AffiVET® Foot And Mouth Disease Virus (FMDV) Type A Antibody Elisa Test Kit For Bovine And Ovine

Foot-and-mouth disease virus (FMDV)Type A Antibody ELISA Test Kit

For Cattle and Sheep  

1. Principle

This kit is used to detect specific antibody against foot and mouth disease virus (FMDV) Type A Antibody in serum of cattle and sheep qualitatively, for monitoring antibody after FMDV Type A immune.

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 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 content in sample, use ELISA reader at 450nm wavelength to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

2.Reagents and contents

3.Materialrequired notprovided

1) Micropipette: 0.5-10ul, 10ul-100ul, 100ul-1000ul.

2) Disposable pipette tips.

3) Graduate: 500ml.

4) Microplate Reader: 96 wells with 450/630nm wavelenth.

5) Distilled water or deionized water.

6) Microplate Washer

4. Samplepreparation

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. Sample dilution

Dilute serum at 1:100 on Serum dilute plate.

Note: both positive control and negative control do not need dilute, change tips after taking every sample, mark the sample location on plate accurately, mix every sample evenly before adding to the pre-coated micro-wells.

7. Notes

1) Return all reagents into room temperature before use, put the reagents at room temperature for at least 1 hour. 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) FMD-Ag coated plates should be sealed and moisture-proof. Put back unused Micro-Well 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 dilute plate is disposable, do not repeat use; the max volume of the plate is 300ul/well.

8.Test procedure

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 2 wells for negative control, take 100ul directly and add into its well, mix gently(do not overflow);

2) Cover it with Adhesive Foil,incubateat 37 ℃ for30 minutes;

3) Open the adhesive foil, 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 50ul/well, Cover it with Adhesive Foil,incubateat 37 ℃ for30 minutes;

5) Open the adhesive foil, discard the liquid of the well, washing for 4-6 times as step 4), 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 Foil,incubateat 37 ℃in darkfor10 minutes;

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

9. Results judgement

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

Resultsinterpretation

IRPC<0.3: Negative

0.3≤IRPC<0.4: Skeptical

IRPC≥0.4: Positive

9. Storage and expire date

Store at 2~8℃ in dark, no frozen, expiry date: 12 months.