Instructions for Quantitative Detection Kit (ELISA) for Canine Tumor Necrosis Factor α (TNF-α)

This kit can only be used for scientific research, not for medical diagnosis.
Canine tumor necrosis factor alpha (TNF-α) quantitative detection kit (ELISA)
user's Guide
【Kit name】
Canine tumor necrosis factor alpha (TNF-α) quantitative detection kit (ELISA)
【Use of the kit】
Quantitative detection of tumor necrosis factor alpha (TNF-α) in dog serum, plasma and related liquid samples.
【Detection principle】
This kit uses double-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the test sample to the pre-coated canine tumor necrosis factor alpha (TNF-α) monoclonal antibody clear enzyme label coated plate, after incubation for a sufficient time, wash to remove unbound components, and then add the enzyme label After incubating the working solution for a sufficient period of time, wash to remove unbound components. Substrates A and B are added in sequence, and the substrate (TMB) is converted into a blue product under the catalysis of horseradish peroxidase (HRP), which turns yellow under the action of an acid. The shade of the color and the canine tumor necrosis factor in the sample The concentration of α (TNF-α) was positively correlated. The OD value was measured at a wavelength of 450 nm. Based on the OD value of the standard and the sample, the content of canine tumor necrosis factor α (TNF-α) in the sample was calculated.
【Composition of the kit】
Enzyme coated plate
12 holes × 8
Developer A liquid
0.3mL × 6 tubes
Developer B liquid
20 times concentrated washing liquid
Stop solution
Sample diluent
1 serving
Special diluent
Sealing film
2 sheets
Enzyme reagent
sealed bag
Remarks: The concentration of standard products (No. 1 → No. 6) is: 80, 40, 20, 10, 5, 2.5ng / L.
[Reagents and equipment needed but not provided]
1. 37 ℃ thermostat
2. Standard specification microplate reader
3. Precision pipettes and disposable tips
4. Distilled water
5. Disposable test tubes
6. Absorbent paper
1. Preparation: Remove the reagent kit from the refrigerator and re-equilibrate at room temperature for 30 minutes.
2. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water to the original one.
3. Add standard products and samples to be tested: take a sufficient number of enzyme-coated plates and fix them to the frame. Set up standard wells, sample wells to be tested and blank control wells, record the positions of each well in the standard wells. Add 50μL of standard product; first add 10μL of the sample to be tested, then add 40μL of sample diluent (that is, the sample is diluted 5 times); the blank control well is not added.
4. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes.
5. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat the washing plate 4 times (you can also use the washing machine to press Instructions for washing the board).
6. Add enzyme-labeled working solution: add 50μL of enzyme-labeled working solution to each well, without adding blank control wells.
7. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes.
8. Plate washing: Discard the liquid, pat dry on the absorbent paper, fill each hole with the washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat the plate washing 4 times (you can also use the washing machine to press Instructions for washing the board).
9. Color development: add 50μL of developer A solution to each well, and then add 50μL of developer B solution. Mix with a plate mixer for 30s (or shake gently by hand for 30s), and avoid color development at 37 ℃ for 15min .
10. Termination: Remove the enzyme labeling plate and add 50μL of stop solution to each well to stop the reaction (the color changes from blue to yellow).
11. Determination: Zero the blank holes, and within 15 minutes after termination, measure the absorbance (OD value) of each well with a wavelength of 450 nm.
12. Calculation: According to the concentration of the standard product and the corresponding OD value, calculate the linear regression equation of the standard curve, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration times the dilution factor.
[Sample requirements]
1. The sample cannot contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP).
2. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
3. The sample should be fully centrifuged, without hemolysis and particles.
1. The experiment is carried out in strict accordance with the instructions, and the result of the experiment must be determined by the reading of the microplate reader.
2. If the enzyme-labeled coated board is not used up after opening, it should be put into a sealed bag and added with desiccant immediately.
3. It is recommended that all standards, samples and blank controls be tested in duplicate, and the average value is taken to reduce the experimental error.
4. Keep in mind that the sample has been diluted 5 times. The calculation result is multiplied by 5 to obtain the actual concentration of the sample.
5. The quantitative range of this kit is 2.5-80ng / L, beyond this range, it is calculated by the extension of the standard curve, not as an accurate quantitative result, please dilute it with a special diluent to determine the accurate result (within the range of 2.5-80ng / L) ), Multiplied by the total dilution factor is the final concentration of the sample.
6. If the color is too light, the substrate incubation time can be extended properly.
7. In order to avoid cross-contamination, the tips, samples and blank controls should be replaced with a new one for each addition; the common components such as enzyme working solution, sample diluent and substrate should be cantilevered, and they should not touch the microwells. ; Do not reuse the sealing film.
8. The kits are used within the warranty period, and different batches of reagents should not be mixed.
9. Substrate B is sensitive to light and avoid prolonged exposure to light.
[Summary of operating procedures]
Prepare reagents, samples and standards

Add prepared samples and standards, and react at 37 ℃ for 30 minutes
Wash the plate 4 times, add the enzyme reagent, and react at 37 ℃ for 30 minutes
Wash the plate 4 times, add color developing solutions A and B, and develop at 37 ℃ for 15 minutes
Add stop solution
Read OD value within 15 minutes
【examination range】
2.5ng / L-80ng / L
96T / box
Store at 2-8 ℃, protected from light and moisture.
6 months

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