1. Usage

This kit is used to detect Avian Marek's disease virus antibody in chicken serum, to assess antibody condition by Avian Marek's disease vaccine in chicken farm and assist diagnosis of serological infected chicken.

2. Principle

The Avian Marek's disease 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

1)         Antigen coated microplate................................................ 1 piece (96 wells)

2)         Enzyme conjugate.................................................... 1 bottle (12 mL/bottle)

3)         10×concentrated washing buffer…………………………1 bottle (50 mL/bottle)

4)         Substrate A /B........................................................ 1 tube each (6 mL/tube)

5)         Sample diluent……………………………………………. 1 bottle (50 mL/bottle)

6)         Stop solution................................................................. 1 tube (6 mL/tube)

7)         Negative control serum................................................ 1 tube (800μL /tube)

8)         Positive control serum................................................. 1 tube (800μL /tube)

9)         Serum diluent plate……………………………………………………..…..1 piece

10)     Adhesive film……………………………………………………………….2 pieces

11)     Instruction……………………………………………………………………1 piece

4. Materials required but not provided

1) Micropipettors and disposable tips: 0.5μL~10μL、10μL~100μL、100μL~1000μL

2) 37 ℃ Incubator

3) Measuring cylinder: 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 buffer to room temperature before use, if there is salty crystals, shake to make the crystals dissolve, then use distilled water or deionied water to diluent it at 10 times. The diluent washing buffer can store for 1 week at 4 ℃.

7. Sample dilution

At serum diluent plate, dilute serum at 1:101 with sample dilution (for example: 200μL sample diluent + 2μL serum)

Notice: Negative control serum and Positive control serum 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 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 including pipetting, timing and washing etc. must be accurate.

8) Serum diluent 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 for Negative control serum, Positive control serum and blank control wells separately. Add 100 μL Negative/Positive control serum to its wells, only add 100μL sample diluent buffer in the blank control wells. Shake softly, incubate at 37℃ for 30 min.

2) Pour the liquid out of the wells, add 350 μL diluted washing solution to each well, static for 1 min, pour out. Repeat 3 times, then pat to dry on absorbent paper.

3) Add 100 μL Enzyme conjugate (except blank wells) to each well, and incubate at 37℃ for 30 min.

4) Repeat the step 2(washing). Remember pat to dry on absorbent paper at last.

5) Add 50 μL substrate A, then substrate B (50 μL) to each well, mix properly, react for 10 min at room temperature at dark.

6)       Add stop solution one drop (50 μL) in each well, and measure the result within 10 min.

10. Results

Set zero for the blank well, and test the OD450nm (recommend 450nm and 630 nm) value on the microplate-reader. The conditions for the test to be tenable are that the positive control wells’ average OD450nm value is greater than or equal to 0.6, and the negative control wells’ average OD450nm value is less than 0.15.(all value should minus the average value of blank control)

If the sample’s A450 value is greater than 0.2+ absorbance of negative control, it is judged to be positive; and if less than 0.2, negative. If absorbance of negative control is less than 0.05, calculate as 0.05

Specifications: 96 wells/kit.

Expiry date: 6 months.

Storage: Storing at 2-8℃, in the dark.