1. Introduction

The BIOKAU^® FMD-Ab ELISA is developed to detect the O type antibodies of Porcine Foot and Mouth Disease in porcine serum samples and assess the immunity conditions against FMD in the pig farms.

2. Principle

The kit is based on an indirect enzymatic immunoassay.The antigen is coated on plates. When a sample serum contains specific antibodies against virus, they will bind to the antigen on plates. Wash the unbound antibodies and other components. Then add a specific peroxidase conjugate(IgG-HRP). After incubation and washing, add the TMB substrate. A colorimetric reaction will appear, measured by a spectrophotometer(630 nm).

3. The kit components

1  FMD coated microplate................................................. 2 pieces (192 wells)

2  FMD Negative control serum (green lid).................................. 1 tube (1 mL)

3  FMD Positive control serum (red lid)....................................... 1 tube (1 mL)

4  FMD Enzyme conjugate (yellow lid)...................................... 1 bottle (22 mL)

5  20×concentrated washing buffer (white lid)........................... 1 bottle (30 mL)

6  Substrate A (purple lid)/B (yellow lid)......................................... (12 mL/each)

7  Stop solution (blue lid)......................................................... 1 bottle (12 mL)

8  Sample diluent (transparent lid)............................................ 1 bottle (50 mL)

4. Precautions and warnings for users

1.         Read the use instructions carefully.

2.         Bring all reagents to room temperature prior to use.

3.         Do not use components after expiration dates and do not mix components from different lots.

4.         If the dry bag is red, don’t use.

5.         Don’t pollute any components.

6.         Reseal the plates after use and avoid to wet.

7.         Do not pipette by mouth

8.         Use gloves and Be careful with reagents

9.         Clean the discarded reagents

5. Materials Required But Not Provided

Micropipettes, Distilled water, Incubator, Reservoir, Spectrophotometer, Absorbent paper.

6. Reagent preparation

Washing buffer: dilute 20×concentrated washing buffer with deionized water.

7. Test procedure

1.         Take out the coated plates(Can be detached) and record the sample position on a worksheet. Dilute the sample at 1:39 with the sample diluent and add to the wells, 100 μL each. Set 2 wells for negative control serum and 2 wells for positive control serum, 100 μL/well. Set one blank control well, add 100 μL sample diluent. Mix gently, incubate at 37℃ for 30 min.

2.         Wash each well with 300 μL washing buffer for 3 times, and each static for 3 min. The last time, pat to dry on absorbent paper.

3.         Add 100 μL enzyme comjugate into each well, and incubate at 37℃ for 30 min.

4.         Repeat step 2.

5.         Add 50 μL substrate A and 50 μL substrate B into each well, mix properly, Color for 10 min at room temperature in the dark.

6.         Add one drop stop solution (50 μL) into each well, and determine the result within 10 min.

8. Results

Set zero for the blank well, and test the OD630 value on the spectrophotometer. For the assay to be valid, the positive control wells’ average OD630 value must be greater than or equal to 0.4, and the negative control wells’ average OD630 value is less than 0.2.

If the sample’s OD630 value is greater than 0.4, it is judged to be positive; from 0.2 to 0.4, doubtful; and if less than 0.2, negative.

Specifications: 96 wells×2.

Expiry date: 6 months.

Storage: Storing at 4℃, in the dark.