PEDV-Ab ELISA Kit Instructions for Use This reagent is for research purposes only: This kit is used to detect infectious gastritis antibody (PEDV-Ab) levels in swine serum, plasma and related liquid samples.
Experimental principle:
This kit uses double-antigen sandwich enzyme-linked immunoassay (ELISA) to determine the swine infectious gastritis antibody (PEDV-Ab) in the specimen. The microplate is coated with purified antigen to make a solid-phase antigen, which can be combined with the antibody (PEDV-Ab) in the sample. After washing to remove unbound antibody and other components, it is combined with the HRP labeled antigen to form an antigen -Antibody-antigen 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 450 nm, and compared with the CUTOFF value to determine the presence or absence of porcine infectious gastritis antibody (PEDV-Ab) in the specimen.
Kit composition:
Kit composition 48-well configuration 96-well configuration preservation manual 1 copy 1 copy
2 pieces of sealing film (48) 2 pieces (96)
One sealed bag
Enzyme-labeled coating plate 1 × 48 1 × 96 2-8 ° C Store negative control 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C Store positive control 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C Enzyme label reagent 3 ml × 1 bottle 6 ml × 1 bottle Store sample diluent at 2-8 ℃ 3 ml × 1 bottle 6 ml × 1 bottle Store reagent A solution at 2-8 ℃ 3 ml × 1 bottle 6 ml × 1 Bottle 2-8 ° C Store Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C Store concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle stored at 2-8 ℃
Sample processing 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. After mixing for 10-20 minutes, centrifuge 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 the cells, the cell suspension is diluted with PBS (PH7.2-7.4), and the cell concentration reaches 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 ° C. 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. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
7. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Steps:
1. Numbering: Number the samples corresponding to the microwells in sequence. Each plate should be provided with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the 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 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 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 it stand for 30 seconds and then discard, repeat this 5 times, 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 for 15 minutes in the dark at 37 ℃
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 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 ≤0.10
Calculation of cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15
Negative judgment: samples with OD value <critical value (CUT OFF) are negative for infectious gastritis antibody (PEDV-Ab) positive judgment: samples with OD value ≥ critical value (CUT OFF) are positive for infectious gastritis antibody (PEDV-Ab) 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.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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