PorcineFoot-and-Mouth Disease Virus 3ABCAntibodyELISA Test Kit
1.Usage
The Green® Porcine Foot-and-Mouth Disease Virus 3ABC ELISA Test Kit is used for detection of FMD type O non-structural 3ABC-IgG antibody in porcine serum qualitatively; it is used to distinguish Porcine FMD antibody between wild virus infection and produced by inactivated vaccine.
2. Principle
The test kit use microtiter plate which coated with pig FMD non-structural 3ABC gene expression product, when testing, add diluted control serum and serum sample, after incubation, if there is pig FMD non-structual 3ABC specific antibody in the sampl, it will combine with antigen coated on plate, after washing and removing unbonded antibodies and other components; then add enzyme conjugate, specific binding with antigen-antibody complex on plate; after washing to remove unbound enzyme conjugate, add TMB substrate, form blue product by enzyme reaction, stop by 2M H2SO4, read OD value of each well by ELISA reader at double-wave length 450/630nm, the OD value will reflect the level of 3ABC antibody in porcine serum.
3. The kit components
| 1 | FMD-3ABC antigencoated microplate | 96T X 2 | |
| 2 | Enzyme conjugate | 22ml | yellow lid |
| 3 | Sample diluent | 50ml | transparent lid |
| 4 | 3ABC-IgGNegativecontrol serum | 1.5ml | green lid |
| 5 | 3ABC-IgG Positivecontrol serum | 1.5ml | red lid |
| 6 | Substrate | 12mlX2 | orange lid |
| 7 | Stop solution | 12ml | blue lid |
| 8 | 20×concentrated washing buffer | 50ml | white lid |
| 9 | Adhesive Foil | 2pieces |
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| 10 | Instruction | 1 piece |
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Note: do not mix use reagents from different lot no.
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.
3) The EDTA, heparin sodiun and other anticoagulants will not affect the results.
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(both single-well and double-well test are OK).
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 0.15. 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≥0.3, it is positive; less than 0.3, it is negative.
9.Interpretation of the result
1) Severe hemolysis, fiber protein in the serum separation is not sufficient, containing erythrocytes, a precipitate, a sample with bacteria may lead to false positive.
2) Negative results may occur on individual pigs after vaccines due to individual differences or immune duration.
3) Positive results for serological diagnosis and epidemiological investigation of swine to be combined with other methods and clinical data.
10.Product performance
1) Specificity: to test 30 negative control serums, no false positive.
2) Sensitivity: to test 30 positive control serums, no negative.
3) Precision: CV（%）no bigger than 15%（n=10）
4) Stability: Store at 2℃～8℃ for 12 months or store at 37℃ for 6 days, the result can reach the above 3 standards.
11. Precautions and warnings for users
1. This test kit is for research use only.
2. Wear glove and working clothes when operate, treat the test kit as containing infectious material.
3. 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.
4. 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 4 ℃. Unused liquid reagent should cover caps, store at 2-8 ℃ in dark with other group components.
5. If the 20×concentrated washing buffer appears crystal, it is normal, put at 37℃until been dissolved.
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.