Fish Tumor Necrosis Factor (TNF) ELISA Kit, Lot 21AO-158 [Cancer Immune Checkpoint Assay Kit]

The kit is designed for in vitro quantitative measurement of Fish Tumor Necrosis Factor (TNF) in Serum, Plasma, Tissue Homogenate.

For the quantitative determination of fish tumor necrosis factor alpha (TNF-alpha) concentrations in serum, plasma, tissue homogenates.

ELISA

The supplier is only responsible for the kit itself, but not for the samples consumed during the assay.
Please predict the concentration before assaying.
If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
Owing to the possibility of mismatching between antigens from another resource and antibodies used in this supplier's kits, some native or recombinant proteins from other manufacturers may not be recognized by this supplier's products.
Influenced by factors including cell viability, cell number and cell sampling time, samples from cell culture supernatant may not be recognized by the kit.
Fresh samples without long time storage are recommended for the test.

Detection wavelength: 450 nm
Information on standard material: Depending on the antigen to be detected, standards can be either native or recombinant protein.
Information on reagents: In most cases the stop solution provided is 1 N H2SO4.
Information on antibodies: The antibodies provided in different kits vary in regards to clonality and host.

Tumor Necrosis Factor (TNF)

Fish

Colorimetric

Competition ELISA

Quantitative

Serum, Plasma, Tissue Homogenate

This assay has high sensitivity and excellent specificity for detection of fish TNF-alpha.

Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between the target antigen and all analogues for other species. Therefore, cross reaction may still exist.

Assay plate (12 × 8 coated Microwells)
Standard (freeze dried)
Biotin-antibody (100 × concentrate)
HRP-avidin (100 × concentrate)
Biotin-antibody Diluent
HRP-avidin Diluent
Sample Diluent
Wash Buffer (25 × concentrate)
TMB Substrate
Stop Solution
Adhesive Strip (for 96 wells)
Instruction manual

Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.
An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.
Squirt bottle, manifold dispenser or automated microplate washer.
Absorbent paper for blotting the microtiter plate.
100mL and 500mL graduated cylinders.
Deionized or distilled water.
Pipettes and pipette tips.
Test tubes for dilution.

125 pg/mL

50 μL

1 - 4.5 h

Pre-coated

Biotin-antibody (1×) - Centrifuge the vial before opening.
HRP-avidin (1×) - Centrifuge the vial before opening.
Wash Buffer (1×) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved.
Standard - Centrifuge the standard vial at 6000-10000rpm for 30s.

1.Add 100µL of standard or sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C.
2.Remove the liquid of each well, don't wash.
3.Add 100µL of Biotin-antibody (1×) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C.
4.Aspirate each well and wash, repeating the process two times for a total of three washes.After the last wash, remove any remaining wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5.Add 100µL of HRP-avidin (1×) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
6.Repeat the aspiration/wash process for five times as in step 6.
7.Add 90µL of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Protect from light.
8.Add 50µL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
9.Determine the optical density of each well within 5 minutes using a microplate reader set to 450nm.

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.

Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.
Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.
Intra-assay: CV% less than 8%
Inter-assay: CV% less than 10%

The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.
Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

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.

6 months

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

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

The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Standards or samples are added to the appropriate microtiter plate wells with an antibody specific for TNF-alpha and Horseradish Peroxidase (HRP) conjugated TNF-alpha. The competitive inhibition reaction is launched between with HRP labeled TNF-alpha and unlabeled TNF-alpha with the antibody. A substrate solution is added to the wells and the color develops in opposite to the amount of TNF-alpha in the sample. The color development is stopped and the intensity of the color is measured.