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Interferon (IFN) ELISA Kit

Details for Product No. ABIN612715, Supplier: Log in to see
Methode Type
Sandwich ELISA
Minimum Detection Limit
16 pg/mL
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Purpose The AssayMax Human IFN- ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human IFN- in plasma, serum and cell culture samples
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Components Human IFN- Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human IFN- . 1 Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human IFN- Standard: Human IFN- in a buffered protein base (2 ng, lyophilized). Biotinylated IFN- Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against IFN- (80µl). 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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water
Alternative Name Interferon- (IFN- )
Background Interferon- (IFN- ) 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- is a homodimer consisting of two 143-amino-acid polypeptides with 20 kDa and 25 kDa. By binding to the receptors IFNGR1 & IFNGR2, IFN- activates the tyrosine kinase JAK-STAT pathway. While protecting against tumor development and cancer immunoediting, IFN- function is significant in tumor surveillance. Aside from functions in host defense, IFN- may contribute to autoimmune pathology. In humans, IFN- is implicated in pathology of diseases such as systemic lupus erythematosus , multiple sclerosis , and insulin-dependent diabetes mellitus. Therapeutically, IFN- administration enhances bone resorption and leukocyte function in patients with osteopetrosis.
Sample Volume 50 μL
Assay Time < 4 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IFN- in less than 4 hours. A polyclonal antibody specific for human IFN- has been pre-coated onto a 96-well microplate with removable strips. IFN- in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for IFN- , which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 2 ng of IFN- Standard with 2 ml of EIA Diluent to generate a solution of 1 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting 2 the standard solution (1 ng/ml) 1:2 with EIA Diluent to produce 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.016 ng/ml solutions. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [IFN- ] (ng/ml) P1 Standard (1 ng/ml) 1.000 P2 1 part P1 + 1 part EIA Diluent 0.500 P3 1 part P2 + 1 part EIA Diluent 0.250 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 0.000 Biotin IFN- Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.

Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. 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 anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. 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 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Assay Procedure

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of IFN- standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated IFN- Antibody to each well and incubate for two hours. Wash a microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 15 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. 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 3 unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision Intra-assay and inter-assay coefficients of variation were 4.4 % and 7.3 % respectively.
Restrictions For Research Use only
Handling Advice The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
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).