1. Principle of the Test
Newcastle disease antibody Elisa kit is used to detect specific antibody against Newcastle disease Virus (NDV) in serum, for monitoring antibody after NDV immune and serological diagnosis of infection in Avian.
This kit use block ELISA method, NDV antigen is pre-coated on microplate. When testing, add diluted serum sample, after incubation, if there is NDV 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 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.
Notes
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) pre-coated plates should be sealed and moisture-proof. Put bac 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) pre-coated plates is disposable, do not repeat use.
2. Test procedure
1) For every test, set 1 well for positive control and 2 wells for negative control, negative, positive control without dilution, directly take 100ul added to the reaction well.
2) The sample diluent 90ul /well was added to the microplate reaction plate, then Adding sample, 10ul/well (Do not mix use the tips), shake gentle to mix it evenly, cover it with Adhesive plate sealer, incubate for 60 minutes at 37℃.
3) Open the adhesive plate sealer, discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, discard the liquid, repeat the above step for 4-6 times, at last flap to dry with the absorbent paper;
4) Adding Enzyme Conjugate, 100ul/well, Cover it with Adhesive plate sealer, incubate at 37℃ for 60 minutes;
5) Open the adhesive plate sealer, discard the liquid of the well, washing 4-6 times as step 3, remember at last flap to dry with the absorbent paper;
6) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive plate sealer, incubate at 37 ℃ in dark for 15 minutes;
7) Add stop solution, 50ul/well to stop the reaction, measure the result in 10 minutes.
3. Results judgement
Set zero at blank control well, read the OD value at 450nm (630nm as reference).
For the assay to be valid:
OD value of negative control(N) > 0.4, meanwhile positive value S/N<0.4.
Calculate method:
S/N value=
Results interpretation：
S/N value < 0.5 Positive
S/N value≥0.5 Negative
4. Storage and expire date
Store at 2~8℃ in dark, no frozen, expiry date: 12 months.