1. Usage
Porcine Toxoplasmosis IgG antibody test kit is used for the detection of the Toxoplasmosis antibodies in the serum.
2. Principle
The test kit is enzyme immunoassay for the detection toxoplasmosis antibodies in porcine serum. Toxoplasmosis antigens are coated on 96-well plate, add sample into wells and then antibodies bound specifically by antigens, the unbound antibodies and other components will be removed by washing. Next add enzyme conjugation to specifically bind with the compound of antibodies and antigens on the microplate. The unbound enzyme conjugation will be removed by washing. Add the TMB substrate in the well, react to a blue product. At last, end the reaction by adding stop solution.
3. Reagents of the Test Kit
1 Antigen coated microplate....................................... 2 pieces (96 wells each)
2 Negative serum(green lid)..................................................... 1 tube (1.5 mL)
3 Positive serum (red lid)......................................................... 1 tube (1.5 mL)
4 Enzyme conjugate (yellow lid).............................................. 1 bottle (22 mL)
5 20×Wash Concentrate (white lid)........................................... 1 bottle (50 mL)
6 Substrate A (red lid)/B (black lid)......................... 1 bottle each (12 mL/bottle)
7 Stop solution(blue lid).......................................................... 1 bottle (12 mL)
8 Sample diluent solution(transparent lid)................................ 1 bottle (25 mL)
9 Adhesive Foil................................................................................. 6 pieces
10 Instruction...................................................................................... 1 piece
4. Material Required not Provided
1 50μL-,100μL-,1000-micropipettes,adjustable.
2 Distilled water.
3 Constant temperature box or incubator.
4 Microtiter plate spectrophotometer
5 Absorbent paper.
5. Precautions and warnings for users
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. Others are wells for samples, add 100μL/well of Sample diluent solution, then add 10 μL serum sample seperately.
2 Mix gently, incubate at 37℃ for 30 min.
3 Remove adhesive foil. Pour the liquid out of the wells, add Washing solution(dillute the Wash Concentrate at 1:19 with distilled water) into each well, 300 μL/well, stand for 1 min. Repeat 5 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 15 min at 37 ℃ 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 5-30 min.
9 Measure the optical density(OD) with a photometer at 450 nm(Reference-wavelength: 630 nm). Set zero for the blank well, and read the OD 450 value of samples on the Microplate reader.
7. ELISA analysis
For the assay to be valid the following specifications must be met. The TOX-Positive control mean (PC:OD450) must be greater than 0.70, the TOX-Negative control mean must be less than 0.10.
Cut-Off Value (C.O.) = 0.15
If the S/C.O. ≥1, the sample is classified as POSITIVE for TOX antibodies;
If the S/C.O. <1, the sample is classified as NEGATIVE for TOX antibodies.
8. Specification
Packing : 96 wells×2.
Expiry date: 12 months.
Storage: Storing at 2~8 ℃ in the dark, not freeze.
9. Warning and Procautions
1. The test kit is only for laboratory use, not for medical.
2. The waste should be treated by High-pressure steam sterilization for 30 min before discard.
3. Never mix up reagents from test kits in different batch number.
4. If the Stop solution splash on skin or clothes, wash with plenty of water immediately.
5. Do not pollute on the test procedure.