1. Introduction
Porcine Parvovirus (PPV) is a major cause of reproductive failure in swine. The PPV is a very resistant virus that can survive without a host for several months, which makes it difficult to remove the virus from the pig herd and its environment. PPV does not cause clinical signs at any other time than during pregnancy when it may cross the placenta and infect developing embryos and foetuses resulting in resorption and mummification, abortion, stillbirth, neonatal death and reduced neonatal vitality. A vaccination programme in combination with herd monitoring provides the best method for preventing PPV-induced reproductive failure.
The BIOKAU™ PPV-Ab ELISA is developed to detect Porcine Parvovirus specific antibodies in porcine serum samples and assess the immunity conditions against porcine parvovirus in the pig farms.
2. Principle
The kit is based on an indirect enzymatic immunoassay(Indirect ELISA).The antigen is coated on plates. When a sample serum contains specific antibodies against virus, they will bind to the antigen on plates. Wash the unbound antibodies and other components. Then add a specific peroxidase conjugate(IgG-HRP). After incubation and washing, add the TMB substrate. A colorimetric reaction will appear, measured by a spectrophotometer(630 nm).
3. The kit components
1 Antigen coated microtiter plate....................................... 2 pieces (192 wells)
2 Negative control serum (green lid)........................................ 1 tube (1.5 mL)
3 Positive control serum (red lid)............................................ 1 tube (1.5 mL)
4 Anti-pig IgG-HRP conjugate(yellow lid)................................. 1 bottle (22 mL)
5 20×concentrated washing buffer (white lid)........................... 1 bottle (50 mL)
6 Substrate A (purple lid)/B (yellow lid)......................................... (12 mL/each)
7 Stop solution (blue lid)......................................................... 1 bottle (12 mL)
8 Sample diluent (transparent lid)............................................ 1 bottle (50 mL)
4. 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.
5. Sample requirement
1 The samples are porcine serum, which should be collected with no bacteria. The storage time should be less than 1 week at 2-8 ℃, if for long term, it should be kept at -20℃.
2 Avoid to use the samples with severe hemolysis, precipitate, contaminated by bacteria or protein suspension.
3 The EDTA, heparin sodiun and other anticoagulants will not affect the results.
6. Procedure
1 Take out the coated plates(Can be detached) and record the sample position on a worksheet. Set one blank control well, add nothing. Set 2 wells for negative control serum and 2 wells for positive control serum, 100 μL/well. Dilute the sample at 1:39 with the sample diluent and add to the wells, 100 μL each.
2 Mix gently, incubate at 37℃ for 30 min.
3 Remove adhesive foil. Pour the liquid out of the wells, add Washing buffer(dillute the 20×concentrated washing buffer at 1:19 with distilled water) into each well, 300 μL/well, stand for 3 min. Repeat 3 times, at last time pat to dry on absorbent paper.
4 Add 100 μL enzyme comjugate into each well(except the blank well).
5 Cover plate with new adhesive foil. Incubate at 37 ℃ for 30 min.
6 Repeat step 3(washing).
7 Add substrate A one drop (50 μL) and substrate B one drop (50 μL) into each well, mix properly, incubate for 10 min at 25 ℃ in the dark with new adhesive foil.
8 Add stop solution one drop (50 μL) into each well, mix gently and determine the result within 10 min.
9 Measure the optical density(OD) with a photometer at 630 nm(Set zero for the blank well, and read the OD 630 value of samples on the Microplate reader).
7. Results
Set zero for the blank well, and test the OD630 value on the spectrophotometer. For the assay to be valid, the positive control wells’ average OD630 value must be greater than or equal to 0.4, and the negative control wells’ average OD630 value is less than 0.2.
If the sample’s OD630 value is greater than 0.4, it is judged to be positive; from 0.2 to 0.4, doubtful; and if less than 0.2, negative.
Specifications: 96 wells×2.
Expiry date: 6 months.
Storage: Storing at 4℃, in the dark.