The Toxoplasma gondii (TO) Antibody Test Kit is designed to detect Toxoplasma gondii antibodies in pig serum.
The kit uses an indirect ELISA method in which purified TO antigen is pre-encapsulated on the microtiter wells of an enzyme lath. In the test, the diluted serum to be tested is added, and after warming, if the sample contains TO-specific antibody, it binds to the antigen on the coated plate, after washing to remove unbound antibody and other components; then the enzyme secondary antibody is added to specifically bind to the antigen-antibody complex on the detection plate; then the unbound enzyme conjugate is removed by washing; the TMB substrate solution is added to the microtiter wells, which is catalyzed by the enzyme to form After the reaction was terminated by adding the termination solution, the absorbance A value in the reaction wells was measured at 450nm by the enzyme standard.
Equipment and reagents to be provided
1.Micro pipettes: 0.5µl-10µl, 10µl-100µl, 100µl-1000µl.
2. Disposable pipette tips.
3. Measuring cylinder: 500ml.
4. 96-well plate enzyme marker.
5. Distilled water or deionized water.
6. Wash bottle or plate washer.
Take the whole blood of the animal and prepare the serum according to the conventional method, requiring the serum to be clear and free of hemolysis.
Preparation of washing solution:
Concentrated washing solution should be restored to room temperature before use. If salt crystallizes, shake to dissolve the crystallized salt, and then dilute 10 times with distilled or deionized water. The diluted washing solution can be stored at 4℃ for about a week.
Dilution of samples:
Dilute the serum to be tested in serum dilution plates at a volume ratio of 1:100.
Note: Negative and positive controls do not need to be diluted. Change the pipette tips after taking each sample and record accurately the position of each sample on the plate. Each sample should be well mixed before being added to the microtiter wells of the coated plate.
1. Each reagent of the kit should be equilibrated to room temperature before use, and each component of the kit should be left at room temperature for at least 1 hour. It should be shaken well before use and put back to 2-8℃ after use.
2. The reagent components of different varieties and batches of kits should not be mixed, and reagent contamination should be prevented when using reagents.
3. The substrate and termination solution may be irritating to the skin and eyes, and care should be taken to protect them when using.
4. TMB (color developing solution) should not be exposed to strong light and avoid contact with oxidizing agents.
5. Avoid moisture or water after unpacking the assay plate (unused antigen-coated plates with desiccant should be placed in self-sealing bags and placed at 2-8°C as soon as possible).
6. All waste should be disposed of properly before discarding to avoid contamination.
7. Strictly follow the operating instructions to obtain the best results. The entire process of pipetting, timing and washing during the operation must be precise.
8. Serum dilution plates are disposable and should not be reused; the maximum capacity of serum dilution plates is 300 μl/well.
1. Take the pre-wrapped assay plate (according to the number of samples, it can be disassembled for sub-use) and add 100μl of the diluted serum to be tested into the wells of the assay plate. At the same time, set up 2 wells for negative control and 1 well for positive control. Add 100 μl of each negative and positive control to the wells and gently shake the sample in the wells (do not overflow).
2. Cover with sealing film (can be cut according to actual needs) and incubate at 37℃ for 30 minutes.
3. Carefully remove the sealing membrane, shake off the solution in the wells, add 250ul per well using working concentration washing solution, then shake off the liquid in the wells.
4. Add 50μl of enzyme conjugate to each well, cover with sealing membrane and incubate at 37℃ for 30 minutes.
5. Carefully remove the sealing membrane, shake off the solution from the wells and wash 4-6 times as in 3. Remember to pat dry on clean blotting paper at the end.
6. Add 100 µl of color development solution to each well, shake lightly and mix well, cover the sealing membrane for 10 minutes at 37°C with closed light.
7. Add 50μl of termination solution to each well to terminate the reaction and determine the results within 10 minutes.
Determination of results:
Read the absorbance OD value at 450nm (630nm as reference wavelength) with an enzyme standard.
The test was set up under the following conditions:
The OD value of negative control (N) is <0.20, while the OD value of positive control (P) is >0.4.
sample OD value ÷ positive control OD mean value = S/P value
Negative and positive judgment:
S/P value ≥ 0.4 judged as positive;
S/P value <0.4 is judged as negative.
Storage method: 2 - 8ºC.
Expiration date: 12 months.