Swine Influenza H1N1 ELISA Kit Instructions for Use This reagent is for research use only: This kit is used to determine the expression of influenza H1N1 in swine serum, plasma, and related liquid samples.
Experimental principle:
This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine swine influenza H1N1 in specimens. The purified swine flu H1N1 antibody is used to coat the microplate to make a solid phase antibody, which can be combined with the swine flu H1N antigen in the sample. Combine to form antibody-antigen-enzyme-labeled antibody complex. After thorough washing, add substrate TMB to develop color. 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 450nm and compared with the CUTOFF value to determine the presence or absence of swine influenza H1N1 in the specimen.
Kit composition:
Kit composition 48 well configuration 96 well configuration storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96)
Sealed bag 1 x 1 microplate coated plate 1 × 48 1 × 96 2-8 ℃ Store negative control 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ℃ Store positive control 0.5ml × 1 bottle 0.5ml × 1 Bottle 2-8 ℃ Store enzyme label reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ Store sample dilution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ Store Developer A solution 3 ml × 1 bottle of 6 ml × 1 bottle at 2-8 ℃ Store Developer B solution 3 ml × 1 bottle of 6 ml × 1 bottle at 2-8 ℃ Store stop solution 3ml × 1 bottle of 6ml × 1 bottle of 2-8 ℃ store concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ℃ Storage sample handling and requirements:
1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.
2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components in cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ℃. Add a certain amount of PBS (PH7.4) and homogenize the specimen by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
6. 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.
7. The samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Steps:
1. Numbering: Samples corresponding to the microwells are numbered in sequence, 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 samples and enzyme reagents, the remaining steps are the same)
2. Add sample: add 50 μl of negative control and positive control to the negative and positive control wells respectively. Then add 40 μl of sample diluent to the sample well to be tested, and then add 10 μl 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 the sealing film and incubate at 37 ℃ for 30 minutes.
4. Mixing solution: add 30 times (20 times of 48T) 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 stand for 30 seconds and then discard, repeat 5 times, pat dry.
6. Add enzyme: add 50 μl of enzyme label reagent 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 50 μl of developer A to each well, then add 50 μl of developer B, mix gently, and develop at 37 ℃ in the dark for 15 minutes
10. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner zero and 4 50 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Result judgment:
Test effectiveness: the average value of positive control wells ≥ 1.00; the average value of negative control wells ≤ 0.15 cut-off value (CUT OFF) calculation: cut-off value = average value of negative control wells + 0.20 negative judgment: the sample OD value <cut-off value (CUT OFF) For swine flu H1N1 negative positive judgment: the sample OD value ≥ critical value (CUT OFF) is swine flu H1N1 positive note
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 period: 6 months 4
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