Human ifn γ elisa max deluxe kit

Manufactured by BioLegend

The Human IFN-γ ELISA MAX Deluxe kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the detection and quantification of human interferon-gamma (IFN-γ) in cell culture supernatants, serum, and plasma.

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8 protocols using human ifn γ elisa max deluxe kit

Quantifying Nebulized IFN-gamma Aerosol Delivery

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A known concentration (500, 250, 125 pg/mL) in 1 ml IFN-γ was nebulized using either the Aerogen Solo or PDAP mesh technology. The respective aerosols produced were captured directly in a sealed 50 mL tube. Following complete nebulization of the dose, the device was powered off, removed and the tube centrifuged (Hettich Rotina 35R Bench centrifuge, 5 mins, 10,000 rpm) in order to pool the sample prior to refrigeration at 4-8 °C. The concentration of delivered dose was determined using a Human IFN-γ ELISA MAX™ Deluxe kit (BioLegend, San Diego, CA 92121) as per kit instructions. The medication cup of both the Solo and PDAP were washed and residual volume calculated.

Sweeney L., McCloskey A.P., Higgins G., Ramsey J.M., Cryan S.A, & MacLoughlin R. (2019). Effective nebulization of interferon-γ using a novel vibrating mesh. Respiratory Research, 20, 66.

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Immunophenotyping and Cytokine Quantification

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APC anti-human CD56, FITC anti-human CD3, PE anti-human CD16, murine isotype controls (IgG1κ-PE, IgG1κ-FITC, IgG2a –APC) and human IFN-γ ELISA MAX™ Deluxe kit were purchased from BioLegend Inc. (San Diego, CA). The recombinant human IL-2 protein was obtained from PeproTech (Rehovot, Israel). Calcein-AM was purchased from Sigma-Aldrich (St, Louis, MO). CCK8 kit was purchased from YESAN (Shanghai, China).

Gong C., Ni Z., Yao C., Zhu X., Ni L., Wang L, & Zhu S. (2015). A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells. Biological Procedures Online, 17, 12.

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Cytokine Secretion Quantification by ELISA

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IFN-γ, IL2, and IL12
secretion was measured by collecting the supernatant from the respective
cell-based assay and frozen at −20 °C prior to analysis
by ELISA. IFN-γ, IL2, and IL12 ELISAs were carried out using
the Human IFN-γ ELISA MAX Deluxe kit, Human IL2 ELISA MAX Deluxe
kit, and Human IL12 (p70) ELISA MAX Deluxe kit in accordance with
the manufacturer’s instructions (BioLegend).

Jha R., Kinna A., Hotblack A., Bughda R., Bulek A., Gannon I., Ilca T., Allen C., Lamb K., Dolor A., Scott I., Parekh F., Sillibourne J., Cordoba S., Onuoha S., Thomas S., Ferrari M, & Pule M. (2024). Designer Small-Molecule Control System Based on Minocycline-Induced Disruption of Protein–Protein Interaction. ACS Chemical Biology, 19(2), 308-324.

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Effect of ADI-PEG 20 on PBMC Activation

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PBMCs from five different donors were tested in six repeat experiments. PBMCs were thawed, washed and rested overnight (18 h). The next day cells were collected, resuspended in AIM-V medium supplemented with Glutamax and counted. Cell concentration was adjusted to 2 × 10^6 cells per mL and 2 mL of cells were added per well of a 6-well plate. PBMCs were treated with 0, 0.8, 4 or 20 nM of ADI-PEG 20 in the presence or absence of CD3/CD28 Dynabeads or PHA. CD3/CD28 Dynabeads were prepared according to manufacturer's instructions. Cells were analyzed after 24 h, 48 h and 72 h of treatment.
Citrulline and arginine levels in the media were measured by LC/MS/MS.
IL-2 was measured by human IL-2 ELISA kit and IFNγ was measured by human IFNγ ELISA MAX Deluxe kit (BioLegend).

Brin E., Wu K., Lu H.T., He Y., Dai Z, & He W. (2017). PEGylated arginine deiminase can modulate tumor immune microenvironment by affecting immune checkpoint expression, decreasing regulatory T cell accumulation and inducing tumor T cell infiltration. Oncotarget, 8(35), 58948-58963.

