Instruction Manual of Quantitative Detection Kit (ELISA) for Rat Nitric Oxide (NO)

This kit can only be used for scientific research, not for medical diagnosis.
Rat Nitric Oxide (NO) Quantitative Detection Kit (ELISA)
user's Guide
[Rat Nitric Oxide (NO) Elisa Kit Kit Name]
Rat Nitric Oxide (NO) Quantitative Detection Kit (ELISA)
[Use of rat nitric oxide (NO) Elisa kit kit]
Quantitative detection of nitric oxide (NO) in rat serum, plasma and related liquid samples.
【Test principle of rat nitric oxide (NO) Elisa kit】
This kit uses double-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the test sample to the pre-coated rat nitric oxide (NO) monoclonal antibody transparent enzyme label coating plate. After incubation for a sufficient time, wash to remove unbound components, and then add the enzyme label working solution. After sufficient incubation time, wash to remove unbound components. Substrates A and B were added in sequence, and the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), which turned yellow under the action of acid. Nitrogen (NO) concentration is positively correlated, and 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 rat nitric oxide (NO) in the sample is calculated.
[Composition of rat nitric oxide (NO) Elisa kit kit]
1
Enzyme coated plate
12 holes × 8
7
Developer A liquid
6mL
2
Standard
0.3mL × 6 tubes
8
Developer B liquid
6mL
3
20 times concentrated washing liquid
25mL
9
Stop solution
6mL
4
Sample diluent
6mL
10
Instructions
1 serving
5
Special diluent
6mL
11
Sealing film
2 sheets
6
Enzyme reagent
6mL
12
sealed bag
1
Remarks: The concentration of standard products (No. 1 → No. 6) is: 40, 20, 10, 5, 2.5, 1.25 μmol / L.
[Reagents and equipment not required by the rat nitric oxide (NO) Elisa kit]
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 of rat nitric oxide (NO) Elisa kit]
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, test sample wells and blank control wells respectively. 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 for rat nitric oxide (NO) Elisa kit]
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.
[Notes for Rat Nitric Oxide (NO) Elisa Kit]
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 1.25-40μmol / L, beyond this range, it is calculated from the extension of the standard curve. It is not used as an accurate quantitative result. Please dilute it with a special diluent to determine the accurate result (in the range of 1.25-40μmol / 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 of rat nitric oxide (NO) Elisa kit]
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
Calculation
[Range of Nitric Oxide (NO) Elisa Kit for Rats]
1.25μmol / L-40μmol / L
【Specification of rat nitric oxide (NO) Elisa kit】
96 servings / box
【Storage of Rat Nitric Oxide (NO) Elisa Kit】
Store at 2-8 ℃, protected from light and moisture.
【Validity of rat nitric oxide (NO) Elisa kit】
6 months

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