1. Usage

The Porcine Japanese B Encephalitis Virus Antibody ELISA Test Kit (JEV Ab) is used for the detection of the porcine encephalitis virus antibodies in swine serum; assessment the immunity conditions against porcine encephalitis virus in the pig farms and investigation of the epidemiology of the porcine encephalitis virus.

2. Principle

The kit is based on an indirect enzymatic immunoassay(Indirect ELISA).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  Antigen coated microtiter plate......................................... 2 pieces (96 wells)

2  Negative (green lid)/positive (red lid)serum................ 1 tube each (1 mL/tube)

3  Anti-pig IgG-HRP conjugate (yellow lid)....................... 1 bottle (22 mL/bottle)

4  20×concentrated washing buffer (white lid).................. 1 bottle (30 mL/bottle)

5  Substrate A (purple lid)/B (black lid)..................... 1 bottle each (12 mL/bottle)

6  Stop solution(blue lid)................................................ 1 bottle (12 mL/bottle)

7  Sample diluent solution(transparent lid)....................... 1 bottle (50 mL/bottle)

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. Preparation

Washing buffer: dilute 20×concentrated washing buffer with deionized water at 1:19.

7. Test Procedure

1.   Take out the coated plates(Can be detached) and record the sample position on a worksheet.  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. Similarly, dilute the samples at 1:39 with the sample diluent, 100 μL each well. Mix gently, incubate at 37℃ for 30 min.

2.    Wash each well with 300 μL washing buffer 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 , incubate for 10 min at room temperature in the dark.

6.  Add stop solution one drop (50 μL) to 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: Store at 2~8℃, in the dark.

Production Date: On outer-packing of the test kit.

This kit is only used in research or experiment, not used in human or animal diagnosis.