Infectious Bronchitis Virus (IBV) antibody ELISA kit
Catalog No. LSY-30017
1. Usage
This kit is used to detect Infectious Bronchitis Virus (IBV) antibody in chicken serum, to assess antibody condition by Infectious Bronchitis vaccine in chicken farm and assist diagnosis ofserological infected chicken.
2. Principle
The Infectious Bronchitis Virus (IBV) antibody ELISA kit is based on an indirect enzymatic immunoassay (Indirect ELISA).The antigen is coated on plates. When a sample serum contains specific antibodies against virus, they will bind to the antigen on plates. Wash the unbound antibodies and other components. Then add a specific enzyme conjugate. After incubation and washing, add the TMB substrate. A colorimetric reaction will appear, measured by a spectrophotometer (450 nm).
3. Reagents
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1
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IBV antigen coated microplate
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2 plates of 96 wells
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7
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Stop solution
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12 ml
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|
2
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Enzyme conjugate
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24 ml
|
8
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Negative control
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1.5ml
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3
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10×concentrated washing buffer
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100 ml
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9
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Positivecontrol
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1.5ml
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4
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Substrate A solution
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12 ml
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10
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Serum dilution plate
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2 piece
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5
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SubstrateB solution
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12 ml
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11
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Adhesive film
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4 pieces
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|
6
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Sample dilution
|
100 ml
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12
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Instruction
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1 piece
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4.Materials required but not provided
1)Micropipettors and disposable tips: 0.5μL~10μL、10μL~100μL、100μL~1000μL
2) 37℃Incubator
3) Measuringcylinder: 500 ml
4) 96 wells microplate reader
5) Distilled water or deionied water
6)Bottle or microplate washing machine
5. Sample preparation
Take animal whole blood, make serum according to regular methods, the serum should be clear, have no hemolysis.
6. Preparation of washing buffer
Return washing solution to room temperature before use, if there is salty crystals, shake to make the crystalsdissolve, then use distilled water or deionized water to dilute it at 10 times. The diluted washing solution can store for 1 week at 4℃.
7. Sample dilution
At serum dilution plate, dilute serum at 1:101 with sample dilution (for example: 200μL sampledilution + 2μL serum)
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 temperatureand shake evenlybefore 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 B) to light and avoid it contact with antioxidants.
5) The wells should avoid damp or touching water after unsealing (Put the un-using microplate 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 includingpipetting, 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. ELISA procedure
1) Take pre-coated microplate (Can unseal for several time use as per sample quantity), add 100μL diluted serum to a well, meanwhile set 1 wells forNegative control, Positivecontrol and blank control wells separately. Add 100μL Negative/Positivecontrol to its wells, only add 100μL sample dilution in the blank control wells.Shake softly (do not spill),
2) Cover and incubate at 37℃ for 30 min.
3)Pour the liquid out of the wells, add about 350 μL diluted washing solution to each well fully, static for1 min, pour out. Repeat3 times, then pat to dry on absorbent paper.
4)Add 100 μLEnzyme Conjugate to each well,coverandincubate at 37℃ for 30 min.
5)Repeatthestep3(washing). Rememberpat to dry on absorbent paper at last.
6)Add 50 μL substrate A, then substrateB (50 μL) to each well, mix properly,cover andreact for 10 min at 37℃ in dark.
7)Add 50 μL stop solution in each well, and measure the result within 10 min.
10. Results
Set zero for the blank well, and test theA450nm(630 nm as a reference)value on the microplate-reader. The conditions for the test to be tenable are that the positive control wells’A450nm value isgreater than or equal to 0.5, and the negative control wells’A450nm value isless than0.15. If the test is invalid, the operation is in doubt, retest and observe all the reagents carefully.
If the sample’s A450 value is greater than 0.15+absorbance of negative control mean, it is judged to be positive; and if less than 0.15+absorbance of negative control mean,negative. Ifabsorbance of negative control mean is less than 0.05, calculate as 0.05
Specifications: 96 wells/kit or 96*2 wells/kit
Expiry date:12months.
Storage: Storing at 2-8℃, in the dark.