Competitive ELISA Kit for Detecting Antibody of Foot and Mouth Disease Virus type O

Catalog No. LSY-30031

1. Usage

It is used for detection of FMD Type O antibody in serum of pig, bovine and goat. It is suitable for the detection of animal vaccine immune antibodies.

2. Principle

This kit adopts solid-phase competitive ELISA method, which is one of the designated methods of OIE for foot-and-mouth disease serological examination in international trade, and is also the reference method for the evaluation of the immune effect of foot-and-mouth disease vaccine in China.

The ELISA plate was coated with FMDV-O VP1 recombinant protein antigen, and anti-VP1 protein monoclonal antibody was used as the enzyme label. Add the serum to be tested to the antigen-coated plate, and then add the enzyme-labeled monoclonal antibody. If there is FMDV-O-specific antibody in the serum to be tested, it will compete with the enzyme marker for the antigen coated on plate, and the unbound components will be washed away. The color is light or no color after adding the substrate. Conversely, the color was darker after adding the substrate. Use a microplate reader to measure the OD_450nm value, and determine the experimental results according to the calculation formula.

3. The kit components

1	FMDV-O antigen coated microplate	96 T	96 T×2
2	FMDV-O Enzyme conjugate	6 ml	12 ml
3	Sample diluent	6 ml	12 ml
4	FMDV-O Negative control	0.8 ml	1.5 ml
5	FMDV-O Positive control	0.8 ml	1.5 ml
6	Substrate	6 ml	12 ml
7	Stop solution	6 ml	12 ml
8	10×concentrated washing buffer	25 ml	50 ml
9	Adhesive Foil	1 piece	2 pieces
10	Instruction	1 piece

4. Materials Required But Not Provided

1) Microplate Reader (single-wave length: 450 nm).

2) Precise micropipette (single-channel 10-100ul、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

Try to use freshly collected serum samples; It cannot be used to detect samples with serious pollution or hemolysis; If the serum sample is turbid, take the supernatant after centrifugation for detection; The test sample can be stored at 2 ~ 8 ℃ for 5 days. If it is stored for a long time, it needs to be transferred to - 20 ℃ or lower temperature.

6. Preparation

1) Bring ELISA reagents to the room temperature （25±3℃） for at least 30 min to get best results. Microplate should return to room temperature and dry before open package.

2) Washing buffer preparation: Dilute the 10×concentrated washing buffer with deionized water at 10 times. (for example: 10mL 10×concentrated washing buffer + 90mL deionized water ).

7.Procedure

1). Add sample: add 50 µL of negative control serum (NC) and 50 µL of positive control serum (PC) to the control wells; firstly add 30 µL of sample diluent to the sample wells, then 20 µL of serum to each well; add 50 μL of Enzyme conjugate to each well, gently shake to mix (do not spill), cover with sealing film, and incubate at 37℃ for 30 min.

2). Wash the plate: discard the liquid in the well, add 300 μL of  diluted washing buffer to each well, and wash three times. After the last wash, gently pat the ELISA plate dry on absorbent paper. It is strictly forbidden for the well plate to dry between steps.

3). Add Substrate: Add 50μL of Substrate to each well, mix gently, cover with sealing film, and incubate at 37℃ for 15 min in the dark.

4). Termination: Add 50μL of stop solution to each well, shake for 10 seconds, mix well, and read after termination.

5). Reading: Use a microplate reader to measure the optical density value (OD value) at a wavelength of 450nm.

8. Results

1)     For the assay to be valid, average OD value of Positive control ＜0.4, average OD value of Negative control ＞0.6.

2)     Calculation method: OD_NC Average value = (OD_NC1＋OD_NC2)/2

3)        Sample S/N value calculation formula: S/N＝Sample OD value/ OD_NC Average value

4)     Judgment standard: S/N＜0.5, judged as positive; S/N≥0.5, judged as 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 2-8 ℃. 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 T、96 T×2.

Expiry date: 12 months.

Storage: Store at 2~8℃, in the dark, no freezing.

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

www.lsybt.com