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TNF alpha ELISA Kit

TNF alpha Reactivity: Mouse Colorimetric Sandwich ELISA 16-2000 pg/mL Cell Lysate, Plasma, Serum
Catalog No. ABIN924831
  • Target See all TNF alpha ELISA Kits
    TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))
    Reactivity
    • 14
    • 12
    • 9
    • 5
    • 4
    • 4
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Mouse
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    16-2000 pg/mL
    Minimum Detection Limit
    16 pg/mL
    Application
    ELISA
    Purpose
    The OmniKine? Murine TNF-? ELISA Kit contains the components necessary for quantitative determination of natural or recombinant Murine TNF-? 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 Murine TNF-? 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.
    Brand
    OmniKine™
    Sample Type
    Cell Lysate, Serum, Plasma
    Analytical Method
    Quantitative
    Specificity
    The Murine TNF-? ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Murine TNF-? proteins.
    Cross-Reactivity (Details)
    The Murine TNF-α ELISA is capable of recognizing both recombinant and naturally produced Murine TNF-α proteins. The antigens listed below were tested at 50 ng/mL and exhibited 100% cross reactivity. Rat: TNF-α The antigens listed below were tested at 50 ng/mL and did not exhibit significant cross reactivity or interference. Human: 4-1BBL, CD40L, FasL, IL-2, sRANKL, TNF-α, TNF-β, sTNF-RI, sTNF-RII, sTRAIL, TWEAK, VEGF Murine: IL-2, CD40L, sRANKL, sTRAIL, VEGF
    Characteristics
    The Murine TNF-alpha ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Murine TNF-alpha proteins within the range of 16-2000 pg/mL.
    Components
    • 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
    Material not included
    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)
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  • Plate
    Pre-coated
    Protocol
    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 Murine TNF-alpha 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.
    Sample Preparation

    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.

    Assay Procedure

    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

    Calculation of Results

    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.

    Restrictions
    For Research Use only
  • Precaution of Use
    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.
    Handling Advice
    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.
    Storage
    4 °C
    Storage Comment
    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
  • Target See all TNF alpha ELISA Kits
    TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))
    Alternative Name
    TNF-alpha (TNF alpha Products)
    Synonyms
    DIF ELISA Kit, TNF-alpha ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit, RATTNF ELISA Kit, Tnfa ELISA Kit, tnf ELISA Kit, TNF-a ELISA Kit, TNFalpha ELISA Kit, Tnfsf1a ELISA Kit, TNFa ELISA Kit, cTNF ELISA Kit, Tnf-alpha ELISA Kit, tnfa-like ELISA Kit, TNF-ALPHA ELISA Kit, dif ELISA Kit, tnfa ELISA Kit, xtnf ELISA Kit, tnfsf2 ELISA Kit, tnf-alpha ELISA Kit, Cachectin ELISA Kit, tumor necrosis factor ELISA Kit, tumor necrosis factor b (TNF superfamily, member 2) ELISA Kit, tumor necrosis factor alpha ELISA Kit, tumor necrosis factor a (TNF superfamily, member 2) ELISA Kit, TNF ELISA Kit, Tnf ELISA Kit, tnf ELISA Kit, tnfb ELISA Kit, tnf-alpha ELISA Kit, LOC103694380 ELISA Kit, tnfa ELISA Kit
    Background
    Murine TNF-α or Tumor Necrosis Factor α, also known as Cachectin, is a 235 amino acid cytokine protein expressed from the Tnf gene located on chromosome 17. The 235 residue chain exists primarily as the membrane form of TNF-α whereas the 156 residue form, created by proteolytic processing from the N-terminal end, exists primarily as the soluble form. The membrane form contains a 35 residue cytoplasmic topological domain, a 21 residue helical membrane domain, and a 179 residue extracellular topological domain. TNF-α is a cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. The cytokine is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia. Under certain conditions, it can stimulate cell proliferation and induce cell differentiation. Source: Entrez Gene: Tnf tumor necrosis factor [Mus musculus], Swiss- Prot: P06804
    Gene ID
    21926
    UniProt
    P06804
    Pathways
    NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, 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, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome
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