This kit is for research use only. 48T
Drug Name:
Generic name: Human rotavirus IgG (RV-IgG) enzyme-linked immunoassay kit Purpose of use:
This kit qualitatively determines rotavirus IgG (RV-IgG) in human blood or other related tissues.
Experimental principle:
This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine the human rotavirus IgG (RV-IgG) in the specimen.
The microplate is coated with purified rotavirus IgG (RV-IgG) antibody to make a solid-phase antibody, which can be used with the rotavirus in the sample
IgG (RV-IgG) antigen is combined, washed to remove unbound antigen and other components, and then combined with HRP-labeled rotavirus IgG (RV-IgG) antibody to form an antibody-antigen-enzyme-labeled antibody complex. Add substrate TMB after thorough washing
Color rendering. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. Measure the absorbance (OD value) at 450nm with a microplate reader and compare it with the CUTOFF value to determine the human rotavirus in the specimen
The presence or absence of IgG (RV-IgG).
Kit composition:
1 20 times concentrated washing solution 20ml × 1 bottle 7 Stop solution 3ml × 1 bottle
2 Enzyme label reagent 3ml × 1 / bottle 8 Positive control 0.5ml × 1 vial
3 Enzyme label coated plate 12 well × 4 strips 9 negative control 0.5ml × 1 bottle
4 Sample diluent 3ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 3ml × 1 bottle 11 2 sealing film
6 Developer B solution 3ml × 1 bottle 12 sealed bag 1 specimen requirement:
1. Specimen processing: serum and plasma specimens can be directly detected
2. Perform the experiment as soon as possible after the specimen is prepared as required. If it can not be detected in time, the specimen can be stored at -20 ℃ for one month,
However, repeated freezing and thawing should be avoided.
3. 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 (the blank control wells do not add samples and enzyme reagents, the remaining steps are the same)
2. Add sample: add 50μl of negative control and positive control (standard) to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. Add 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: add 30 times concentrated washing liquid to distilled water to 600ml and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds, then discard, 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 avoid color development at 37 ℃
15 minutes
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Result judgment:
Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.15
Calculation of critical value (CUT OFF): critical value = average value of negative control wells + 0.15
Negative judgment: samples with OD value <critical value (CUT OFF) are negative for rotavirus IgG (RV-IgG) positive judgment: samples with OD value ≥ critical value (CUT OFF) are positive for rotavirus IgG (RV-IgG) Precautions
1. The operation is strictly in accordance with the instructions. The components of different batches of this reagent must not be mixed.
2. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, the strip should be stored in a sealed bag.
3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a 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 results 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 liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, you must pay attention to safety when using it.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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