Foot and mouth disease virus type A(FMDV) Ab Elisa Test kit can be used to detect type A foot and mouth disease virus antibody in serum of pigs, cattle and sheep to evaluate the immune status of type A FMD vaccine situation.

This kit use indirect ELISA method, pured FMD antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is FMD 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-FMD 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.

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) 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) Serum dilution plate is disposable, do not repeat use.The  max volume of the plate is 300ul/well

 

2. 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. 

 

3.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 > 0.3;

Calculate method:

(Sample OD value - Negative control OD average value)/(Positive control OD average value-Negative control OD average value)= IRPC value 

Results interpretation

IRPC value< 0.4   Negative

S/P value≥0.45    Positive 

0.4≤IRPC < 0.45   disputability

 

4. 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.