LSY-30007 Porcine Circovirus (PCV2) ELISA diagnostic kit192wells/kit

Green Spring Porcine Circovirus (PCV2) ELISA test Kit

Catalog No. LSY-30007

1. Usage

The GreenSpring^® porcine circovirous(PCV2) ELISA Test kit is used for detection of porcine circovirus type 2 antibody in porcine serum; assessment of immunity conditions against porcine circovirus in the pig farm and investigation of the epidemiology of the porcine circovirus.While the result can not distinguish immune antibody and infectious antibody, to confirm that is antibody by vaccine or wild virus infection needs to combine clinical data, immune status, and other diagnostic methods.

2. Principle

The GreenSpring® PCV2 antibody ELISA test kit is made from the antigen coated microtiter plate(coated with PCV antigen) and other reagents. It applies the Solid-phase ELISA principle to PCV-Ab in serum, then add enzyme conjugate to specifically bind with complex of coated antigen+PCV-Ab+enzyme labeled anti-pig-IgG antibody on the microplate. With the TMB substrate, it will generate an amount of color. The depth of color is relative with the content of the PCV-Ab, when the value of color is greater than the cut-off value, the pigs are vaccinated well or natural infected exist.

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 Microplate Washer.

6 Disposable tips (10ul, 200ul)

7   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.

3 The EDTA, heparin sodiun and other anticoagulants will not affect the results.

6. Preparation

1) Bring ELISA reagents to the room temperature (20-25 ℃) for 30 min to get best results.

2) Sample dilute: Dilute sample with the sample diluent at 40 times.(There will be color change after adding solution), the diluted sample need to mix evenly to get better results.

3) Washing solution preparation: Dilute the 20×concentrated washing buffer with deionized water at 20 times.

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. Others are sample wells, add the diluted sample, 100μL each(both single- well and double-well test is OK).

2 Mix gently, cover and incubate 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, directly pour out, no need to be static. 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 ℃ for 30 min.

6 Repeat step 3(washing).

7 Add substrate 100ul into each well, mix properly, incubate for 10 min at 37 ℃ 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, 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.  Interpretation of the result

1. Severe hemolysis, fiber protein in the serum separation is not sufficient, containing erythrocytes, a precipitate, a sample with bacteria may lead to false positive.

2. Negative results may occur on individual pigs after vaccines due to individual differences or immune duration.

3. Positive results for serological diagnosis and epidemiological investigation of swine to be combined with other methods and clinical data.

10. Product performance

1. Specificity: use this kit to detect reference serum, the compliance rate reach 100%.

2. Sensitivity: can reach max 1:5120.

3. Precision: CV(%)no bigger than 8%.

4. Stability: Store at 2℃～8℃ for 12 months or store at 37℃ for 3 days, the result can reach the above 3 standard.

11. Precautions and warnings for users

1. This test kit is for research use only.

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

3. Read the Manual carefully before use.

4. 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.

5. 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.

6. If the 20×concentrated washing buffer appears crystal, it is normal, put at 37℃ until been dissolved.

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

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

9. 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.

Shenzhen Lvshiyuan Biotechnology Co., Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788

Fax 86-755-28938800

Email:  info@lsybt.com

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