Newcastle disease virus (NDV) antibody ELISA kit  

Catalog No. LSY-30013

1. Brief

This kit is used to detectNewcastle disease virus(NDV) antibody in serum of poultry, to assess antibody condition byNewcastle disease virus(NDV) vaccine poultry and assist diagnosis ofserological infected poultry.

This kit use blocking ELISA method, it can detect Newcastle disease antibodies in the serum of chickens, ducks, pigeons and other poultry. NDV antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there isNDV specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labeled anti-NDV 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 wavelength 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 platesshould be sealed andmoisture-proof. Put backunused Micro-Well plateintodry foil bagandsealedat 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) NDV 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,90ul/well, then add serum sample to the reaction wells,10ul/well, blow and mix evenly(Do not mix using tips)

3) Cover it with Adhesive Foil, incubate at37℃ for 60minutes;

4) Open the adhesive foil,discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, thendiscard the liquid, repeat the above step for4-6 times, at lastflap to dry with the absorbent paper;

5) Adding EnzymeConjugate 100ul/well, Cover it with Adhesive Foil,incubateat37℃ for 60 minutes;

6) Open the adhesive foil,discard the liquid of the well, washing for4-6 times as step 4), remember at lastflap to dry with the absorbent paper;

7) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat37℃ in darkfor15 minutes;

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

8. Results

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

For the test to be valid:

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≥0.5, Negative;

S/N < 0.5, Positive.

Specifications: 96 or 96*2 wells/kit.

Expiry date:12months.

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