Indirect ELISA Kit for Classical swine fever virus
Instructions for use
[Introduction]
The kit is made of antigen-coated plate, serum dilution plate, enzyme-labeled secondary antibody and other reagents, and the use of indirect ELISA method to detect the presence of swine fever specific antibodies in pig serum, so as to evaluate the immune effect of swine fever vaccine.
[Indication]
It is suitable for post-immunization evaluation of swine fever vaccine and serological diagnosis of unimmunized pigs.
[Principle]
The kit uses mixed antigen consisting of two main specific antigens of classical swine fever virus to coat a microplate. In the assay, if the serum sample to be tested contains the corresponding antibody, it will bind to the antigen on the test plate to form an antigen-antibody complex. The enzyme labeled secondary antibody added later will specifically bind to the antigen-antibody complex on the detection plate, and can make the TMB substrate solution form a blue product. The color reaction was yellow after the termination of the liquid, and finally the result was obtained by measuring the absorbance at 450nm wavelength.
Kit main components]
1
antigen coated plate
2 pieces 96 wells/block
2
negative control serum
1 tube 50 µl/tube
3
positive control serum
1 tube 50 µl/tube
4
enzyme labeled secondary antibody
2 tubes 0.2ml/tube
5
25 times concentrated washing liquid
1 bottle 25ml/bottle
6
Substrate solution A
1 bottle 10ml/bottle
7
Substrate solution B
1 bottle 10ml/bottle
8
Stop fluid
1 bottle 10ml/bottle
9
Sample Diluent
2 bottles of 30ml/bottle
10
serum dilution plate
2 pieces 96 wells/block
11
Instructions
1 sheet
sample preparation]
Take animal whole blood and prepare serum according to the regular method. The serum is required to be clear and free of hemolysis.
reagent preparation]
Before use, all reagents in the kit except the enzyme labeled secondary antibody should be returned to room temperature (about 25 ˚C). The 25-fold concentrated washing liquid is returned to room temperature or slightly heated and shaken to dissolve the precipitated salt, and then diluted 25 times with deionized water. The diluted washing liquid can be stored at 2~8 ˚C for 7 days. The enzyme-labeled secondary antibody is diluted 1:100 times with sample diluent before use, and is now prepared for use.
sample dilution]
The standard sample and the sample to be tested are diluted in the serum dilution plate at a volume of 1:50 times (recommend dilution method: add 5µL to 245µL of sample dilution
serum samples to be tested).
operation steps]
1. Take the antigen-coated plate, add 50µL of sample diluent first, then add 50µL of diluted sample to be tested and control sample to the wells of the antigen-coated plate, and gently blow and mix with a pipette. The negative control and the positive control were each set up in 2 wells and incubated at 37 ˚C for 30 minutes.
2. Shake off the solution in the plate, add 200 µL of diluted washing solution to each hole, let stand for 1 minute and pour it out, then pat it dry on absorbent paper for 3 times.
3. Add diluted enzyme-labeled secondary antibody 100 µL to each well and incubate at 37 ˚C for 30 minutes.
4. Shake off the solution in the plate, add diluted washing liquid 200 L to each hole, let stand for 1 minute and pour it out, then pat it dry on absorbent paper for 3 times.
5. Add 100µL of mixed substrate solution A and substrate solution B in equal volume to each well, and color in the dark for 10 minutes.
6. Add 50 µL of stop solution to each well, and measure the absorbance at 450nm wavelength within 10 minutes.
result judgment]
The notice of the Ministry of Agriculture on the issuance of the "2013 National Animal Disease Compulsory Immunization Plan" clearly pointed out that the swine fever antibody positive indirect hemagglutination test antibody titer ≥ 25 is qualified. According to the notice, the result judgment standard is formulated:
1 The experimental results are valid only if they meet the following conditions:
Standard positive control wells mean OD450nm ≥ 0.6±0.02.
Standard negative control wells mean OD450nm <0.3±0.02.
2 Determination of antibody level test results after vaccine immunization:
Sample OD450nm ≤ 0.3, judged as swine fever antibody negative.
Sample OD450nm>0.3, judged as swine fever antibody positive.
The sample OD450nm is within the 0.3±0.02 critical range, and it is recommended to repeat the test to ensure accurate results.
3 Determination of the test results of the immune protection of classical swine fever virus:
The OD450nm of the sample is less than or equal to 0.3, which is judged as no protection, and the tested sample has no protection against classical swine fever virus infection.
The OD450nm of the sample is between 0.3 and 0.6, which is judged as low protection, and the sample antibody level is low, which has certain protection against the epidemic strain.
The OD450nm of the sample ≥ 0.6 is judged to be protective. The antibody level of the tested sample is relatively high, and it can provide more than 90% resistance in the attack test.
The sample OD450nm is within 0.3±0.02 or 0.6±0.02 of the two critical values, and it is recommended to repeat the test to ensure accurate results.
[Note]]
1 The background is too high can lead to the overall level of the result is too high, the experimental process needs to ensure that the secondary anti-cleaning clean, or add blank control in the experiment.
2 The ambient temperature below 15 ˚C can lead to a low overall level of the result. The experimental process needs to ensure that the ambient temperature is as close to 25 ˚C as possible, or appropriately extend the color development time of the substrate solution.
3 Substrate liquid A and substrate liquid B should not be exposed to strong light and avoid contact with oxidants.
4 Concentrated washing liquid is diluted with deionized water. If crystals are found, they should be dissolved by heating in a water bath.
5 Enzyme-labeled secondary antibody should be used now.
6 4 ˚C overnight incubation of samples and enzyme labeled secondary antibody can obtain better results.
storage]2~8 C protected from light
[validity period]]9 months
production enterprise]Chongqing Aolong Biological Products Co., Ltd.
[address]]No.5, 4th Branch Road, Banqiao Industrial Park, Rongchang District, Chongqing
The amount of antigen in scientific compatibility is high. Each vaccine contains not less than 30000RID of swine
Need help? Talk to our experts.
In the process of browsing our products, is there anything that makes you feel particularly satisfied or needs to be improved? Your advice or suggestions will directly help us improve the quality of our products and services, and bring you a more intimate experience.
* Please fill in the above fields (* fields are required) and we will reply to your feedback as soon as possible.
渝公网安备50022602000038号