Instructions for mouse rotavirus (RV) Elisa kit
This kit is for research use only.
96T
purpose of usage:
This kit is used to determine the expression of rotavirus (RV) in mouse serum, plasma and related liquid samples.
Experimental principle
Mouse rotavirus (RV) Elisa kit instructions This kit uses the double antibody sandwich method to determine the expression of mouse rotavirus (RV) in the specimen. Purified mouse rotavirus
(RV) antibody coats microplate to make solid phase antibody, which can be combined with rotavirus (RV) in the sample and removed by washing
Unbound antigen and other components are then combined with HRP-labeled rotavirus (RV) antibody to form antibody-antigen-enzyme
The target antibody complex, after thorough washing, is added with substrate TMB for color development. TMB turns into blue under the catalysis of HRP enzyme,
And converted into the final yellow under the action of acid. Measure the absorbance (OD value) at 450nm with a microplate reader, and
The CUTOFF values ​​are compared to determine the presence or absence of mouse rotavirus (RV) in the specimen.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 Positive control 0.5ml × 1 bottle
3 Enzyme label coated plate 12 well × 8 strips 9 negative control 0.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 2 sealing film
6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag
Specimen requirements
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If not
Perform the test immediately. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Numbering: The microwells corresponding to the samples are numbered in order. Each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 blank control.
Wells (Blank control wells do not add samples and enzyme reagents, the remaining steps are the same)
2. Add sample: add negative control and positive control 50μl to the negative and positive control wells respectively. Then in the sample hole to be tested
Add 40μl of sample diluent, then add 10μl of the sample to be tested. Add the sample to the bottom of the well of the microplate, try not to
Touch the wall of the hole, shake gently to mix,
3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated
Within 15 minutes after the solution.
Summary of operating procedures:
Calculation and result judgment:
Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.10
Calculation of cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15
Negative judgment: samples with OD value <cut-off value (CUT OFF) are negative for rotavirus (RV)
Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for rotavirus (RV)
.
Precautions
1. The operation is carried out in strict accordance with the instructions. The components of different batches of this reagent must not be mixed.
2. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment.
After use, the slats should be stored in sealed bags.
3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the results will not be affected during washing.
4. The sealing film is limited to one-time use to avoid cross-contamination.
5. Please keep the substrate away from light.
6. The test result must be determined based on the reading of the microplate reader. When using dual wavelength detection, the reference wavelength is 630nm
7. All samples, washing liquid and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, which must be used
be careful.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months instructions for mouse rotavirus (RV) Elisa kit
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