1. Usage
The BIOKAU® porcine circovirous ELISA Test kit is used for detection of porcine circovirus antibodies in the serum; assessment of immunity conditions against porcine circovirus in the pig farm and investigation of the epidemiology of the porcine circovirus.
2. Principle
The BIOKAU® PCV2 ELISA 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 (96 wells)
2 Negative (green lid)/positive (red lid)serum............. 1 tube each (1.5 mL/tube)
3 Anti-pig IgG-HRP conjugate (yellow lid)....................... 1 bottle (22 mL/bottle)
4 20×concentrated washing buffer (white lid).................. 1 bottle (50 mL/bottle)
5 Substrate A (purple lid)/B (yellow lid)................... 1 bottle each (12 mL/bottle)
6 Stop solution(blue lid)................................................ 1 bottle (12 mL/bottle)
7 Sample diluent solution(transparent lid)....................... 1 bottle (50 mL/bottle)
8 Adhesive Foil................................................................................. 6 pieces
9 Instruction....................................................................................... 1 piece
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
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: Store at 2~8℃, in the dark.
Production Date: On outer-packing of the test kit.