Anti-gE Porcine Pseudorabies Virus Antibody Elisa Kit

Specification

PRV-gE Ab Elisa

Intended Use:The Porcine Pseudorabies Virus gE Blocking

Antibody ELISA Detection Kit is for the determination of gE antibody in pig's serum of porcine pseudorabies virus in vitro. The test is used to distinguish infected wild

virus and vaccinated from immunization with gE gene deletion vaccine.

Specimen: serum

Materials Required But Not Provided:

Purified water

Pipettor

96-well Microplate reader (450nm)

Timer

Centrifuge (4000rpm)

Washing machine

Oven (37℃)

 

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 sample 1:1 (add 70μl serum sample to 70μl sample diluent) and mix thoroughly.

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 and positive samples, and add 100μl each for negative and positive controls 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 60 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.

③Add 100µl of Enzyme Conjugate to each well, cover the plate, and incubate at 37℃ for 30 minutes.

④Open 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.

⑤Add 100µl substrate solution to each well, cover the plate and incubate for 15 minutes at 37℃ away from light.

⑥Add 50µl stop solution to each well, and determine the result within 5 minutes.

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 ≥ 0.5 and the blocking rate of positive control was >50%, the experimental results were valid. Otherwise, the test should be repeated.

2.2 Calculation Method

For each sample, calculate the PI (Blockingr ate) as follows using the corrected sample and control values:

2.3 Result Interpretation

When PI < 40%, it is judged as negative.

When PI > 50%, it is judged as positive.

When 40% ≤ PI ≤ 50%, it is judged as suspicious, and suggest to repeat sampling and test one week later.