Interferon gamma ELISA Kit

Produktnummer ABIN924768

Produktdetails Interferon gamma ELISA Kit

Target: Interferon gamma (IFNG)

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 46-3000 pg/mL

Untere Nachweisgrenze: 46 pg/mL

Applikation: ELISA

Verwendungszweck: The OmniKine? Human IFN-? ELISA Kit contains the components necessary for quantitative determination of natural or recombinant Human IFN-? concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a "Sandwich" Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a "sandwich" format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on Human IFN-? while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non- specific binding between proteins to other proteins or to the solid phase. After incubation and "sandwiching" of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3', 5, 5'-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Marke: OmniKine™

Proben: Cell Lysate, Serum, Plasma

Analytische Methode: Quantitative

Spezifität: The Human IFN-? ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human IFN-? proteins.

Kreuzreaktivität (Details): The Human IFN-γ ELISA is capable of recognizing both recombinant and naturally produced Human IFN-γ proteins. The antigens listed below were tested at 50 ng/mL and did not exhibit significant cross reactivity or interference. Human: IFN-λ1, IFN-λ2, IFN-β, IL-10, IL-12, IL-16, IL-17A, IL-17B, IL-17D, IL-17E, IL-17F Murine: IFN-λ2, IFN-γ, IL-10, IL-12 Rat: IFN-γ, IL-10

Produktmerkmale: The Human IFN-gamma ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human IFN-gamma proteins within the range of 46-3000 pg/mL.

Bestandteile:

• Microstrips Coated w / Capture Antibody: 12 x 8-Well Microstrips
• Protein Standard: Lyophilized (100 ng), Red container
• Biotinylated Detection Antibody: Lyophilized, Yellow container
• 400x Streptavidin-HRP: 30 μL, Blue container
• Wash Buffer (10x): 50 mL, Clear containter
• Assay Diluent: 50 mL, Clear container
• Ready-to-Use Substrate: 12 mL, Brown container
• Stop Solution: 12 mL, Clear container
• Adhesive Plate Sealers: 4 Sheets
• Technical Manual 1 Manual

Benötigtes Material: The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
Microplate reader able to measure absorbance at 450 nm (with correction wavelength set to 540 nm or 570 nm)
Micropipettes with capability of measuring volumes ranging from 1 μl to 1 mL
Deionized or sterile water
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent paper or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 mL
Bench
top centrifuge (optional)
Bench
top vortex (optional)
Orbital shaker (optional)

Anwendungsinformationen

Plattentyp: Pre-coated

Protokoll: This particular immunoassay utilizes the quantitative technique of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a sandwich format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on the Human IFN-gamma cytokine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and sandwiching of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3’, 5, 5’- Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Aufbereitung der Proben:

If samples are to be used within 24 hours, aliquot and store at 4 °C. If samples are to be used over a long period of time, aliquot and store between -20 °C and -80 °C, depending on the duration of storage.
Note: Samples containing a visible precipitate or pellet must be clarified prior to use in the assay.
Caution: Avoid repeated freeze/thaw cycles to prevent loss of biological activity of proteins in experimental samples.

• Cell Lysate and Supernatants:
  Remove large cell components via centrifugation and perform the assay. Cell lysates and supernatants require a dilution using Assay Diluent. A serial dilution may be performed to determine a suitable dilution factor for the sample. For future use of the sample, follow the sample storage guidelines stated above.
• Serum:
  Allow samples to clot in a serum separator tube (SST) for 30 minutes. After sufficient clotting, centrifuge at 1000 x g for 15 minutes and remove serum from SST in preparation for the assay. Serum samples require at least a 1:50 dilution using Assay Diluent. For future use of the sample, follow the storage guidelines above.
• Plasma:
  Use heparin, citrate or EDTA as an anticoagulant to gather plasma from original biological sample. After collection of the plasma, centrifuge for 15 minutes at 1000 x g. This step must be performed within 30 minutes of plasma collection. Plasma samples require at least a 1:50 dilution using Assay Diluent. Afterwards, perform the assay or for future use of the sample, follow the storage guidelines stated above.

