Mouse Tumor Necrosis Factor (TNF) ELISA Kit, Lot 21AO-137 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P048)

1.Microplate: 96 breakable wells (12strips x 8wells) coated with anti-mouse TNF alpha.
2.20x Wash Buffer Concentrate: 1 Vial, 25 ml.
3.5x Assay Diluent: 1vial, 15 ml.
4.Standards: 2 vials, recombinant mouse TNF alpha.
5.Detection Antibody: 2 vials, biotinylated anti-mouse TNF alpha.
6.HRP-Streptavidin Concentrate: 1vial.
7.TBM Substrate solution: 1 Vial, 12 ml.
8.Stop Solution: 1 Vial, 8 ml of 0.2 M sulfuric acid.

1.Distilled or deionized water. 2.Precision pipettes, with disposable plastic tips. 3.Beakers, flasks, cylinders necessary for prepacanineion of reagents. 4.Microplate washing device (multichannel pipette or automated microplate washer). 5.Microplate shaker. 6.Microplate reader capable of reading at 450 nm.

40pg/mL

100 μL

4.5 h

Pre-coated

1.Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water.
2.Standard: Add 400 μL 1x Assay Diluent to prepare a 80 ng/mL standard. Add 400 μL 1x Assay Diluent to 7 tubes.Detection Ab: Add 100 μL of 1X Assay Diluent into the vial to prepare a detection antibody concentrate. The detection antibody concentrate should be diluted 80-fold with 1X Assay Diluent.
3.Streptavidin-HRP: HRP-Streptavidin concentrate should be diluted 350-fold with 1X Assay Diluent.
4.Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1: 2.

1.All reagents must be brought to room temperature (18-25 °C) prior to use.
2.Add 100 μL of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
3.Decant or aspirate contents of wells. Wash wells by filling with at least 300 μL/well prepared wash buffer followed by decanting/aspirating. Repeat wash 4 times for a total of 5 washes. After the last wash, blot plate on absorbent paper to remove residual buffer.
4.Add 100 μL of 1X prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking. Discard the solution. Repeat the wash as in step
5.Add 100 μL of prepared Streptavidin solution to each well. Incubate for 45 minutes at room temperature with gentle shaking.
6.Discard the solution. Repeat the wash as in step
7.Add 100 μL of TMB One-Step Substcaninee Reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking. Add 50 μL of Stop Solution to each well. Read absorbance at 450nm within 30 minutes of stopping the reaction.

Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with target concentration on the y-axis and absorbance on the x-axis.

Intra CV<10%, inter CV <15%

1.All reagents must be at room temperature (18 °C to 25 °C) before running assay.
2.Do not expose kit reagents to strong light during storage or incubation.
3.Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results.
4.Avoid contact of stop solution with skin or eyes. If contact occurs, immediately flush area with copious amounts of water.
5.Do not use TMB substance solution if it has begun to turn blue.
6.Do not expose bleach to work area during actual test procedure because of potential interference with enzyme activity.

4 °C,-20 °C

4°C/-20°C,May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.

4°C/-20°C,May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.

For Research Use only

Tumor Necrosis Factor Alpha (TNFa)

DIF; TNF-alpha; TNFA; TNFSF2; RATTNF; Tnfa; tnf; TNF-a; TNFalpha; Tnfsf1a; TNFa; cTNF; Tnf-alpha; tnfa-like; TNF-ALPHA; dif; tnfa; xtnf; tnfsf2; tnf-alpha; Cachectin; tumor necrosis factor; tumor necrosis factor b (TNF superfamily; member 2); tumor necrosis factor alpha; tumor necrosis factor a (TNF superfamily; member 2); TNF; Tnf; tnf; tnfb; tnf-alpha; LOC103694380; tnfa

21926

P06804

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

An antibody specific for canine TNF alpha was coated on a 96-well plate. Standards and samples are added to the wells and any TNF alpha present binds to the immobilized antibody. The wells are washed and biotinylated anti-canine TNF alpha antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is added to the wells. The wells are again washed and TMB substcaninee solution is added, which produces a blue color in direct proportion to the amount of TNF alpha present in the initial sample. The Stop Solution changes the color from blue to yellow, and the microwell absorbances are read at 450 nm.