1. Usage
Aujeszky’s disease or pseudorabies is caused by a herpes virus and affects mainly pigs which are the only known reservoir of the disease. It is an important disease of pigs causing severe economic losses. The BIOKAU® Pseudorabies virus gE antibody ELISA test kit can be used to detect anti PRV-gE antibody in the serum and distinguish naturally infected virus from vaccined swine.
2. Brief
The BIOKAU® PRV gE ELISA test kit is made from the antigen coated microplate, enzyme conjugate(HRP goat-anti-pig IgG) and other reagents. It applies the indirect ELISA principle to test the antibody against PRV-gE antibodies in the serum.
3. Principle
The test kit is the antigen coated microplate made through the protein expressed by pseudorabies virus gE gene. In the test, the diluted control serum and sample are added, then incubate. If PRV-gE specific antibodies exist in the sample, it will be bound with the antigens of the microplate. Then the unbound antibodies and other components are removed by washing. Next, add IgG-HRP to specifically bind with the compound of antibody and antigen on the microplate. The unbound conjugation will be removed by washing, add the TMB substrate in the well, react with HRP to a blue product. At last, end the reaction by adding stop solution,.
4. Components
1 PRV Antigen coated microplate........................................ 2 pieces (96 wells)
2 PRV Negative control (green lid)............................................. 1 tube (1 mL)
3 PRV Positive control (red lid)................................................. 1 tube (1 mL)
4 PRV Enzyme Conjugate (yellow lid)...................................... 1 bottle (22 mL)
5 Wash Concentrate (20×) (white lid)...................................... 1 bottle (30 mL)
6 Substrate A (purple lid)/B (yellow lid)................... 1 bottle each (12 mL/bottle)
7 Stop solution (blue lid)......................................................... 1 bottle (12 mL)
8 Sample diluent solution (transparent lid)............................... 1 bottle (50 mL)
5. Material Required But not Provided
1 Microplate Reader(wave length: 450/630 nm).
2 Microplate Washer.
3 Micropipettes, adjustable.
4 Constant temperature box or incubator.
5 Oscillator.
6. Warning and Procautions for the Users
1 All reagents should be adjusted to the room temperature before using the test kit.
2 Never mix up reagents from different test kits in batch number.
3 Prevent the microplate to get damp.
7. Preparation of Washing Solution
Washing solution: dilute Wash Concentrate (20×) solution with distilled water at 1:19 (1 μL Wash Concentrate (20×) solution + 19 μL distilled water), mix properly.
8. Test Procedure
1 Take out the pre-coated microtiter plate(just to the required quantity). Dilute the sample at 1:39 with the sample diluent solution, then add the diluted sample into appropriate wells, 100 μL each. Set 2 wells for negative control serum, add negative control serum, 100 μL/well; 2 wells for positive control serum, add positive control serum, 100 μL/well. Set one blank control well, add 100 μL sample diluent solution. Mix gently by shaking the plate manually, incubate at 37 ℃ for 30 min.
2 Pour the liquid out of the wells, add washing solution to each well, 300 μL/well, static for 1 min. Repeat 5 times, then pat to dry on absorbent paper.
3 Add 100 μL PRV enzyme conjugate(anti-pig IgG-HRP combination) in each well, and incubate at 37 ℃ for 30 min.
4 Repeat step 2(washing).
5 Add 50 µL substrate A(one drop) and 50 µL substrate B(one drop) into each well, mix gently by shaking the plate manually , incubate for 10 min at 37 ℃ in the dark.
6 Add 50 μL stop solution (one drop) in each well, and measure the result within 10 min.
9. Results
l Generally speaking, the PRV-Positive control OD value should be greater than 0.40, the PRV-Negative control OD value should be less than 0.1.
l Cut-Off Value (C.O.) = 0.15+ PRV-Negative control mean(PC:OD450).
Note: All OD450 value of positive, negative control or sample should be minus the value of blank control, then the value is used for calculation;
If the PRV-Negative control OD value is greater than 0.1, the result should not use. If the two PRV-Negative control OD value are greater than 0.1, please test again;
If the PRV-Negative control OD value is less than 0.05, count as 0.05.
l Results:
If the S/C.O. ≥1, the sample is classified as POSITIVE for PRV antibodies;
If the S/C.O. ＜1, the sample is classified as NEGATIVE for PRV antibodies.
(Note: S is the OD450 value of sample)
Specifications : 96 wells × 2
Expiry date: 12 months.
Storage: Storing at 2-8 ℃, don’t expose in strong light.
Production Date: See outer-packing of the test kit.