AffiVET® Porcine Parvovirus Antibody Elisa Test Kit

Porcine Parvovirus(PPV) antibody ELISA test Kit

1.Usage

The Green® Porcine Parvovirus(PPV) antibody ELISA Test kit is used for detection of Porcine Parvovirus(PPV) IgG antibody in porcine serum Qualitatively; assessment of immunity conditions against porcine Parvovirus in the pig farm and investigation of the epidemiology of the porcine Parvovirus.

2. Principle

The Green® Porcine Parvovirus(PPV) antibody ELISA test kit is made from the antigen

The Green® Porcine Parvovirus(PPV) antibody ELISA test kit uses the principle of solid-phase enzyme-linked immunosorbent assay (ELISA), and consists of a microwell reaction plate coated with high-purity PPV antigen, anti-pig IgG labeled with horseradish peroxidase, and other reagents. The reaction mechanism is that the coated antigen is combined with the PPV-Ab in the sample, and then combined with the enzyme-labeled anti-pig IgG antibody to form a "coated antigen+PPV-Ab+enzyme-labeled anti-pig IgG antibody" complex. Add adding Substrate, Catalytic reaction color development. The color depth is directly proportional to the amount of PPV-Ab. When the color reaction of the sample is detected, the result obtained by the microplate reader exceeds the set threshold, and the result is positive, which indicates that the immune has produced antibodies or there is a natural infection.

3. The kit components 

4. Materials Required But Not Provided

1 Microplate Reader (double-wave length: 450/630 nm).

2 Precise micropipette (single-channel 1-100ul、0.5-10ul、multi-channel 30-300ul)

3 Constant temperature box or water bath box.

4 Oscillator.

5 Disposable tips (10ul, 200ul)

6   Deionized water

5. Sample requirement

1 The samples are porcine serum, which should be collected with no bacteria. The storage time should be less than 1 week at 2-8 ℃, if for long term, it should be kept at -20℃.

2 Avoid to use the samples with severe hemolysis, precipitate, contaminated by bacteria or protein suspension.

6. Preparation

1) Bring ELISA reagents to the room temperature (20-25 ℃) for 30 min to get best results. Microplate should return to room temperature and dry before open package.

2) Sample dilute: Dilute sample with the sample diluent at 40 times.(5ul serum + 195ul sample diluent), the diluted sample need to mix evenly to get better results.

3) Washing solution preparation: Dilute the 10×concentrated washing buffer with deionized water at 10 times. (10ml 10×concentrated washing buffer + 90ml deionized water ) It is normal if there is crystallization in the 10×concentrated washing buffer, put at 37℃until completely dissolved.

7.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, add undiluted negative control serum, 2 wells for positive control serum, add undiluted positive control serum, 100μL/well(Not set blank well is OK). Others are sample wells, add the diluted sample, 100μL each.

2 Mix gently, cover andincubate at 37℃ for 30 min.

3 Remove adhesive foil. Pour the liquid out of the wells, add the diluted Washing buffer into each well fully, be static for 10s,pour out. Repeat 3 times, at last time pat to dry on absorbent paper.

4 Add 100μL enzyme conjugate into each well.

5 Cover plate with new adhesive foil.Incubate at 37 ℃ for30 min.

6 Repeat step 3(washing).

7 Add substrate 100ul into each well, mix properly,incubate for 10 min at37 ℃ in the dark with new adhesive foil.

8 Add stop solution 50μL into each well, mix gently and determine the result.

9 Measure the OD value of each well with a photometer at dual-wave length 450nm/630nm.

8.Results

For the assay to be valid, the positive control wells’ average OD value must be greater than or equal to 0.6, and the negative control wells’ average OD value is less than 0.1. Otherwise the test is invalid, need test again.

The result is judged by S/P value,

S/P=(Sample OD450/630- NCx(—))/( PCx(—)- NCx(—)), NCx(—) means Negative control’s average OD450/630 value(calculate as 0.05 when the value is less than 0.05), PCx(—) means Positive control’s average OD450/630 value

If S/P≥0.2, it is positive; less than 0.2, it is negative.

9. Precautions and warnings for users

1. Read the Manual carefully before use.

2. Do not use reagents expired, do not mix reagents from different lots.

3. Experiment rubbish should be dealt with high pressure steam sterilization at 121 ℃ for 30 minutes, or treated with 5.0g/L sodium hypochlorite disinfectant for 30 minutes, then discard.

4. MicroWell plate removed from the refrigerated environment should be balanced moisture to dry at room temperature, then can be opened. Put back unused MicroWell plate into dry foil bag and sealed at 4 ℃. Unused liquid reagent should cover caps, store at 2-8 ℃ in dark with other group components.

5. Should use Micropipettor to add sample and reagents, and often proof its accuracy.

6. When adding washing buffer, should be full but no overflow, avoid appearing free enzyme at mouth of well or cross pollution between wells.

7. Stop solution is corrosive, use large amount of water to wash immediately when touch the skin or clothes.

Specifications:96 wells×2.

Expiry date:12 months.

Storage:Store at 2~8℃, in the dark.

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