Human tumor necrosis factor alpha (TNF-α) quantitative detection kit (ELISA)

This kit can only be used for scientific research, not for medical diagnosis.
Human tumor necrosis factor alpha (TNF-α) quantitative detection kit (ELISA)
Instruction Manual [Reagent Kit Name]
Human tumor necrosis factor alpha (TNF-α) quantitative detection kit (ELISA)
【Use of the kit】
Quantitative detection of tumor necrosis factor alpha (TNF-α) in human 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 human tumor necrosis factor alpha (TNF-α) monoclonal antibody transparent 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. The substrate (TMB) is converted into a blue product catalyzed by horseradish peroxidase (HRP) and turns yellow under the action of acid. The shade of the color and the human tumor necrosis factor The concentration of α (TNF-α) is positively correlated. The OD value is measured at a wavelength of 450 nm. Based on the OD value of the standard and the sample, the content of human tumor necrosis factor α (TNF-α) in the sample is calculated.
【Composition of the kit】
1 Enzyme label coated plate 12 well × 8 strips 7 Developer A solution 6mL
2 Standard (80ng / L) 0.5mL 8 Developer B solution 6mL
3 20 times concentrated washing solution 25mL 9 stop solution 6mL
4 Sample diluent 6mL 10 Instructions 1 copy
5 Standard diluent 6mL 11 sealing film 2 sheets
6 Enzyme-labeled reagent 6mL 12 sealed bag 1 Remarks: The standard product is diluted with the standard product dilution solution as follows: 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 [operation steps]
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 label coating plates, fix them on the frame, and set standard product holes,
Sample wells to be tested and blank control wells, record the positions of each well, add 50μL of the standard to the wells of the standard; add 10μL of the sample to be tested to the well, then add 40μL of the sample diluent (that is, the sample is diluted 5 times); blank control Holes are 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 hole with the washing solution, let stand for 1min, shake off the washing solution, pat dry on the absorbent paper, repeat the washing 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. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each hole with 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, then add 50μL of developer B solution, and mix for 30s with a plate mixer
(Or mix gently by hand for 30s), and develop color at 37 ° C in the dark 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: Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, 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. 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.
【Precautions】
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. If the color is too light, the substrate incubation time can be extended properly.
6. In order to avoid cross-contamination, the tips, samples and blank control should be replaced with a new one each time;
The sample diluent and substrate and other public components should be loaded with cantilever, and should not touch the micropores; the sealing film should not be reused.
7. The kit is used within the warranty period, and reagents of different batches should not be mixed.
8. Substrate B is sensitive to light and avoid prolonged exposure to light.

[Summary of operating procedures]
Prepare reagents, samples and standards Add the prepared samples and standards, wash the plate 4 times at 37 ° C for 30 minutes, add the enzyme reagent, wash the plate 4 times at 37 ° C for 30 minutes, add the coloring solutions A, B, 37 Develop for 15 minutes at ℃ and add stop solution
Calculate OD value within 15 minutes 【Detection range】
2.5ng / L- 80ng / L
【specification】
96 servings / box 【Storage】
Store at 2-8 ℃, protected from light and moisture.
【Validity】

6 months

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