TNF alpha ELISA Kit

Catalog No. ABIN612749

Product Details TNF alpha ELISA Kit

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 15 pg/mL

Application: ELISA

Purpose: The AssayMax Mouse TNF-alpha ELISA kit is designed for detection of TNF-alpha in mouse plasma, serum or cell culture supernatants

Brand: AssayMax

Sample Type: Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Specificity: This assay recognizes both natural and recombinant mouse TNF-alpha.

Components: TNF-alpha Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against TNF-alpha. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. TNF-alpha Standard: Mouse TNF-alpha in a buffered protein base (2 ng, lyophilized). Biotinylated Mouse TNF-alpha Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against mouse TNF-alpha (80µl). MIx Diluent Concentrate (10x): A 10-fold 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

Application Details

Sample Volume: 50 μL

Assay Time: < 5 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures TNF-alpha in less than 5 hours. A polyclonal antibody specific for mouse TNF-alpha has been pre-coated onto a microplate. TNF-alpha in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for mouse TNF-alpha, 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. MIx Diluent Concentrate (20x): Dilute the MIx Diluent Concentrate 1:20 with reagent grade water. Store for up to 1 month at 2-8°C. 2 Standard Curve: Reconstitute the 2 ng of mouse TNF-alpha Standard with 1 ml of MIx Diluent to generate a 2 ng/ml of standard solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the TNF-alpha standard solution (2ng/ml) twofold with equal volume of MIx Diluent to produce 1, 0.5, 0.25, 0.125, 0.0625 and 0.0313 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [TNF-alpha] (ng/ml) P1 1 part Standard (2ng/ml) 2.000 P2 1 part P1 + 1 part MIx Diluent 1.000 P3 1 part P2 + 1 part MIx Diluent 0.500 P4 1 part P3 + 1 part MIx Diluent 0.250 P5 1 part P4 + 1 part MIx Diluent 0.125 P6 1 part P5 + 1 part MIx Diluent 0.063 P7 1 part P6 + 1 part MIx Diluent 0.031 P8 MIx Diluent 0.000 Biotinylated Mouse TNF-alpha Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx 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 MIx 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. Store the remaining samples at -20°C or below. 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 and assay. 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. The samples can be stored 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 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 Mouse TNF-alpha Antibody to each well and incubate for two hours. Wash a microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate per 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 approximately 10 minutes or till the optimal blue color density develops. Gently tap the 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. 3 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.

Calculation of Results:

Calculate the mean value of the 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 4.5% and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: Prepare all reagents (working diluent buffer, wash buffer, standards, 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 protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. 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 MIx 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 with zip-seal. May be stored for up to 1 month in a vacuum desiccator.

References for TNF alpha ELISA Kit (ABIN612749)

Sellmann, Priebs, Landmann, Degen, Engstler, Jin, Gärttner, Spruss, Huber, Bergheim: "Diets rich in fructose, fat or fructose and fat alter intestinal barrier function and lead to the development of nonalcoholic fatty liver disease over time." in: The Journal of nutritional biochemistry, (2015) (PubMed).

Sellmann, Jin, Degen, De Bandt, Bergheim: "Oral Glutamine Supplementation Protects Female Mice from Nonalcoholic Steatohepatitis." in: The Journal of nutrition, (2015) (PubMed).

Target Details for TNF alpha

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Alternative Name: Tumor Necrosis Factor alpha (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: Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine with a myriad of innate immune anti-tumor properties. TNF-alpha has a critical role in the bone and cartilage damage associated with rheumatoid arthritis (RA)[1]. TNF-alpha may be involved in the pathogenesis and/or progression of gestational diabetes mellitus (GDM) [2]. TNF-alpha is expressed in myocardium during compensated pressure-overload hypertrophy and contributes to postischemic myocardial dysfunction [3]. The serum levels of TNF-alpha were also significantly elevated in active WG (Wegener's granulomatosis) [4], in the late stages of HIV-associated disease [5], and in the spinal cord of arthritic patients [6].

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