ELISA test kit for Actinobacillus pleuropneumoniae ApxIVA antibody
user's manual
ã€product name】
Generic name: ApxIVA antibody detection kit for porcine pleuropneumoniae (enzyme-linked immunosorbent assay)
English name: Test Kit for Antibodies to Porcine Actinobacillus Pleuropneumoniae ApxIVA (ELISA)
[Package Specification] 96T×1/Box, 96T×2/Box
[Intended use] Porcine infectious pleuropneumonia is a highly contagious respiratory disease caused by Actinobacillus Pleuropneumoniae (APP), characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic fibrinous necrotizing pleuropneumonia. . The most important virulence factor of APP is Apx toxin, in which ApxIVA toxin is present in all serotypes.
This kit detects ApxIV antibodies in pig serum and plasma samples and can be used for the diagnosis of Actinobacillus pleuropneumoniae infection.
[Original]
The kit consists of an enzyme-labeled plate, an enzyme label and other supporting reagents pre-coated with Actinobacillus pleuropneumoniae (APP) antigen. The enzyme-linked immunosorbent assay (ELISA) principle is used to detect pleural pneumonia in pig serum and plasma samples. A. faecalis ApxIVA antibody. During the experiment, the sample to be tested is added to the well of the microplate. After the incubation, if the sample contains Actinobacillus pleuropneumoniae ApxIVA antibody, it binds to the antigen on the plate; after washing, the unbound other components are removed, and the enzyme label is added. The substance specifically binds to the antigen-antibody complex on the plate; then the unbound enzyme label is removed by washing, and the substrate liquids A and B are added to react with the enzyme to form a blue product, which is dark in color and in the sample. The specific antibody content was positively correlated; the product turned yellow after the termination solution was added; the absorbance in each reaction well was measured by a microplate reader at a wavelength of 450 nm, and the sample was confirmed to contain an Actinobacillus pleuropneumoniae ApxIVA antibody.
[kit composition]
Serial number | name | Specifications (96T×1) | Specifications (96T × 2) |
1. | Enzyme plate | 96T×1 | 96T×2 |
2. | Enzyme label (red cover) | 11ml × 1 | 11ml×2 |
3. | Sample dilution (yellow cover) | 50ml × 1 | 50ml × 1 |
4. | 20X concentrated washing solution | 40ml × 1 | 40ml × 1 |
5. | Substrate A (white cover) | 6ml×1 | 11ml × 1 |
6. | Substrate B (black cover) | 6ml×1 | 11ml × 1 |
7. | Stop solution (yellow cover) | 6ml×1 | 11ml × 1 |
8. | Positive control (red cover) | 1.0 ml×1 | 1.5 ml × 1 |
9. | Negative control (green cover) | 1.0 ml×1 | 1.5 ml × 1 |
10. | Cover film | 1 piece | 2 sheets |
11. | Ziplock bag | 1 | 1 |
12. | Instruction manual | 1 serving | 1 serving |
[Storage and expiration date] It is stored at 2～8°C in the dark and valid for 12 months.
After the cover plate is opened, please keep it at 2~8°C to avoid moisture. The period of use is 2 months.
[Applicable instrument] Microplate reader with wavelength of 450nm and 630nm, constant temperature equipment of 37 °C, adjustable micropipette.
[sample preparation]
1. Take the whole blood of the animal and prepare the serum according to the conventional method. The serum is required to be clear, no hemolysis and no pollution. The sample can be stored at 2 to 8 ° C for 1 week and stored at -20 ° C for a long time.
2. Dilute the test serum by 40-fold with a sample dilution (eg, 5 μl of serum is added to 195 μl of sample dilution and mix). The positive control was diluted 10-fold and the negative control was not diluted.
3. The concentrated washing solution should be returned to room temperature before use to dissolve the precipitate, and then diluted with distilled water or deionized water for 20 times.
ã€Testing method】
1. Set the kit to room temperature for 30 minutes before use and return to room temperature.
2. Take the required amount of the enzyme label, set 2 wells of blank control and 2 holes of negative/positive control. The unused strips should be sealed as soon as possible and stored at 2-8 °C.
3. The blank control well was added with 100 μl of the sample dilution; the negative control well was added with 100 μl of the negative control; the positive control well was added with the diluted positive control 100 μl; and the sample well was added with 100 μl of the diluted sample per well.
4. Mix and react at 37 ° C for 30 minutes.
5. Deduct the liquid in the well, fill each well with the washing solution, let stand for 30 seconds, discard it, repeat the washing 5 times, and pat dry.
6. Add 100 μl of enzyme label per well (except for blank wells). The reaction was carried out at 37 ° C for 30 minutes.
7. Wash, same as step 5.
8. Add 50 μl of each of substrate A and substrate B to each well, mix well, and react at 37 ° C for 10 minutes in the dark.
9. Add 50 μl of stop solution to each well, mix well, adjust to zero with a blank well, and measure the absorbance (A value) of each well at 450 nm (using 630 nm as the reference wavelength).
[Reference value] In the case of normal experiment, the negative control was ≤ 0.15, and the positive control was ≥ 0.4.
[Explanation of test results]
The (positive) absence (negative) of the Actinobacillus pleuropneumoniae ApxIVA antibody in the porcine serum sample was determined by calculating the ratio of the absorbance of the sample to the positive control (S/P value).
1. S/P = sample A value / positive control ratio.
2. S/P ≥ 1 is positive for ApxIVA antibody; S/P < 1 is negative for ApxIVA antibody.
[Limitations of test methods]
This test is only used as a qualitative test for Actinobacillus pleuropneumoniae ApxIVA antibody.
ã€Precautions】
1. Wear gloves and work clothes during the operation. Strictly improve and implement the disinfection and isolation system. All kinds of experimental waste should be treated as infectious materials.
2. The stop liquid is corrosive and should be avoided from contact with skin and clothing. If it is inadvertently contacted, please rinse it with plenty of tap water immediately.
3. When the enzyme plate is taken out from the refrigerated environment, it should be returned to room temperature before the bag can be opened. The unused enzyme plate should be stored in a sealed bag with desiccant.
4. Concentrated washing liquid is easy to crystallize at low temperature, and needs to be returned to room temperature to completely dissolve when used.
5. When washing, each hole should be filled with liquid to prevent the free enzyme in the orifice from being washed.
6. The sample used for testing should be kept fresh.
7. The determination of the test results must be based on the reading of the microplate reader.
8. Different batches of reagent components should not be mixed.
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