The Rotavirus (RV) antibody ELISA kit is used to detect RV antibodies in pig serum, and to evaluate the immune status of pig farm RV vaccine and serologically assisted diagnosis of infected pigs.

This kit use indirect ELISA method, pured RV antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is Rota 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-Rota 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 contentin sample, use ELISA reader at 450nm wavelenth to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

 Procedure of the Test 

1) Take the antigen coated plate(the plate can be open and used for several times according to sample quantity each time), add the diluted serum to reaction wells, 100ul/well; meanwhile, set 2 wells for positive control and 1 well for negative control, both positive control and negative control do not need dilute, take 100ul directly and add into its well, mix gently(do not overflow);

2) Cover it with Adhesive plate sealer, incubate at 37 ℃ for 30 minutes;

3) Open the adhesive plate sealer, discard the liquid of the well

Add diluted washing buffer to each well, 250ul/well,be static for 1min, then 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 3, remember at last flap to dry with the absorbent paper;

6) Add substrate: 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. 

Interpretation of the Results 

Read the OD value with ELISA Reader at 450nm (630nm as reference).

For the assay to be valid:

Negative control (N) OD value< 0.2, meanwhile positive control (P) OD value > 0.4;

Calculate method:

Sample OD value / Positive control OD average value= S/P value 

Results interpretation

S/P value < 0.4   Negative

S/P value ≥ 0.4   Positive 

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.