Poultry disease diagnostic Bird Flu H5 subtype antibody ELISA test kit (Work for Chicken, duck, goose, etc.)

Bird Flu H5 subtype antibody ELISA test kit

(Work for Chicken, duck, goose, etc.)

Catalog No. LSY-30011

1. Brief

This kit is used to detect Bird Flu H5 subtype antibody in serum of poultry, wild birds etc., to assess antibody condition by Bird Flu H5 vaccine poultry and assist diagnosis of serological infected poultry.

This kit use blocking ELISA method, H5 antigen is pre-coated on enzyme micro-well strips, indirect detection of anti-H5 antibodies in serum by anti-monoclonal antibodies compete with antibodies in serum for binding sites in solid-phase vectors. When testing, add diluted serum sample, after incubation, if there is H5 virus specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-H5 virus monoclonal antibody, antibody in sample block the combination of monoclonal antibody and pre-coated antigen; discard the uncombined enzyme conjugate with washing; Add TMB substrate in micro-wells, the blue signal by Enzyme catalysis is in inverse proportion of antibody content in sample, use ELISA reader at 450nm wavelenth to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

2. Reagents

3. Materials required but not provided

1) Micropipette: 10ul-100ul, 100ul-1000ul.

2) Disposable pipette tips.

3) Graduate: 500ml.

4) Microplate Reader: 96 wells with 450/630nm wavelength.

5) Distilled water or deionized water.

6) Bottle washer or Microplate Washer

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 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.

6. Notes

1) Return all reagents into room temperature before use, put the reagents at room temperature for at least 1 hour. Shake it evenly before use, and store back to 2-8℃ after usage.

2) Do not mix use reagents from different kits and different lot no., prevent the reagents been polluted when using.

3) Substrate and stop solution may have irritation to skin and eyes, be careful to use.

4) Do not expose Substrate to strong light and avoid contact with the oxidant.

5) H5 Ag coated plates should be sealed and moisture-proof. Put back unused Micro-Well plate into dry foil bag and sealed at 2-8 ℃.

6) All wastes should be treated well to avoid pollution before discarding.

7) Strict compliance with the operating instructions can get the best results. Pipetting operation, timing, and washing of the whole process must be precise.

8) H5 Ag Coated plates is disposable, do not repeat use.

7. ELISA procedure

1) Take the antigen coated plate(the plate can be open and used for several times according to sample quantity each time), for every test, set 1 well for positive control and 2 wells for negative control, positive control and negative control do not need dilute, take 100ul directly and add into its well;

2) Add Sample dilution to reaction wells, 50ul/well, then add serum sample to the reaction wells, 50ul/well, blow and mix evenly(Do not mix use tips)

3) Cover it with Adhesive Foil, incubate at 37℃ for 120minutes;

4) Open the adhesive foil, discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, then discard the liquid, repeat the above step for 6 times, at last flap to dry with the absorbent paper;

5) Adding Enzyme Conjugate 100ul/well, Cover it with Adhesive Foil, incubate at 37℃ for 30 minutes;

6) Open the adhesive foil, discard the liquid of the well, washing for 6 times as step 4), remember at last flap to dry with the absorbent paper;

7) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil, incubate at 37℃ in dark for 15 minutes;

8) Add stop solution 50ul/well to stop the reaction, measure the result in 10 minutes.

8. Results

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

For the test to be valid:

Average OD value of Negative control (N) >0.4;

meanwhile OD value of Positive control (P)/ Average OD value of Negative control (N)<0.4.

Calculation method:

OD value of samples/ Average OD value of Negative control = S/N value

Result judge:

S/N ≥ 0.55, Negative;

S/N < 0.55, Positive.

Specifications: 96 or 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