Manual of Human Parainfluenza Virus (HPIV) ELISA Kit

Manual of Human Parainfluenza Virus (HPIV) ELISA Kit
This kit is for research use only.
Drug Name:
Common name: Human Parainfluenza Virus (HPIV) Antigen ELISA Diagnostic Kit
purpose of usage:
This kit qualitatively determines the parainfluenza virus (HPIV) antigen in human blood or other related tissues
Experimental principle:
This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine the human parainfluenza virus (HPIV) in the specimen. Coat the microplate with purified human parainfluenza virus (HPIV) antibody to make a solid-phase antibody, which can be combined with the human parainfluenza virus (HPIV) antigen in the sample, after washing to remove unbound antigen and other components It is combined with HRP-labeled goat anti-human antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm and compared with the CUTOFF value to determine the presence or absence of human parainfluenza virus (HPIV) antigen in the specimen.
Kit composition:
1
20 times concentrated washing liquid
50ml × 1 bottle
7
Stop solution
6ml × 1 bottle
2
Enzyme reagent
6ml × 1 / bottle
8
Standard (positive control)
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 8
9
Standard dilution (negative control)
0.5ml × 1 bottle
4
Sample diluent
6ml × 1 bottle
10
Instructions
1 serving
5
Developer A liquid
6ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B liquid
6ml × 1 bottle
12
sealed bag
1
Specimen requirements:
1. Specimen processing: (1) Serum and plasma specimens: can be directly detected
(2) Tissue specimens; after wiping the mucosa with a cotton swab, immerse the cotton swab in 1ml saline, soak for 10 minutes, centrifuge to take the supernatant for use; or prepare according to relevant literature.
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, but repeated freezing and thawing should be avoided.
3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps:
1. Numbering: serially number the corresponding microwells of the sample, each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control well does not add sample and enzyme reagent, the rest of the 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 the sample to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix,
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: add 20 times concentrated washing liquid to distilled water to 1000ml
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 and then discard, repeat this 5 times, pat dry.
6. Add enzyme: add 50μl of enzyme label reagent (or one drop) to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl (or one drop) of developer A to each well, then add 50μl (or one drop) of developer B, mix gently, and develop color at 37 ℃ for 15 minutes in the dark
10. Termination: Add 50μl of stop solution (or one drop) to each well to stop the reaction (in this case, the blue will turn yellow)
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm 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 + 0.30
Negative judgment: samples with OD value <cut-off value (CUT OFF) are negative for human parainfluenza virus (HPIV) antigen
Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for human parainfluenza virus (HPIV) antigen
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 equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, 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, and you must pay attention to safety when using it.
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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