher Scientific, MA) and interferon γ (IFNγ) levels using human IFNγ ELISA kit (Biolegend, CA), while pellets were either evaluated for CAR T cell activation by staining with anti-human CD69 APC (1:100 dilution, Biolegend, catalog#: 310910) or examined for CAR T cell proliferation by culturing for 5 days in TexMACS^TM medium (Miltenyi Biotech Inc, CA) containing 1% penicillin and streptomycin sulfate and quantitating by flow cytometry using the intrinsic GFP fluorescence and staining with anti-human CD3 APC antibody (1:100 dilution, Biolegend, catalog#: 300312).

Lee Y.G., Chu H., Lu Y., Leamon C.P., Srinivasarao M., Putt K.S, & Low P.S. (2019). Regulation of CAR T cell-mediated cytokine release syndrome-like toxicity using low molecular weight adapters. Nature Communications, 10, 2681.

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Inducing CAR T cell activity

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Anti-CD19 CAR T cells coexpressing either FITC-FR or FKBP-FR were cocultured with CD19-expressing Raji cells at an effector : target cell ratio of 5:1. FITC-FK506 or FK506-FK506 was then added to the coculture and the incubation was continued for an additional 2 hours in the absence or presence of competing ligands (FITC-glucosamine or FK506-glucosamine). Cells were then washed, supplemented with fresh medium and the incubation was continued for an additional 12 hours. Culture plates were then centrifuged at 350 × g for 10 min, and supernatants were assayed for both cancer cell death by evaluating released lactate dehydrogenase activity using CytoONE (Promega) and IFNγ levels using human IFNγ ELISA kit (BioLegend).

Zhang B., Napoleon J.V., Liu X., Luo Q., Srinivasarao M, & Low P.S. (2020). Sensitive manipulation of CAR T cell activity using a chimeric endocytosing receptor. Journal for Immunotherapy of Cancer, 8(2), e000756.

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Activation of CD4+ T cells by VSIG3

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For PBMCs or human CD4⁺ T cell cultures, 96-well flat-bottom plates (ThermoFisher, Cat#AB0751) were coated with anti-human CD3 antibody (clone OKT3, Biolegend, Cat#317326) at 1 μg/mL mixed together with 0 μg/mL, 2.5 μg/ml, 5 μg/mL, 10μg/mL or 20μg/mL of the human VSIG3-ECD protein in PBS at 4°C overnight. The PBMCs or human CD4⁺ T cells were plated at a density of 1×10⁵ cells/well in complete RPMI medium. The cell supernatants were harvested after 48 h for the cytokine measurement by human IFN-γELISA kit (Biolegend, Cat#430104) and human TNF-αELISA kit (Biolegend, Cat#430204).

Xie X., Chen C., Chen W., Jiang J., Wang L., Li T., Sun H, & Liu J. (2021). Structural Basis of VSIG3: The Ligand for VISTA. Frontiers in Immunology, 12, 625808.

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Quantify IFNγ in Breast Cancer Co-culture

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IFNγ produced in conditioned media after co-culture of various breast cancer cells with PBMC in the presence of trastuzumab or control IgG was measured in a 96-well plate using a human IFNγ ELISA kit from BioLegend as we recently described [14 (link)]. In brief, properly diluted conditioned media were added onto the wells coated with anti-IFNγ capture antibodies and then incubated for 2 h, followed by additional sequential incubations with an avidin-labeled IFNγ detection antibody and horseradish peroxidase-conjugated biotin. TMB (3,3′,5,5′-tetramethylbenzidine) was used as the substrate of horseradish peroxidase for color development.

Chaganty B.K., Qiu S., Gest A., Lu Y., Ivan C., Calin G.A., Weiner L.M, & Fan Z. (2018). Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNγ secretion. Cancer letters, 430, 47-56.

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Cytotoxicity Assay of NK Cells

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Target cells (K562, H929, MM.1R, U266, RPMI8226) were plated into a 96-well U-bottom plate at a density of 2 × 10⁴ cells/well and co-cultured with effector cells (Coc NK92, CAR NK92) at a ratio of 1:1 for 4 h at 37 °C. After 4 h of co-culturing, plates were centrifuged at 1000× g for 5 min and supernatants were harvested for ELISA. The concentration of IFN-γ in each well was determined using a human IFN-γ ELISA kit (#421203, Biolegend, San Diego, CA, USA). The concentration of granzyme B was determined using a human granzyme B ELISA kit (#DY2906, R&D system, Minneapolis, MN, USA). All groups were analyzed in duplicate.

Park E., Mun H.J., Seo E., Hwang S., Lee J.H., Song S., Sung H., Kim H.Y, & Kwon M.J. (2024). CAR NK92 Cells Targeting BCMA Can Effectively Kill Multiple Myeloma Cells Both In Vitro and In Vivo. Biomedicines, 12(1), 248.

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Measuring Serum IFN-γ Levels

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The concentration of serum IFN-γ was evaluated using Human IFN-γ ELISA Kit (BioLegend).

Ghannam K., Martinez Gamboa L., Kedor C., Spengler L., Kuckelkorn U., Häupl T., Burmester G, & Feist E. (2020). Response to abatacept is associated with the inhibition of proteasome β1i expression in T cells of patients with rheumatoid arthritis. RMD Open, 6(3), e001248.

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Measuring Cytokine Secretion by ELISA

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Human IFN-γ ELISA kit (Biolegend or MabTech) and granzyme B ELISA kit (MabTech) were used to measure cytokines secretion. Supernatants were collected from TICS, followed by centrifugation at 700 × g for 4 min to remove cell debris. After preparation of samples, ELISAs were conducted according to the manufacturers’ protocols. After measuring the absorbance at a wavelength of 450 nm and 570 nm, subtraction of 570 nm readings from those at 450 nm was performed on a CLARIOstar Plus instrument (BMG Labtech), followed by subtraction of an averaged background signal. IFN-γ and granzyme B concentrations were then calculated and plotted against different number of cancer cells using GraphPad software.

Papakyriacou I., Kutkaite G., Rúbies Bedós M., Nagarajan D., Alford L.P., Menden M.P, & Mao Y. (2024). Loss of NEDD8 in cancer cells causes vulnerability to immune checkpoint blockade in triple-negative breast cancer. Nature Communications, 15, 3581.

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Quantifying IFN-γ and IL-2 in T-cell Assays

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Interferon (IFN)-γ and IL-2 levels in supernatants from T-cell proliferation assays were quantified using the Human IFN-γ ELISA Kit (BioLegend 430107) and IL-2 ELISA kit (BioLegend 431815) based on provided directions. Samples were diluted to an appropriate concentration such that they fell within the linear range of the corresponding standard curves, which were used to calculate cytokine levels in these samples.

Kim Y.S., Lee S.H., Park A.H., Wu C., Hong B.K., Jung H., Lin S.H, & Yoo S.S. (2024). BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1. Journal for Immunotherapy of Cancer, 12(3), e008303.

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