ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof
A porcine epidemic diarrhea and detection kit technology, applied in the field of porcine epidemic diarrhea virus detection, can solve the problems of unexplainable antibodies, recurrent infection and infection, and increased operational difficulty, so as to reduce the incidence of false positives, increase sensitivity, and improve specific effect
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Embodiment 1
[0037] Example 1: Anti-PEDV specific egg yolk antibody was extracted after PEDV virus immunization laying hens
[0038] according to figure 1 The shown procedure prepares anti-PEDV specific egg yolk antibody:
[0039] (1) PEDV culture and purification: commercially purchased African green monkey kidney (vero) cells were cultured in DMEM growth medium (pH 7.2) containing 10% newborn bovine serum at 37°C, and after the cells were cultured for 48 hours, the culture flask was completely covered , pour off the supernatant gently, wash the cells 3 times with serum-free DMEM maintenance solution, and inoculate 1 / 50 volume of PEDV virus (PEDV CV777 strain (gifted by the Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau)) , adsorb the cells for 1 hour, then add 10 mL of maintenance solution without newborn bovine serum (FBS) and trypsin, and place the cell culture flask at 37°C with 5% CO 2 Continue to grow in the inc...
Embodiment 2
[0045] Embodiment 2: PEDV detection kit assembly
[0046] Coating microtiter plate: Dilute the anti-PEDV specific egg yolk antibody to a concentration of 5 μg / mL, take 100 μL of the diluted specific egg yolk antibody solution and coat the 96-well microtiter plate, and coat overnight at 4°C. After taking it out the next day, wash the plate 3 times with PBST, 3 minutes each time. 1% BSA prepared in PBST solution was used as a blocking solution, and 100 μL of blocking solution was added to each well, and blocked at 37°C for 1 h. After sealing, the plate was washed 3 times with PBST, 3 min each time. Put it into a special packaging bag for 96-well microplate plate, seal it with a sealing machine and store it at 4°C.
[0047] Detection antibody (PEDV polyclonal antibody): PEDV polyclonal antibody antibody and enzyme-labeled secondary antibody can be obtained through technical service outsourcing or commercially purchased, and can also be prepared by conventional methods in the fi...
Embodiment 3
[0060] Embodiment 3: The operation method of using the PEDV detection kit to detect the sample
[0061] according to image 3 The method shown is to operate:
[0062] (1) On-site use a disposable sampling spoon to take fresh pig vomit or feces into sterile sampling tubes and store them in ice boxes;
[0063] (2) Weigh 1 g of the vomitus or feces sample to be tested and dissolve it in 10 mL of sample diluent, centrifuge at 3000 rpm at 4°C for 15 minutes to take the supernatant;
[0064] (3) Add 100 μL / well of the supernatant to the antigen detection ELISA plate, incubate at 37°C for 30 minutes, shake off the solution in the plate well, wash the plate 3 times with 200 μL / well, and let stand for 3 times each time. Drain in minutes and pat dry on absorbent paper for the last time;
[0065] (4) Add 100 μL of primary antibody to each well, incubate at 37°C for 30 minutes, wash 3 times, the method is the same as step (3);
[0066] (5) Add 100 μL of enzyme-labeled secondary antibo...
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