IFNG ELISA Kit (Interferon gamma)

Details for Product IFNG ELISA Kit No. ABIN612715, Supplier: Log in to see
Antigen
  • IFNG2
  • IFN-g
  • Ifg
  • IFN-G
  • IFN-gamma
  • IFNG
  • IFG
  • IFI
  • interferon gamma
  • interferon gamma-like
  • interferon, gamma
  • Ifng
  • IFNG
  • LOC101337436
Alternatives
Human IFNG ELISA Kit
Reactivity
Human
Alternatives
Kits with alternative reactivity to:
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Method Type
Sandwich ELISA
Detection Range
0.016-1 ng/mL
Minimum Detection Limit
0.016 ng/mL
Application
ELISA
Options
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Purpose The AssayMax™ Human Interferon-gamma ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of IFN-gamma in human plasma, serum, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IFN-gamma in approximately 5 hours. A polyclonal antibody specific for human IFN- gamma has been pre-coated onto a 96-well microplate with removable strips. IFN-gamma in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human IFN- gamma, which is recognized by a streptavidin-peroxidase (SP) conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Brand AssayMax™
Sample Type Cell Culture Cells, Plasma, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Components Human IFN-gamma Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human IFN- gamma. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human IFN-gamma Standard: Human IFN-gamma in a buffered protein base (2 ng, lyophilized). Biotinylated Human IFN-gamma Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human IFN-gamma (120 μl). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C)
Alternative Name Interferon-gamma (IFN-gamma) (IFNG ELISA Kit Abstract)
Background Interferon-gamma (IFN-gamma) is a highly pleiotropic protein secreted mainly by activated T-lymphocytes and natural killer cells. It is involved in a wide range of physiological processes, including antiviral, immunoregulatory and anti-tumour properties, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes (1-3). IFN-gamma is a homodimer consisting of two 143-amino-acid polypeptides [20 kDa and 25 kDa, respectively] (4). By binding to the receptors IFNGR1 & IFNGR2, IFN- gamma activates the tyrosine kinase JAK-STAT pathway (5). While protecting against tumor development and cancer immunoediting, IFN-gamma function is significant in tumor surveillance (6). Aside from functions in host defense, IFN-gamma may contribute to autoimmune pathology (7-10).
Gene ID 3458
UniProt P01579
Pathways Interferon-gamma Pathway, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, ER-Nucleus Signaling, Regulation of Carbohydrate Metabolic Process, Protein targeting to Nucleus, Autophagy
Sample Volume 50 μL
Assay Time 4 h
Plate Pre-coated
Protocol
  • Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
  • Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 2 hours.
  • Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
  • Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 25 minutes.
  • Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human IFN-gamma Standard: Reconstitute the Human IFN-gamma Standard (2 ng) with 2 mL of EIA Diluent to generate a 1 ng/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (1 ng/mL) 2-fold with equal volume of EIA Diluent to produce 0.5, 0.25, 0.125, 0.063, 0.031, and 0.016 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be stored at -20 °C and used within 7 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [IFN-gamma] (ng/mL) P1 1 part Standard (1 ng/mL) 1.0 P2 1 part P1 + 1 part EIA Diluent 0.5 P3 1 part P2 + 1 part EIA Diluent 0.25 P4 1 part P3 + 1 part EIA Diluent 0.125 P5 1 part P4 + 1 part EIA Diluent 0.063 P6 1 part P5 + 1 part EIA Diluent 0.031 P7 1 part P6 + 1 part EIA Diluent 0.016 P8 EIA Diluent 0.0 Biotinylated Human IFN-gamma Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with EIA Diluent to produce a 1x solution. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with EIA Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °C. 5

Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and collect plasma. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles.
Assay Procedure

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human IFN-gamma Standard or sample to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human IFN-gamma Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Wash the microplate as described above. Add 50 l of SP Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 25 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings. 6

Calculation of Results
  • Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
  • To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
  • Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.
Assay Precision Intra-assay and inter-assay coefficients of variation were 4.4 % and 7.3 % respectively.
Restrictions For Research Use only
Handling Advice This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. 2 Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent. 3
Supplier Images
ELISA image for IFNG ELISA Kit (Interferon gamma) (ABIN612715) Interferon gamma (IFNG) ELISA Kit
Product cited in: Shawki, Gaafar, Erfan, El Khateeb, El Sheikhah, El Hawary: "Immunomodulatory effects of umbilical cord-derived mesenchymal stem cells." in: Microbiology and immunology, Vol. 59, Issue 6, pp. 348-56, 2015 (PubMed).

Taylan, Sari, Akinci, Bilge, Kozaci, Akar, Colak, Yalcin, Gunay, Akkoc: "Biomarkers and cytokines of bone turnover: extensive evaluation in a cohort of patients with ankylosing spondylitis." in: BMC musculoskeletal disorders, Vol. 13, pp. 191, 2012 (PubMed).

Background publications Schoenborn, Wilson: "Regulation of interferon-gamma during innate and adaptive immune responses." in: Advances in immunology, Vol. 96, pp. 41-101, 2007 (PubMed).

Schroder, Hertzog, Ravasi, Hume: "Interferon-gamma: an overview of signals, mechanisms and functions." in: Journal of leukocyte biology, Vol. 75, Issue 2, pp. 163-89, 2004 (PubMed).

Baechler, Batliwalla, Karypis, Gaffney, Ortmann, Espe, Shark, Grande, Hughes, Kapur, Gregersen, Behrens: "Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, Issue 5, pp. 2610-5, 2003 (PubMed).

Ikeda, Old, Schreiber: "The roles of IFN gamma in protection against tumor development and cancer immunoediting." in: Cytokine & growth factor reviews, Vol. 13, Issue 2, pp. 95-109, 2002 (PubMed).

Boehm, Klamp, Groot, Howard: "Cellular responses to interferon-gamma." in: Annual review of immunology, Vol. 15, pp. 749-95, 1997 (PubMed).

Kaplan, Greenlund, Tanner, Shaw, Schreiber: "Identification of an interferon-gamma receptor alpha chain sequence required for JAK-1 binding." in: The Journal of biological chemistry, Vol. 271, Issue 1, pp. 9-12, 1996 (PubMed).

Key, Ries, Rodriguiz, Hatcher: "Recombinant human interferon gamma therapy for osteopetrosis." in: The Journal of pediatrics, Vol. 121, Issue 1, pp. 119-24, 1992 (PubMed).

Sarvetnick, Liggitt, Pitts, Hansen, Stewart: "Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MHC and interferon-gamma." in: Cell, Vol. 52, Issue 5, pp. 773-82, 1988 (PubMed).

Panitch, Hirsch, Haley, Johnson: "Exacerbations of multiple sclerosis in patients treated with gamma interferon." in: Lancet, Vol. 1, Issue 8538, pp. 893-5, 1987 (PubMed).

Rinderknecht, OConnor, Rodriguez: "Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation." in: The Journal of biological chemistry, Vol. 259, Issue 11, pp. 6790-7, 1984 (PubMed).