Avian Infectious Bursal Disease virus VP2 antibody (IBDV-VP2 Ab) ELISA kit
Catalog No. LSY-30014
1. Introduction
This kit is used to detect Avian Infectious Bursal Disease virus Neutralizing antibody in chicken serum, to assess antibody condition by Avian Infectious Bursal Disease virus VP2 vaccine in chicken farm and assist diagnosis of serological infected chicken.
Chicken infectious bursal disease virus VP2 protein has a conformational (discontinuous) neutralizing antigenic determinant. Studies have found that antibodies against this antigenic determinant can passively protect chickens from infectious bursal disease virus infection.This kit use competitive ELISA method, composed by the reaction Micro-plate coated with high purity IBDV-VP2 antigen, horseradish peroxidase-labeled anti-chicken IgG and other reagents. The reaction mechanism is the coated antigen binding with IBDV-VP2-Ab in sample, and then with the enzyme-labeled anti-chicken IgG antibody to form a "coated antigen + IBDV-VP2-Ab + anti-chicken IgG HRP antibody" complex, add substrate, it will have coloration by the enzyme catalytic reaction. Color depth is proportional to the amount of IBDV-VP2-Ab, when the sample chromogenic reaction, the results detected by the microplate reader exceeds a set threshold value result judged as positive, indicating that the immune produced antibodies or natural infection exists.
2. The kit components
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1
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IBD antigen coated microplate
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2 plates of 96 wells
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7
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Substrate B solution
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12 ml
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|
2
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Enzyme conjugate
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24 ml
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8
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Stop solution
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12 ml
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3
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101X Concentrated VP2 Monoclonal antibody
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210 uL
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9
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Negative control
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100 uL
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4
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Monoclonal dilution
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24 ml
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10
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Positive control
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100 uL
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5
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10×concentrated washing buffer
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100 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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Substrate A solution
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12 ml
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12
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Instruction
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1 piece
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3. Materials Required But Not Provided
1) Micropipettors and disposable tips: 0.5μL~10μL、10μL~100μL、100μL~1000μL、1mL-10mL
2) Disposable tips
3) 37 ℃ Incubator
4) Measuring cylinder: 500 ml
5) 96 wells microplate reader
6) Distilled water or deionied water
7) Bottle or microplate washing machine
4. Sample preparation
Take animal whole blood, make serum according to regular methods, the serum should be clear, have no hemolysis.
5. Preparation of washing buffer
Return washing buffer to room temperature before use, if there is salty crystals, shake to make the crystals dissolve, then use distilled water or deionized water to dilute it at 10 times. The diluted washing buffer can store for 1 week at 4 ℃.
6. Preparation of VP2 Monoclonal antibody working solution( Prepare for immediately usage)
Firstly calculate the required quantity of VP2 Monoclonal antibody working solution, then dilute the 101X Concentrated VP2 Monoclonal antibody with Monoclonal dilution at 1:101 (For example, 900ul Monoclonal dilution + 9uL 101X Concentrated VP2 Monoclonal antibody, which can meet the quantity of 10 wells; It should dilute 100ul more for each dilution to avoid the loss during adding sample and cause insufficient liquid).
7. Notes
1) All reagents should be adjusted to the room temperature and shake evenly before using, store back 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 be excitant to skin and eyes, pay attention when using.
4) Do not expose Substrate 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 including pipetting, timing and washing etc. must be accurate.
8. ELISA procedure
1) Take pre-coated microplate (Can unseal for several time use as per sample quantity), add 10μL serum sample tosample wells, meanwhile set 1 well forNegative control, Positivecontrol and blank control well separately. Add 10μL Negative/Positivecontrol to its wells,then immediately add 90μL Monoclonal dilution; only add 100μL Monoclonal dilution in the blank control well.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 buffer 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,coverand incubate 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.
9. Results
Set zero for the blank well, and test theA450nm(630 nm as a reference)value on the microplate-reader.
For the test to be valid:
OD value of Negative control (N) >1.0;
OD value of Positive control (P)<0.7.
If the test is invalid, the operation is in doubt, retest and observe all the reagents carefully.
Calculation method:
OD value of samples/ Average OD value of Negative control = S/N value
Result judge:
S/N ≥ 0.7, Negative;
S/N＜0.7, Positive.
Specifications:96*2 wells/kit.
Expiry date:12 months.
Storage: Storing at 2-8℃, in the dark.