Competitive ELISA Kit For Foot and Mouth Disease Virus Type O Antibodies
1.Usage
The Green® Competitive ELISA Kit For Foot and Mouth Disease Virus Type O Antibodiesis usedfor detection ofFMD Type Oantibody in serum of pig, bovine and goat.
2. Principle
The coated plate is coated with FMD-type O recombinant protein antigen, and the specific antibody in the sample to be tested competes with the enzyme-labeled monoclonal antibody for binding to the recombinant protein antigen on theplates. If the sample to be tested contains foot-and-mouth disease type O antibody, the color is light,it ispositive, otherwise it is negative.
3. The kit components
| 1 | FMDV-O antigencoated microplate | 96T X 2 | |
| 2 | FMDV-O monoclonal antibody Enzyme conjugate | 12ml | yellow lid |
| 3 | Sample diluent | 50ml | transparent lid |
| 4 | FMDV-O Negativecontrol | 1.5ml | green lid |
| 5 | FMDV-O Positivecontrol | 1.5ml | red lid |
| 6 | Substrate | 12ml X2 | orange lid |
| 7 | Stop solution | 12ml | blue lid |
| 8 | 10×concentrated washing buffer | 50ml | white lid |
| 9 | Adhesive Foil | 2 pieces |
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| 10 | Instruction | 1 piece |
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4. Materials Required But Not Provided
1) Microplate Reader (double-wave length: 450/630 nm).
2)Precise micropipette (single-channel 1-100ul、0.5-10ul、multi-channel 30-300ul)
3)Constant temperature box orwater bath box.
4) Oscillator.
5) Disposable tips (10ul, 200ul)
6) Deionized water
5. Sample requirement
1) The samples areanimal serum, which should be collected with nobacteria. The storage time should be less than3 days at 2-8℃, if for long term, it should be kept at -20℃, avoid repeated freezing and thawing.
2) Avoid to use the samples with severe hemolysis, precipitate, contaminated by bacteria or protein suspension.
6. Preparation
1) BringELISA reagents to the room temperature (20-25℃) for 30 min to get best results. Microplate should return to room temperature and dry before open package.
2) Sample dilute:take animal whole blood,prepare serum according to the conventional method. The serum is required to be clear and without hemolysis.Then diluteserumsample with thesample diluent at 4 times.(for example:25ul serum +75ul sample diluent). Negative and Positive control do not need dilute.
3) Washing solution preparation: Dilute the10×concentrated washing buffer with deionized water at10 times. (for example:10ml10×concentrated washing buffer +90ml deionized water ) It is normal if there is crystallization in the10×concentrated washing buffer, put at 37℃until completely dissolved.
7.Procedure
1)Add sample:Take out the needed coated plates (Can be detached) according to sample quantityand record the sample position on a worksheet. When testing, set 2 wells for negative control, add undilutednegative control serum, 2 wells for positive control, add undilutedpositive control serum, 50μL/well. Others are sample wells, add the diluted serumsample, 50μL each.Meanwhile addFMDV-O monoclonal antibody Enzyme conjugate into each well, shake and mix it evenly, cover plate with adhesive foil,incubate at 37℃ for 30 min. Both single-well and double-well test are OK for serum sample.
2)Washing plate: Remove adhesive foil.Pour the liquid out of the wells, addthe dilutedWashingbuffer into each well, 300ul/well,pour out. Repeat5 times, at last time pat to dry on absorbent paper.
3)Add substrate: Add substrate100ulinto each well, mix properly, cover plate with adhesive foil, incubate for 15 min at37℃ in dark.
4)Stop reaction:Add stop solution 50μL into each well, and determine the result within 10 minutes.
8. Results
Read OD450/630value of each well by Microplate.For the assay to be valid,average OD450/630 value of Positive control≤0.3, average OD450/630 value of Negative control≥1.0.
The result is judged by S/N value,
S meansOD450/630value of serum sample, N means average OD450/630value ofNegative control,
IfS/N≤0.35, it is positive;ifS/N＞0.35, it is negative.
9. Precautions and warnings for users
1).Return all reagents into room temperature, shake to mix the reagents evenly before usage, put it back to 2~8℃after usage.
2). Reagent components of different varieties and different batches shall not be mixed. Reagent pollution shall be prevented when using reagents.
3). Thesubstrate and the stop solution may be irritating to the skin and eyes,take care when using it.
4). TMB (substrate) should not be exposed to strong light and avoid contact with oxidizing agents.
5). After unseal the plate, avoid moisture or water (unused antigen-coated plate plus desiccant should be placed in a self-sealing bag and placed at 2-8℃as soon as possible).
6). All wastes should be treated reasonably before being discarded to avoid pollution.
7). Strictly follow the instruction to get the best result. During the operation, all processes such as pipetting, timing and washing must be precise.
Specifications:96 wells×2.
Expiry date:12 months.
Storage:Store at 2~8℃, in the dark,no freezing.
Production Date:On outer-packing of the test kit.