1. Principle of the Test

The PPRV antibody ELISA test kit use to detection of Peste des petits ruminants virus antibodies in the serum of sheep and goat .

This kit use competitive ELISA method to pre-coated PPRV antigens on microplate wells. When testing, add diluted serum sample, after incubation, if there have PPRV antibody, it will combine with the pre-coated antigen,  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.

2. Materials Provided

3. Material required not provided

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

2) Disposable pipette tips.

3) Cylinder: 500ml.

4) Microplate Reader: 96 wells.

5) Distilled water or deionized water.

6) Microplate Washer

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 week.

6. 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.

7. Test procedure

1) For every test, set 1 well for positive control and 2 wells for negative control;

2) Add sample diluent into each well, 20ul/well;

3) Add sample, negative control and positive control to its corresponding wells, 30ul/well. (Do not mix use the tips);

4) Adding PPRV Mab solution, 50ul/well, shake gentle to mix it evenly, cover it with Adhesive plate sealer, incubate at 37 ℃ for 45 minutes;

5) 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 30 minutes;

5) Open the adhesive plate sealer, discard the liquid of the well, washing 4-6 times as step 5, 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 10 minutes;

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

8. Results judgement

Set zero at blank control well, read the OD value at 450nm (630nm as reference).