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Human IFN-gamma ELISA - Quantikine QuicKit Best Seller

Bio-Techne includes R&D Systems | Catalog # QK285

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QK285

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Key Product Details

Assay Length

80 minutes

Sample Type & Volume Required Per Well

Cell Culture Supernates (50 µL), Serum (50 µL), EDTA Plasma (50 µL), Heparin Plasma (50 µL)

Sensitivity

2.56 pg/mL

Assay Range

31.3-2000 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma)

Product Summary for Human IFN-gamma Quantikine QuicKit ELISA

The Quantikine QuicKit Human IFN-gamma Immunoassay is a one step, 80-minute solid phase ELISA designed to measure human IFN-gamma levels in cell culture supernates, serum, and plasma. It contains HEK293-expressed recombinant human IFN-gamma and antibodies raised against the recombinant protein. Results obtained using natural human IFN-gamma showed linear curves that were parallel to the standard curves obtained using the QuicKit standards. These results indicate that this kit can be used to determine relative mass values for natural human IFN-gamma.

Product Specifications

Measurement

Quantitative ELISA

Detection Method

Colorimetric - 450nm (TMB)

Conjugate

HRP

Reactivity

Human

Cross-reactivity

< 0.5% cross-reactivity observed with available related molecules. < 50% cross-species reactivity observed with species tested.

Interference

No significant interference observed with available related molecules.

Sample Values

Serum/Plasma - Ten serum and plasma samples from apparently healthy volunteers were evaluated for the presence of human IFN-gamma in this assay. No medical histories were available for the donors used in this study. All samples measured less than the lowest standard, 31.3 pg/mL.

Cell Culture Supernates:

Peripheral blood mononuclear cells (multiple donors) (seeded at 1 x 106 /mL) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Cells were left untreated or treated with 10 μg/mL of PHA for 5 days. Aliquots of the cell culture supernates were removed and assayed for levels of human IFN-gamma.

CD4+ T cells were isolated from human PBMCs (single donor) using the MagCellect™ Human CD4+ T cell Isolation Kit (R&D Systems, Catalog # MAGH102). CD4+ T cells were then seeded at 5 x 105 /mL and cultured in ExCellerate Human T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030). T cells were left untreated or treated for 5 days. Treated T cell media was supplemented with 10 ng/mL GMP recombinant human (rh) IL-7 (R&D Systems, Catalog # 207-GMP) and 10 ng/mL GMP rhIL-15 (R&D Systems, Catalog # 247-GMP). TCR stimulation was mediated using either (1) immobilized Human CD3 epsilon Antibody (R&D Systems, Catalog # MAB100, coated at 1 μg/mL) with 5 μg/mL soluble Human CD28 Antibody (R&D Systems, Catalog # MAB342) or (2) 25 μL Cloudz CD3/28 particles (Cloudz™ T Cell Activation Kit-CD3/CD28, R&D Systems, Catalog # CLD001) per mL of culture media. Aliquots of the cell culture supernates were removed and assayed for levels of human IFN-gamma.

Condition(pg/mL)
UnstimulatedND
Stimulated PBMCs111,232
Unstimulated CD4+ T CellND
CD4+ T Cell CD3/CD2852,293
CD4+ T Cell Cloudz191,034
ND=Non-detectable

Precision

Intra-Assay Precision (Precision within an assay) Two samples of known concentration were tested twenty times on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Two samples of known concentration were tested in ten separate assays to assess inter-assay precision. Assays were performed by at least three technicians.

Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Serum

Intra-Assay Precision Inter-Assay Precision
Sample 1 2 1 2
n 20 20 10 10
Mean (pg/mL) 198 1156 211 1233
Standard Deviation 3.76 20.2 20.4 56.9
CV% 1.9 1.8 9.6 4.6

Recovery for Human IFN-gamma Quantikine QuicKit ELISA

The recovery of human IFN-gamma spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type Average % Recovery Range %
Cell Culture Media (n=5) 92 83-102
EDTA Plasma (n=4) 107 88-132
Serum (n=4) 93 74-114

Linearity

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human IFN-gamma were diluted with calibrator diluent to produce samples with values within the dynamic range of the assay. Samples were diluted prior to assay.

Scientific Data Images for Human IFN-gamma Quantikine QuicKit ELISA

Human IFN-gamma QuicKit ELISA Spiked Recovery Competitor Comparison

IFN-gamma is spiked at three known concentrations throughout the range of the assay and run to measure response of the spiked sample matrix. Serum recovery is 115% compared to 144% for the top competitor. Plasma recovery is 120% compared to 135% for the top competitor. Cell Culture Supernates recovery is 118% compared to 79% for the top competitor. In spike and recovery experiments, natural samples are spiked with the recombinant target analyte of interest to identify interference caused by sample matrices.

Human IFN-gamma QuicKit ELISA Spiked Linearity Competitor Comparison

IFN-gamma is spiked at high concentration in various matrices and diluted with appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay. The linearity is between 99%-126% compared to 71%-85% for the top competitor.

Human IFN-gamma Standard Curve

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: IFN-gamma

IFN-gamma (Interferon-gamma) is the prototype proinflammatory cytokine and is produced by a variety of immune cells under inflammatory conditions, notably by T cells and NK cells. It plays a key role in host defense by promoting the development and activation of Th1 cells, chemoattraction and activation of monocytes and macrophages, upregulation of antigen presentation molecules, and immunoglobulin class switching in B cells. It also exhibits antiviral, antiproliferative, and apoptotic effects. In addition, IFN-gamma functions as an anti-inflammatory mediator by promoting the development of regulatory T cells and inhibiting Th17 cell differentiation. IFN-gamma dimers signal through a receptor complex of two IFN-gamma R1 and two IFN-gamma R2 subunits.

Long Name

Interferon gamma

Alternate Names

IFG, IFI, IFNG, IFNgamma

Entrez Gene IDs

3458 (Human); 15978 (Mouse); 25712 (Rat); 396991 (Porcine); 281237 (Bovine); 403801 (Canine); 493965 (Feline)

Gene Symbol

IFNG

Additional IFN-gamma Products

Product Documents for Human IFN-gamma Quantikine QuicKit ELISA

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human IFN-gamma Quantikine QuicKit ELISA

For research use only

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  • Human IFN-gamma Quantikine QuicKit ELISA
    Name: Anonymous
    Sample Tested: Urine
    Verified Customer | Posted 11/16/2022
    Quick, easy to follow protocol. Urine tested at 2-fold dilution and did not observe a matrix effect.
    Human IFN-gamma Quantikine QuicKit ELISA QK285

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Protocols

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