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 Echinococcus specific antibodies in sheep serum, so as to infer whether sheep suffer from Echinococcus granulosus (hydatid) disease.
[Indication]
It is suitable for serological diagnosis of sheep hydatid disease.
[Principle]
This kit uses a mixed antigen consisting of three main specific antigens of Echinococcus 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.
[Main components of the kit]
1
antigen coated plate
2 pieces 96 wells/block
2
negative control serum
1 tube 50μl/tube
3
positive control serum
1 tube 50μl/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 according to the volume of 1:40 times (recommend dilution method: 195µL sample dilution plus 5µL serum sample to be tested).
[Operation steps]
1. Take the antigen-coated plate, respectively add 100µL of diluted sample to be tested and control sample to the wells of the antigen-coated plate, set 2 wells for the negative control and 2 wells for the positive control, and incubate 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 100 µL 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]
Referring to the notice of the Ministry of Agriculture on the issuance of the "2013 National Animal Epidemic Compulsory Immunization Plan", the antibody titer ≥ 25 of the tested antibody is positive in the forward indirect hemagglutination test. 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 Echinococcus granulosus (hydatid) disease serological test results criteria:
The OD450nm of the sample is less than or equal to 0.3, which is negative. The sample has not suffered from Echinococcus granulosus (hydatid) disease.
The OD450nm of the sample was between 0.3 and 0.6, which was judged to be weakly positive. The corresponding sample was in the early stage of echinococcosis infection, and had not yet formed cysts or cysts were not obvious.
The OD450nm of the sample ≥ 0.6 is positive, and the corresponding sample should be in the late stage of Echinococcus granulosus infection and form a more obvious cyst.
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 When the OD450nm of the sample is ≥ 0.6, it may be caused by exposure to a large amount of antigen instead of hydatid disease. Please confirm that the sample is not immunized with antigen.
2 The background is too high can lead to the overall level of the result is too high, and the experimental process needs to ensure that the secondary resistance is cleaned.
3 The ambient temperature lower than 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.
4 Substrate liquid A and substrate liquid B should not be exposed to strong light and avoid contact with oxidants.
5 Concentrated washing liquid is diluted with deionized water. If crystals are found, they should be dissolved by heating in a water bath.
6 Enzyme-labeled secondary antibody should be used now.
7 4 ˚C overnight incubation of samples and enzyme-labeled secondary antibodies can obtain better results.
The amount of antigen in scientific compatibility is high. Each vaccine contains not less than 30000RID of swine
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