1. Principle of the Test

The FMD Ab ELISA is a blocking Enzyme Linked Immunosorbent Assay for the qualitative detection of specific antibodies to Foot and Mouth Disease Virus (FMDV), in Swine ,bovine ,goat and sheep serum.

The FMDV Ab ELISA contains a microplate, which is pre-coated with recombinant VP1 on the well. For testing, ELISA plates are incubated with a serum and control(1:1 dilution with the sample diluents) for 60 minutes at 37℃. During first incubation, anti-FMDV antibodies present in sample bind onto the antigen coated on the well. After incubation and washing step, then mAb-HRP is dispensed into the wells and incubated for 60 minutes at 37℃. Following this incubation, all unbounded materials are removed by washing step. The enzyme linked to the complex is revealed by addition of a substrate. The enzyme activity will thus be in inverse proportion to the anti-FMDV antibodies in specimens and evidenced by incubating the solid-phase with a substrate solution. The reaction is stopped by adding stop solution, and colorimetric reading will be performed by using a spectrophotometer at 450nm and reference wavelength at 620nm.

2. Materials Provided

 3. Precautions for Use

In order to obtain reproducible results, the following rules must be observed.

1) For in vitro diagnostic use only.

2) Store the components at 2-8℃ right after use. Do not reuse microwells or pour reagents back into their original bottles once dispensed.

3) Do not intermix components from kits with different batch numbers.

4) Do not use reagents after the expiry date.

5) Do not reuse containers and residues, so avoid contamination of each reagent with sample or other reagents.

6) Handle all reagents and samples as biohazardous materials.

7) Use fresh samples. Hemolyzed or contaminated samples may give erroneous results.

8) Remove the blood corpuscle in samples clearly. It may give non-specific reaction.

9) Wear the gloves when you handle the potentially infectious materials. After handling, wash hands with sanitizers.

10) Keep all reagents away from skin and eyes. If exposure should occur, immediately rinse with fresh cold water.

11) Dispose of containers and residues safely in accordance with national and local regulations.

12) Substrate and stopping solution can cause irritation or burns to the skin and eyes. In case of accident, rinse immediately with fresh cold water.

4. Collection and Storage of Sample

1) Fresh pig serum samples should be used for this assay. Hemolyzed or contaminated samples may give erroneous results.

2) If samples are not immediately tested, they should be refrigerated at 2~8℃. For longer storage, freeze the samples at -20℃ or below. Avoid repeated freezing and thawing.

3) Heat inactivated serum (for 30min at 56℃) is available.

5. Preparation of Reagent and Samples

1) Allow all reagents and samples to come to room temperature(18~25℃) before use.

2) Unused microplate wells must be sealed with silica gel in enclosed sealing bag and stored at 2~8℃.

3) Mix samples thorough by gentile inversion. If necessary, any visible particulate matters in the samples should be removed by low-speed centrifugation.

4) Wash solution (10X concentrated): Dilute the 10x wash solution by distilled/deionized water(1:9). Add 50㎖ of Wash solution to 450㎖of distilled/deionized water and mix thoroughly. Store at 2-8℃ or room temperature(18~25℃) after use.

6. Procedure of the Test

1) Dispense 100ul of the Positive Control into 1 well of the plate and Negative Control  into 2  well of the plate

2) Dispense 50ul of the sample diluents into other well of the plate Dispense 50ul of the test sample into well containing sample diluents.

3) Cover the wells with plate sealer and incubate for 60 minutes at 37±1℃.

4) Remove all liquid from the wells and rinse the wells five times with 250㎕ of diluted wash solution. Remove any remaining wash solution by inverting the plate and blotting it against a clean paper towel.

5) Dispense 100㎕ of enzyme conjugate(ready to use) into each well.

6) Cover the wells with plate sealer and incubate for 60 minutes at 37±1℃.

7) Wash the wells as described above in Step 4.

8) Dispense 100㎕ of substrate(ready to use) to each well.

9) Cover the wells with plate sealer and incubate for 15 minutes at 37℃ in the dark.

10) Add 50 ㎕ of stopping solution(ready to use) to each well. Mix by gentle shaking.

11) Read the absorbance values of the wells at 450nm in a bichromatic spectrophotometer( with reference wavelength at 620nm) right after from the end of assay, within 30 minutes.

7. Interpretation of the Results

1) Test Validation

② The mean absorbance value of negative control(NCx) is ≥ 0.6.

③ The PI value of positive control is ≥ 60%.

④ If these values are out of range, result should be considered invalid and the samples should be retested.

⑤ If the OD450 of a test sample is higher than the mean OD450 of negative control, the Percentage Inhibition can be interpreted as 0%.

2) Calculation of the Result

For example,

- PCx : 0.028, NCx : 2.013, OD450 of sample : 0.562

- PI value of positive control = [1-(0.028/2.013)] x 100 %= 98.6%(Validation: ≥ 80)

- PI value of sample = [1-(0.562/2.013)] x 100% = 72.1%

3) Interpretation of Results

The status of samples is determined as follows;

- PI value > 35% is considered positive.

- PI value ≤ 35% is considered negative.

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.