AffiVET® Peste Des Petits Ruminants Virus (PPRV) Antibody Elisa Test Kit For Ovine

Peste des petits ruminants virus (PPRV) antibody ELISA kit

1.Usage

It is used to detect PPRV antibody in serum and plasma of sheep and goat qualitatively, evaluate the immune status of Peste des petits ruminants vaccine and assisted serological diagnosis of infected animals.

2. Principle

This kit use competition ELISA method, PPRV antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample and Monoclonal Enzyme conjugate, after incubation, if there is PPR virus specific antibody, it will bind to the PPRV antigen on the coating plate and prevent the enzyme-labeled monoclonal antibody from binding to the antigen on the plate; conversely, if the sample does not contain PPRV-specific antibody, it will not bind to the coating plate. After washing to remove the unbound antibody and other components, add substrate to the microwells to form a blue product through enzymatic catalysis. After adding stop solution to terminate the reaction, use a microplate reader at 450nm/630nm double wavelength to measure the absorbance A value in the reaction well.

3. The kit components 

4.Materialrequired notprovided

1) Micropipette: 0.5-10ul, 10ul-100ul, 100ul-1000ul.

2) Disposable pipette tips.

3) Graduate: 500ml.

4) Microplate Reader: 96 wells with 450/630nm wavelength.

5) Distilled water or deionized water.

6) Microplate Washer

5. Samplepreparation

Take animal whole blood, get serum by using regular method, the serum should bright and no hemolysis.

6. Washing buffer preparation

Return 10X Concentrated washing buffer into room temperature before use, If there is salt crystals, put it in at 37℃water bath for 5~10min to dissolve it, then dilute it with deionized water at 10 times (for example, to prepare 200ml washing buffer: 180ml of deionized water + 20ml of 10X concentrated washing buffer, mix it evenly). The diluted washing buffer can be stored at 4℃for about a week.

7. Notes

1) Return all reagents into room temperature before use, put all reagents at room temperature for at least 30 minutes. Shake it evenly before use, and store back to 2-8℃after usage.

2) Do not mix use reagents from different kits and different lot no., prevent the reagents been polluted when using.

3) Substrate and stop solution may have irritation to skin and eyes, be careful to use.

4) Do not expose Substrate to strong light and avoid contact with the oxidant.

5) PPRV-Ag coated plates should be sealed and moisture-proof. Put back unused MicroWell plate into dry foil bag and sealed at 2-8 ℃.

6) All wastes should be treated well to avoid pollution before discarding.

7) Strict compliance with the operating instructions can get the best results. Pipetting operation, timing, and washing of the whole process must be precise.

8. Test procedure

1) Take the pre-coated microplate (according to the number of samples, it can be disassembled and used separately), set 2 wells for positive control, 2 wells for negative control , add 50µl positive control serum or negative control serum to it’s well accordingly; others are sample well, firstly add 40µl sample dilution, then add 10µl sample into each well. At last, add 50µl Enzyme conjugate to each well, shake gently to mix it evenly, cover the plate with adhesive foil,incubateat 37 ℃ for30 minutes;

2) Open the adhesive foil, discard the liquid of the well, add diluted washing buffer to each well, 300ul/well, be static for 30s, then discard the liquid, repeat the above step for 5 times, at last flap to dry with the absorbent paper;

3) Add substrate solution,100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat 37 ℃in darkfor15 minutes;

4) Add stop solution 50ul/well to stop the reaction, measure the result in 10 minutes.

9.Results judgement

Read the OD value with ELISA Reader at 450nm (630nm as reference).

For the assay to be valid:

OD value of Negative control (N)≥1.0,

OD value of positive control (P)  ≤0.3;

Calculate method:

Sample OD value/average OD value of Negative control (N)= S/N value

Resultsinterpretation

S/N value≥0.5:  Negative

S/N value<0.5:  Positive

10.Storage and expire date

Store at 2~8℃ in dark, no frozen, expiry date: 12 months.