This kit can only be used for scientific research, not for medical diagnosis.
Bovine Pseudorabies Virus (PRV) Qualitative Detection Kit (ELISA)
user's Guide
ã€Kit name】
Bovine Pseudorabies Virus (PRV) Qualitative Detection Kit (ELISA)
ã€Use of the kit】
Qualitative detection of pseudorabies virus (PRV) in bovine serum, plasma and related liquid samples.
ã€Detection principle】
This kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Add negative control, positive control, sample to be tested and enzyme-labeled working solution to the transparent enzyme-labeled plate precoated with bovine pseudorabies virus (PRV) antibody. After incubation for a sufficient time, wash to remove unbound components. Add the developer A and B in sequence. The developer (TMB) is converted into a blue product under the catalysis of horseradish peroxidase (HRP), which turns yellow under the action of acid. The concentration of pseudorabies virus (PRV) was positively correlated. The OD value was measured at a wavelength of 450 nm. Based on the OD values ​​of the negative control, positive control and sample, it was determined whether the sample contained bovine pseudorabies virus (PRV).
ã€Composition of the kit】
1
Enzyme coated plate
12 holes × 8
7
Developer A liquid
6mL
2
Negative control
0.6mL
8
Developer B liquid
6mL
3
Positive control
0.6mL
9
Stop solution
6mL
4
20 times concentrated washing liquid
25mL
10
Instructions
1 serving
5
Sample diluent
6mL
11
Sealing film
2 sheets
6
Enzyme reagent
10mL
12
sealed bag
1
[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
ã€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 control and test samples: take a sufficient number of enzyme-coated plates and fix them on the frame. Set up positive control wells, negative control wells, test sample wells and blank control wells, record the positions of each well. Add 50μL each of the positive control and negative control to the control well; add 10μL of the test sample to the test well, and then add 40μL of the sample dilution (that is, the sample is diluted 5 times); add 100μL of the enzyme solution to each well; blank Control wells are not added.
4. Incubation: Incubate in a 37 ° C water bath or thermostat for 60 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 5 times in this way Instructions for washing the board).
6. Color development: add 50 μL of developer A solution to each well, then add 50 μL of developer B solution, and develop color at 37 ° C in the dark for 15 min.
7. 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).
8. 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.
[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.
ã€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. If the color is too light, the substrate incubation time can be extended properly.
5. In order to avoid cross-contamination, the standard, sample and blank control should be replaced with a tip for each additional one; the common components such as enzyme working solution, sample diluent and substrate should be added by cantilever, and should not touch the microwell ; Do not reuse the sealing film.
6. The kits are used within the warranty period, and different batches of reagents should not be mixed.
7. Substrate B is sensitive to light and avoid prolonged exposure to light.
ã€Result judgment】
1 Test effectiveness: the average value of the OD value of the positive control well ≥1.00;
The average value of OD value of negative control wells was ≤0.15.
2 Cut-off value (Cut off) calculation: cut-off value = average value of negative control well +0.15
3 Negative judgment: the sample OD value <cut-off value (Cut off), the sample is negative
4 Positive judgment: the sample OD value> cut-off value (Cut off), the sample is positive
[Summary of operating procedures]
Prepare reagents, samples and controls
Add the prepared sample, reference substance and enzyme working solution, and react at 37 ℃ for 60 minutes
Wash the plate 5 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
ã€specification】
96 servings / box
ã€Storage】
Store at 2-8 ℃, protected from light and moisture.
ã€Validity】
6 months
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