Mycoplasma hyopneumoniae(MHP) antibody ELISA kit
1.Usage
The Mycoplasma hyopneumoniae(MHP) Antibody ELISA test kit is used for the detection of the MHP antibody in swine serum; assessment the immunity conditions against MHP virus, serological diagnosis of pig infection in the pig farms and investigation of the epidemiology of the MHP virus.
2. Principle
This kit use indirect ELISA method, MHP antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is MHP specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-MHP antibody, combine with the antigen-antibody complex on the plate; discard the uncombined enzyme conjugate with washing; Add TMB substrate in micro-wells, blue product formed enzymatically, after adding stop solution to stop the reaction, use ELISA reader at 450nm wavelength to measure the absorbance A value in reaction wells .
3. The kit components
| Code | Item | Spec. | Code | Item | Spec. |
| 1 | MHP-AgCoatedplates 96 wells | 1/2 plates | 7 | Negativecontrol | 2 ml |
| 2 | EnzymeConjugate | 11/22 ml | 8 | Positive control | 1.6 ml |
| 3 | 10X Concentrated washing buffer | 100 ml | 9 | Serum dilution plate | 1/2 pieces |
| 4 | Substrate | 11/22 ml | 10 | Adhesive Foil | 2/4 pieces |
| 5 | Sample diluent | 100 ml | 11 | Instruction sheet | 1 piece |
| 6 | Stop solution | 15 ml |
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4. Materials Required But Not Provided
1) Micropipettors: 0.5μL~10μL、10μL~100μL、100μL~1000μL
2) Disposable pipette tips
3) Measuring cylinder: 500 ml
4) 96 wells microplate reader
5) Distilled water or deionied water
6) Bottle or microplate washing machine
5. Samplepreparation
Take animal whole blood, get serum by using regular method, the serum should bright and no hemolysis
6. Washing buffer preparation
Return 10X Concentrated washing buffer into room temperature before use, if there is salt crystals, shake to make it dissolved, then dilute it at 10 times with distilled water or deionized water. The diluted washing buffer can store at 4℃for about 1 week.
7. Sample dilution
At serum dilution plate, dilute serum at 1:100 with sample diluent.
Notice: Negative control and Positive control do not need dilute. Exchange tip after taking sample every time, record the situation of the sample on plate accurately. Shake the sample evenly before adding it.
8. Notes
1) All reagents should be adjusted to the room temperature and shake evenly before using, store at 2-8 ℃after using
2) Do not exchange the reagents from the kits of different lot numbers to use. Avoid reagent pollution when using.
3) Substrate and stop solution may have excitant to skin and eyes, pay attention when using.
4) Do not expose TMB (Substrate) to light and avoid it contact with antioxidants.
5) The wells should avoid damp or touching water after unsealing (Put the un-using microtiter strips back to bag with dehydrator in 2~8 ℃soon )
6) Deal all waste reasonable before dumping to avoid pollution.
7) Strictly adhere to instruction to get best result. All procedure including pipetting, timing and washing etc. must be accurate.
8) Serum dilution plate is disposable, do not use for second time; the MAX volume of it is 300μL/well.
9. TestProcedure
1) Take pre-coated microtiter strips (Can unseal for several time use as per sample quantity), add 100μL diluted serum to test well, meanwhile set 1 well for Negative control, 2 wells for Positive control separately. Add 100 μL Negative/Positive control to its well. Shake gently, do not overflow.
2) Cover andincubate at 37℃ for 30 min.
3) Pour the liquid out of the wells, add 250 μL diluted washing solution to each well, pour out. Repeat 4-6 times, at last pat to dry on absorbent paper.
4) Add 100 μL Enzyme Conjugate to each well, cover andincubate at 37℃ for 30 min.
5) Repeat the step 3(washing). Remember pat to dry on absorbent paper at last.
6) Add 100 μL substrate to each well, mix properly, cover andreact for 10 min at 37℃ at dark.
7) Add 50 μL stop solution in each well, and measure the result within 10 min.
10.Results
Read the OD value with ELISA reader at 450nm(630nm as reference)
For the assay to be valid:
OD Value of Negative control (N)< 0.2, meanwhile average OD value of Positive control (P)>0.4.
Calculation method:
Sample OD value / Positive control OD Average value= S/P value
The result is judged by S/P value:
If S/P≥0.5, it is positive; If S/P< 0.5, it is negative.
Expiry date:12 months.
Storage:Store at 2~8℃, in the dark.
Production Date:On outer-packing of the test kit.