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Tracking γδ T Cell Responses

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RPMI-1640, FBS, non-essential amino acids, pyruvate, penicillin streptomycin, HEPES, 2-ME, HMBPP and FITC-γδ TCR antibody (clone 5A6.E91) were obtained through Fisher Scientific. IL-2 and the MACS γδ T cell negative selection kit were obtained through Miltenyi. PE-CD3 (UCHT1), APC-TNFα (MAb11), PE-IFNγ (4S.B3), FITC-IL-2 (MQ1-17H12), APC-CD45RA (HI-100), PerCP-eFluor710-CD27 (LG.7F9), FITC-Annexin V and PerCP-eFluor710-Annexin V were from eBioscience. K562 cells were from ATCC while Research Blood Components (Boston, MA) supplied blood. DiD, calcein AM, pcDNA3.1 and DNA primers were obtained through Life Technologies, while CRISPR/Cas9 repair templates were obtained through IDT. Restriction enzymes including XhoI, EcoRV and NheI were obtained through New England Biolabs. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma. Anti-myc (9E10) antibody was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank at The University of Iowa. POM₂-C-HMBP was a kind gift of Dr. David Wiemer at the University of Iowa.

Kilcollins A.M., Li J., Hsiao C.H, & Wiemer A.J. (2016). HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors. Journal of immunology (Baltimore, Md. : 1950), 197(2), 419-428.

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Evaluating Viral Oncolytic Potency via NK Cell Assays

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In co-culture experiments (CC), 10⁴ target cells (TOV21G and OVCAR4 infected with dl922-947 [MOI 10] or enadenotucirev [MOI 100] for 48 h) were incubated with 10⁵ pNK cells or no-NK culture medium (RPMI with 10% FBS) for 24 h in a 96-well plate at a total volume of 200 μL (E:T ratio 10:1). In the conditioned medium experiments (CM), cell-free conditioned medium from mock-infected or virus-infected cells was added to 10⁵ pNK or no-NK culture medium (RPMI with 10% FBS) for 24 h in a 96-well plate at a total volume of 200 μL. At the end of the incubation period (24 h) for both groups (CC and CM), the plate was centrifuged (430 g, 5 min), and the supernatant collected for IFN-γ assessments by ELISA (Human IFN-γ ELISA MAX Deluxe kit, cat number 430104; Biolegend, San Diego, CA), according to the manufacturer’s instructions.

Leung E.Y., Ennis D.P., Kennedy P.R., Hansell C., Dowson S., Farquharson M., Spiliopoulou P., Nautiyal J., McNamara S., Carlin L.M., Fisher K., Davis D.M., Graham G, & McNeish I.A. (2020). NK Cells Augment Oncolytic Adenovirus Cytotoxicity in Ovarian Cancer. Molecular Therapy Oncolytics, 16, 289-301.

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ELISA-based IFNγ Quantification in ADCC

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Human IFNγ ELISA MAX™ Deluxe Kit (Biolegend, 430104) was used to measure IFNγ in the media four hours post ADCC exposure. ADCCS1 and ADCCR1 cells were plated in 96-well clear bottom plates (Corning, 3300) at 10,000 cells/well and incubated in culture conditions overnight at 37°C in 5% CO₂. The control wells were then exposed to either media, cetuximab (1 μg/mL), or NK92-CD16V cells at the indicated E:T ratios in the absence of antibody. The ADCC wells all were incubated with cetuximab (1 μg/mL) and NK92-CD16V cells reflecting E:T ratios of 0:1, 1:1, 2:1 and 4:1 by adding 0; 20,000; 40,000; and 80,000 NK cells, respectively, to the wells. After 4 hours incubation, the plates were spun down at 1000 × g for 5 minutes, and the supernatant was collected and transferred into a fresh round bottom plate. IFNγ detection in supernatants was done using the ELISA Max Deluxe Kit (Biolegend, Cat. No. 430105) according to the manufacturer’s instructions.

Aldeghaither D.S., Zahavi D.J., Murray J.C., Fertig E.J., Graham G.T., Zhang Y.W., O’Connell A., Ma J., Jablonski S.A, & Weiner L.M. (2018). A Mechanism of Resistance to Antibody-Targeted Immune Attack. Cancer immunology research, 7(2), 230-243.

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CD44+ Tumor Cell Cytokine Assay

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Tumor cells were sorted into CD44⁻ and CD44⁺ subsets on the day of the assay. 5×10⁴ CD8⁺ T cells and 5×10⁴ autologous sorted tumor cells were co-cultured (1:1) in 200µl of media for two days. Supernatants were collected and assayed for IFNγ levels in at least two replicates using the Human IFN-γ ELISA MAX™ Deluxe Kit (BioLegend). A standard curve was constructed and fitted using a sigmoidal 4-parameter logistics curve-fitting algorithm to determine the unknown analyte concentrations.

Lee Y., Shin J.H., Longmire M., Wang H., Kohrt H.E., Chang H.Y, & Sunwoo J.B. (2016). CD44+ cells in head and neck squamous cell carcinoma suppress T cell-mediated immunity by selective constitutive and inducible expression of PD-L1. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(14), 3571-3581.

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