1.Principle of the Test
The FMD Type Asia I antibody ELISA test kit use to detection of foot-and-mouth disease virus antibodies in the serum of pigs, cattle, sheep and goat for the evaluate of FMD vaccine immunity.
This kit use indirect ELISA method, pured FMDV 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 conjugate, discard the uncombined enzyme conjugate with washing. Add TMB substrate in micro-wells, the blue signal by Enzyme catalysis is directly 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.
7.Dilution of sample
Dilute the sample to be tested (198ul sample dilution + 2ul sample serum) by 1:100 on the dilution plate.
Note: negative and positive controls need not be diluted. After taking each sample, replace the Micropipette tip and accurately record the position of each sample on the board. Each sample should be thoroughly mixed before adding to the Antigen coated microplate.
8. Procedure of the Test
1) Take the antigen coated microplate(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 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 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, 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 step3, 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.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:
IRPC value =
Results interpretation
IRPC value< 0.35 Negative
IRPC value≥0.4 Positive
0.35≤IRPC < 0.4 disputability
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.