1. Principle of the Test

This kit is used to detect specific antibody against Newcastle disease virus NDV Antibody in serum qualitatively, for monitoring antibody after NDV immune and serological diagnosis of infection in poultry.

This kit use Blocking ELISA method, Pured NDV antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there have NDV 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-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 wavelenth to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

2. Materials Provided

3. Necessary equipment and reagents

1.  Micropipettor: 0.5µl-10µl、10µl-100µl、100µl-1000µl。

2.  Disposable pipette suction head

3.  Graduated cylinder：500ml。

4.  Enzyme mark with 96 microplate.

5.  Distilled water or deionized water

6.  Bottle washing machine

4. Sample preparation

Take animal whole blood, get serum by using regular method, the serum should bright and no hemolysis

5.Washing buffer preparation

Return 10X Concentrated washing buffer into room temperature before use, if  there is salt crystals, shake to make it dissovled, then dilute it at 10 times with distilled water or deionized water. The diluted washing buffer can store at 4℃ for about 1

6.Note

1) Return all reagents into room temperature before use, 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) Brucellosis-Ag coated plates should be sealed and moisture-proof. Put back unused MicroWell 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) Antigen coated plate is disposable, do not repeat use.

7. Procedure of the Test

1) Each experiment set a positive control 1 hole and negative control 2 hole, negative, positive control without dilution, directly take 100ul added to the reaction well.

2) The sample diluent 90ul / hole was added to the microplate reaction plate, then add sample serum 10ul / well into the hole. (pipette tips do not mix)

3) Cover the wells with plate sealer and incubate for 60mins at 37℃.

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

5)Adding Enzyme Conjugate,100ul/well,  Cover it with Adhesive plate sealer, incubate at 37 ℃ for 60 minutes;

6) Wash the wells as described above in Step 4.

7)Add substrate: add Substrate 100ul/well, mix it evenly then cover it with Adhesive plate sealer, incubate at 37 ℃ in dark for 10 minutes;

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

8. Interpretation of the Results

The absorbance OD value was read with a microplate reader at 450 nm (630 nm as reference wavelength).

The conditions for the test are:

  Negative control (N) OD value> 0.4,

Calculation method:

Sample OD value/Negative control (N) OD value=S/N value

Positive and negative judgment in poultry:

S/N value ≥ 0.5  Positive

S/N value <0.5  Negative

8. Stability and Storage

1) All reagents should be stored at 2~8℃. Do not freeze.

2) Shelf life is 12 months. Use all reagents before the expiry date on the kit.