Intended Use: Infectious Bovine Rhinotracheitis Virus Antibody ELISA Detection Kit is used to determine the infectious rhinotracheitis virus antibodies in bovine serum in vitro.
Specimen: serum
Materials Provided:
Test Procedures & Interpretation
STEP 1 Test Procedure
1.1 Wash Solution Preparation
Working concentration washing liquid: Before use, the wash solution should be restored to room temperature (20-25℃) and shaken evenly to make it uniform. Then dilute 20 times the concentrated wash solution with purified water (for example, 5ml concentrated washing solution is added to 95ml purified water), mix well, and the diluted washing liquid can be stored at 2-8℃ for 7 days.
1.2 Sample Preparation
① Whole animal blood was taken, centrifuged at 4000rpm for 10 minutes, and supernatant was collected. Serum can also be coagulated naturally without hemolysis.
② Dilute the serum to be tested 50 times in the serum dilution plate (for example： add 3μl of serum to be tested to 147μl sample diluent).
Note: Negative and positive controls need not be diluted
1.3 Test Procedure
①Take the enzyme label Microplate (according to the number of samples, can be disassembled and used several times), add 100μl of the diluted serum to the enzyme label Microplate, set two wells each for negative control and positive control, and add 100μl to the reaction well respectively. Gently shake the sample in the well, using the sealing plate film to cover the plate, and incubate at 37℃ for 30 minutes.
②Open the sealing plate film, shake off the solution in the plate well, add 300µl of working concentration washing liquid to each well, and then discard the liquid in the well. Repeat the above steps 5 times, and finally pat dry on a clean absorbent paper.
③Diluted enzyme conjugate：Dilute the enzyme conjugate (80×) 80 times (for example: 10μl enzyme conjugate concentrate + 790μl sample diluent, the specific amount required is calculated according to the number of test samples), mix well and set aside.
④Add 100µl of Enzyme Conjugate to each well, cover the plate, and incubate at 37℃ for 30 minutes.
⑤Washing enzyme plate, the specific operation is the same as the above ②.
⑥Add 100µl substrate solution to each well, cover the plate and incubate for 10 minutes at 37℃ away from light.
STEP 2 Result Interpretation
The OD450 value of each well was read on the 96-well microplate reader.
2.1 Validation The average OD450nm value of negative control was < 0.25 and the average OD450nm value of positive control was ≥ 0.6, the experimental results were valid. Otherwise, the test should be repeated.
2.2 Calculation Method
For each sample, calculate the S/P as follows using the corrected sample and control values:
2.3 Result Interpretation
When S/P < 0.2, it is judged as negative.
When S/P > 0.3, it is judged as positive.
When 0.2 ≤ S/P ≤ 0.3, it is judged as suspicious, and suggest to repeat sampling and test one week later.
