Avian EDS76 antibody ELISA kit egg drop syndrome detection

Avian EDS76 antibody ELISA kit

Catalog No. LSY-30015

1. Usage

The Egg drop syndrome virus 76(EDS76) antibody ELISA kit is used to detect Avian EDS76 virus antibody in chicken serum, to assess antibody condition by Avian EDS76 vaccine in chicken farm and assist diagnosis of serological infected chicken.

2. Principle

The Avian EDS76 antibody ELISA kit is based on an indirect enzymatic immunoassay (Indirect ELISA).The purified EDS76 antigen is coated on plates. When testing, add the diluted serum sample, after incubation, if the serum sample contains specific antibodies against EDS76 virus, they will bind to the antigen coated on plates. Wash the unbound antibodies and other components. Then add a specific enzyme conjugate, it will combine the antigen-antibody combination on plates. After incubation and washing, discard the uncombined enzyme conjugate; add the TMB substrate. A colorimetric reaction will appear, add stop solution, measured OD value by a spectrophotometer (450 nm).

3. Reagents

4. Materials required but not provided

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

2) Disposable tips

3) 37 ℃ Incubator

4) Measuring cylinder: 500 ml

5) 96 wells microplate reader

6) Distilled water or deionied water

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 deionized water to dilute it at 10 times. The diluted washing buffer can store for 1 week at 4 ℃.

7. Sample dilution

At serum dilution plate, dilute serum at 1:100 with sample dilution (for example: 297μL sample dilution + 3μL 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 temperature, put all reagents at room temperature for at least 1 hour, and shake evenly before using, store at 2-8 ℃ after using

2) Disposable tips should be replaced for each sample when aspirating additional samples.

3) Do not exchange the reagents from the kits of different lot numbers to use. Avoid reagent pollution when using.

4) Substrate and stop solution may be excitant to skin and eyes, pay attention when using.

5) Do not expose Substrate to light and avoid it contact with antioxidants.

6) The wells should avoid damp or touching water after unsealing (Put the un-using microplate back to bag with desiccant in 2~8 ℃ soon )

7) Deal all waste reasonable before dumping to avoid pollution.

8) Strictly adhere to instruction to get best result. All procedure including pipetting, timing and washing etc. must be accurate.

9) 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 sample well, meanwhile set 1 well for Negative control, 2 wells for Positive control separately. Add 100 μL Negative/Positive control serum to its wells accordingly. Shake softly, do not overflow, cover plate with adhesive film, incubate at 37℃ for 30 min.

2) Unseal the adhesive film, pour the liquid out of the wells, add 250 μL diluted washing solution to each well, then pour out. Repeat 4-6 times, at last pat to dry on absorbent paper.

3) Add 100μL Enzyme conjugate to each well, shake softly, cover plate with adhesive film and incubate at 37℃ for 30 min.

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

5) Add 100μL substrate to each well, mix properly, cover plate with adhesive film and react for 10 min at dark at 37℃ in dark.

6. Add 50 μl of stop solution in each well, and measure the result within 10 min.

10. Results

Read the OD value with microplate-reader at 450nm (630nm as reference).

For the test to be valid:

OD value of Negative control (N) < 0.2, meanwhile OD value of Positive control (P) 〉0.4.

Calculation method:

OD value of samples/ Average OD value of Positive control = S/P value

Result judge:

S/P ≥ 0.35, Positive.

S/P < 0.35, Negative;

Specifications: 96*2 wells/kit.

Expiry date: 12 months.

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

Shenzhen Lvshiyuan Biotechnology Co., Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788 Fax 86-755-28938800

Email: info@lsybt.com

www.lsybt.com