Swine Foot And Mouth Diseasetype O antibody ELISA Kit
1.Usage
The Green® Porcine Foot And Mouth Disease type O antibody ELISA Test kit is used for detection of Porcine Foot And Mouth Disease Type O antibody in porcine serum; assessment of immunity conditions against porcine FMD in the pig farm and investigation of the epidemiology of the porcine FMD.
2. Principle
This test kit is based on the principle of solid-phase enzyme-linked immunosorbent assay (ELISA), consisting of microwell reaction plate coated with VP1 structural protein of pig FMD Type O, horseradish peroxidase-labeled anti-pig IgG and other reagents. The reaction mechanism is coated antigen combine with FMDV-VP1-Ab in the sample, then combine with enzyme labled anti-pig IgG to form a complex of “coated antigen+FMDV-VP1-Ab+ enzyme-labeled anti-pig IgG antibody”, add substrate, color appear by enzyme catalysis reaction. The color depth is directly proportional to the amount of FMDV-VP1-Ab. When the sample reaction is chromogenic, the result is positive when the results measured by ELISA reader exceed the set threshold value, which means that immunity has produced antibodies or natural infection exist.
3. The kit components
| 1 | FMD-O antigen coated microplate | 96T X 2 | |
| 2 | Enzyme conjugate | 22ml | yellow lid |
| 3 | Sample diluent | 50ml | transparent lid |
| 4 | FMD-O-IgGNegativecontrol serum | 1.5ml | green lid |
| 5 | FMD-O-IgGPositivecontrol serum | 1.5ml | red lid |
| 6 | Substrate | 12ml X2 | orange lid |
| 7 | Stop solution | 12ml | blue lid |
| 8 | 20×concentrated washing buffer | 50ml | white lid |
| 9 | Adhesive Foil | 2 pieces |
|
| 10 | Instruction | 1 piece |
|
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 or water bath box.
4) Oscillator.
5) Disposable tips (10ul, 200ul)
6) Deionized water
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.
6. Preparation
1) Bring ELISA 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: Dilute sample with the sample diluent at 40 times.(for example: 5ul serum + 195ul sample diluent).
3) Washing solution preparation: Dilute the 20×concentrated washing buffer with deionized water at 20 times. (for example: 50ml 20×concentrated washing buffer + 950ml deionized water ) It is normal if there is crystallization in the 20×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 quantity and record the sample position on a worksheet. When testing, set 2 wells for negative control, add undiluted negative control serum, 2 wells for positive control, add undiluted positive control serum, 100μL/well (It is OK not to set blank control). Others are sample wells, add the diluted sample, 100μL each.
2)Incubation: Mix gently, cover andincubate at 37℃ for 30 min.
3)Washing plate: Remove adhesive foil. Pour the liquid out of the wells, add the diluted Washing buffer into each well fully, be static for 10s,pour out. Repeat 3 times, at last time pat to dry on absorbent paper.
4)Add Enzyme Conjugate: Add 100μL enzyme conjugate into each well.
5)Incubation: Cover plate with new adhesive foil.Incubate at 37 ℃ for30 min.
6)Washing plate:Repeat step 3.
7)Add substrate: Add substrate 100ul into each well, mix properly,incubate for 10 min at37 ℃ in dark.
8)Stop reaction:Add stop solution 50μL into each well, mix gently and determine the result.
9)Read results: Measure the OD value of each well with a ELISA reader at dual-wave length 450nm/630nm.
8.Results
For the assay to be valid, the positive control wells’ average OD value must be greater than or equal to 0.6, and the negative control wells’ average OD value is less than 0.1. Otherwise the test is invalid, need test again.
The result is judged by S/P value,
S/P=(Sample OD450/630- NCx(—))/( PCx(—)- NCx(—)), NCx(—) means Negative control’s average OD450/630 value, PCx(—) means Positive control’s average OD450/630 value
If S/P≥0.15, it is positive; less than 0.15, it is negative.
9. Precautions and warnings for users
1. This test kit is for research use only.
2. Do not use reagents expired, do not mix reagents from different lots.
3. Read the Manual carefully before use.
4. Experiment rubbish should be dealt with high pressure steam sterilization at 121 ℃ for 30 minutes, or treated with 5.0g/L sodium hypochlorite disinfectant for 30 minutes, then discard.
5. MicroWell plate removed from the refrigerated environment should be balanced moisture to dry at room temperature, then can be opened. Put back unused MicroWell plate into dry foil bag and sealed at 2~8 ℃. Unused liquid reagent should cover caps, store at 2-8 ℃ in dark with other group components.
6. Should use Micropipettor to add sample and reagents, and often proof its accuracy.
7. When adding washing buffer, should be full but no overflow, avoid appearing free enzyme at mouth of well or cross pollution between wells.
8. Stop solution is corrosive, use large amount of water to wash immediately when touch the skin or clothes.
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.