1. Usage
Classical Swine Fever Virus Antigen ELISA Test Kit  (CSFV Ag), is used for the detection of CSFV antigen in porcine serum,

2. Principle

This kit use block ELISA method,Pured CSFV antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample after incubation, it will combine with the pre-coated antigen if the CSFV virus is specific antibody . Discard the uncombined antibody and other components with washing .Then add enzyme label anti-CSFV virus monoclonal VP1 . Antibody in sample block the combination of monoclonal antibody and pre-coated antigen. Disscard  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 wavelenth to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

3. Materials Provided

4. Necessary equipment and reagents

1.  Micropipettor: 0.5µl-10µl、10µl-100µl、100µl-1000µl。

2.  Disposable pipette suction head

3.  Graduated cylinder：500ml。

4.  Enzyme mark with 96 microplate.

5.  Distilled water or deionized water

6.  Bottle washing machine

5. 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.

6.Preparation of wash solution

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.

7.Note

1. The kit should keep to room temperature and gentle shaking well before using.after using store in temperature 2-8 ℃

2. Different varieties, different batch number kit reagent components shall not be mixed, should prevent reagent pollution when using reagent.

3. The substrate and terminate solution to skin and eyes might be irritating, should pay attention when using.

4. Color liquid not exposed to strong light and avoid contact with antioxidants.

5.Test plate after unpacking should avoid to be damp or touch water (the rest antigen package add desiccant in the self-styled bag, and placed 2-8 ℃ as soon as possible)

6. All waste before discarding should be reasonable processing to avoid pollution.

7. Strictly abide by the instructions you can get the best results.Move fluid, timing, washing, all the process must be accurate in the process

8. Serum dilution plate for disposable supplies, shall not be repeated use;Serum dilution plate of the maximum capacity of 300 mu l/hole

8. Procedure of the Test

1) Dilute the serum sample with sample dilution at 1:50,

2) 100 μl of negative control and positive control respectively, and 80 μl diluted samples and 20μl serum sample each into remaining wells.

3) Cover the wells with plate sealer and incubate for 30 minutes at 37±1℃.

4) Remove all liquid from the wells and rinse the wells 4-6 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 50㎕ of enzyme conjugate(ready to use) into each well.

6) Cover the wells with plate sealer and incubate for 30 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 10 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 10 minutes.

9. Interpretation of the Results

1) Test Validation

② The mean absorbance value of negative control(NCx) is < 0.2.

③ The mean absorbance value of Positive control(PCx) is > 0.5

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

2) Calculation of the Result

3) Interpretation of Results

The status of samples is determined as follows;

- IRPC≥ 0.4 is considered positive.

- IRPC < 0.4 is considered negative.

10. 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.