Testdurchführung:

Note: If possible, all incubation steps should be performed on an orbital shaker to equilibrate solutions when added to the microplate wells. Also, all provided solutions should be at ambient temperature prior to use.
Note: Avoid adding solutions into wells at an angle, always keep pipette tip perpendicular to plate bottom.

Reconstitution of Provided Materials:

1. Reconstitute the Biotin-Conjugated Detection Antibody in 67 µL of ddH₂O for a concentration of 180 µg/ml.
2. Reconstitute the Protein Standard in 100 µL of ddH₂O for a concentration of 340 ng/ml.
3. Dilute the 50 mL of 10x Wash Buffer in 450 mL of ddH2O for 500 mL of 1x Wash Buffer.

Addition of Known Standard and Unknown Sample to Immunoassay:

The OmniKine™ Human CD163 ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human CD163 proteins

Ergebnisberechnung:

Generation of Standard Curve and Interpretation of Data
1. Average the duplicate or triplicate readings for each standard, control and sample and subtract the average zero standard optical density.
2. Generate a standard curve by using Microsoft Excel or other computer software capable of establishing a 4- Parameter Logistic (4-PL) curve fit. If using Excel or an alternative graphing tool, plot the average optical density values in absorbance units (y-axis) against the known standard concentrations in pg/ml (x-axis). Note: Only use the values in which a noticeable gradient can be established. Afterwards, generate a best fit curve or trend-line through the plotted points via regression analysis.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Vorsichtsmaßnahmen: Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Handhabung: This ELISA kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind.
Materials included in this kit should NOT be used past the expiration date on the kit label.
Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.
Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided.
The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.

Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Lagerung: 4 °C

Informationen zur Lagerung: Note: If used frequently, reagents may be stored at 4 °C.

• Unopened Kits: Store at 4 °C for 6 months.
• Microstrips Coated w/ Capture Antibody, 400x Streptavidin-HRP Wash Buffer (10x), Assay Diluent Ready-to-Use Substrate, Stop Solution: 6 Months at 4 °C
• Protein Standard, Biotinylated Detection Antibody: Lyophilized: 6 Months (if Reconstituted: 1 Month) at 4 °C

Details zu Interferon gamma

Target: Interferon gamma (IFNG)

Andere Bezeichnung: IFN-gamma (IFNG Produkte)

Synonyme: IFG ELISA Kit, IFI ELISA Kit, IFN-g ELISA Kit, Ifg ELISA Kit, IFNG2 ELISA Kit, IFN-gamma ELISA Kit, IFN-G ELISA Kit, IFNG ELISA Kit, IFNgamma ELISA Kit, TCRalpha ELISA Kit, INF-G ELISA Kit, ifng ELISA Kit, interferon gamma ELISA Kit, interferon, gamma 1-2 ELISA Kit, IFNG ELISA Kit, Ifng ELISA Kit, ifng1-2 ELISA Kit

Hintergrund: Human IFN-γ, also known as Interferon γ, is a 166 amino acid cytokine protein produced by lymphocytes activated by specific antigens or mitogens. The IFNG gene encodes the cytokine and is a member of the type II interferon family. IFN-γ is known to be a soluble cytokine with antiviral, immunoregulatory and anti-tumor properties and is a potent activator of macrophages. Mutations in this gene are often associated with aplastic anemia. AA is a rare disease in which the reduction of the circulating blood cells results from damage to the stem cell pool in bone marrow. In most patients, the stem cell lesion is caused by an autoimmune attack. T-lymphocytes, activated by an endogenous or exogenous, and most often unknown antigenic stimulus, secrete cytokines, including IFN- gamma, which would in turn be able to suppress hematopoiesis. IFN-γ, released by T lymphocytes, has anti-proliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons. Also, some post-translational modifications that take place with IFN-γ include proteolytic processing to produce C-terminal heterogeneity, with proteins ending alternatively at Gly-150, Met-157 or Gly-161. Source: Entrez Gene: IFNG interferon, gamma [Homo sapiens], Swiss-Prot: P01579

Gen-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, Autophagie