Cow Tumor Necrosis Factor (TNF) ELISA Kit, Lot 21AO-149 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P060)

Pre-coated, ready to use 96-well strip plate
Plate sealer for 96 wells
Standard
Standard Diluent
Detection Reagent A
Detection Reagent B
Assay Diluent A
Assay Diluent B
TMB Substrate
Stop Solution
Wash Buffer (30 x concentrate)
Instruction manual

1.Microplate reader with 450 ± 10nm filter.
2.Precision single or multi-channel pipettes and disposable tips.
3.Eppendorf Tubes for diluting samples.
4.Deionized or distilled water.
5.Absorbent paper for blotting the microtiter plate.
6.Container for Wash Solution
7.0.01Mol/L (or 1x) Phosphate Buffered Saline(PBS), pH 7.0-7.2.

< 3.1 pg/mL

1.Bring all kit components and samples to room temperature (18-25 °C) before use.
2.Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently.
3.Dilute Detection Reagent A and Detection Reagent B to the working concentration 100-fold with Assay Diluent A and B, respectively.
4.Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5.TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

1.Add 100μL each of dilutions of standard, blank and samples into the appropriate wells, respectively. Cover with the Plate sealer. Incubate for 1 hour at 37 °C.
2.Remove the liquid of each well, don't wash.
3.Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and incubate for 1 hour at 37 °C.
4.Aspirate the solution and wash with 350μL of 1x Wash Solution to each well and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. Invert the plate and blot it against absorbent paper.
5.Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and incubate for 30 minutes at 37 °C.
6.Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7.Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at 37 °C. Protect from light.
8.Add 50μL of Stop Solution to each well. Mix the liquid by tapping the side of the plate.
9.Remove any drop of water and fingerprint on the bottom of the plate. Then, run the microplate reader and conduct measurement at 450nm immediately.

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-assay Precision (Precision within an assay): 3 samples with low, middle and high level of target were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level of target were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV < 10%
Inter-Assay: CV < 12%

4 °C,-20 °C

For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.

6 months

For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.

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

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

1.Prepare all reagents, samples and standards,
2.Add 100μL standard or sample to each well. Incubate 1 hours at 37 °C,
3.Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
4.Aspirate and wash 3 times,
5.Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
6.Aspirate and wash 5 times,
7.Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
8.Add 50μL Stop Solution. Read at 450nm immediately.