Porcine epidemic diarrhea virus specific siga ELISA detection kit and its application
A porcine epidemic diarrhea and detection kit technology, applied in the field of medicine, can solve problems such as inability to effectively remove viruses, and achieve the effects of convenient collection, strong sensitivity and specificity
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Embodiment 1
[0050] Embodiment 1 Stable secretion is directed at the preparation of the hybridoma cell line of SIgA molecular secretion sheet (SC) antibody
[0051] 1. Collection and purification of sow colostrum
[0052] 1.1 Colostrum collection and purification
[0053] Collect colostrum within 1-3 days after sow delivery, centrifuge the collected colostrum at 4°C, 15000g for 15min, collect whey, discard sediment and fat layer. Add an equivalent amount of saturated ammonium sulfate dropwise to the whey while stirring to make the saturation of ammonium sulfate reach 50%, stir at 4°C for 30 minutes, and centrifuge at 15,000 g for 30 minutes. After diluting the precipitate with physiological saline, slowly add saturated ammonium sulfate dropwise to make the saturation reach 40%, centrifuge and discard the supernatant. The precipitate was dissolved in PBS with pH 7.5, centrifuged at 15,000 g for 30 min, and the supernatant was the primary extract of porcine SIgA, which was stored at 4°C fo...
Embodiment 2
[0079] Example 2 Establishment of an indirect ELISA method for detecting PEDV-specific SIgA antibodies
[0080] 1. Method
[0081] 1.1. Culture of PEDV-LNCT2 strain
[0082] After the Vero-E6 cells were overgrown to form a monolayer, the nutrient solution was discarded, washed three times with PBS (phosphate buffered saline, pH 7.2), and the PEDV-LNCT2 strain was inoculated. The dose of virus inoculation was inoculated according to the concentration of 5%, and trypsin was added at the same time of virus inoculation, and the final concentration of trypsin was 10 μg / ml. Observe the cell lesions, and when 80% to 90% of the cells have lesions, freeze the virus in a -20 refrigerator, collect the virus, and set aside.
[0083] 1.2. Purification of PEDV
[0084] After the PEDV-LNCT2 virus liquid was centrifuged at 5000g for 5min to remove cell debris, it was centrifuged at 100000g for 2h in an ultracentrifuge, the supernatant was discarded, and the precipitate was collected. The ...
Embodiment 3
[0124] The assembly of embodiment 3 kit, compliance test and preliminary application
[0125] 1. Assembly of kit
[0126] (1) ELISA plate: the ELISA plate has been coated with purified PEDV-LNCT2 virus particles, and the coating concentration is 5.8 ng / well. The preparation method is as follows:
[0127] (a) Add the purified PEDV-LNCT2 virus particles to each well of the microtiter plate, the amount added is 5.8ng / well, and coat overnight at 4°C;
[0128] (b) Washing: take out and pat dry, wash with PBST buffer 3 times (5min / time), and pat dry the liquid;
[0129] (c) Block with 1% BSA-PBST, 100 μL / well, at 37°C for 1 hour, and wash with the same step (b);
[0130] (2) 10× washing solution: the 10× washing solution contains 5v / v% Tween-20, 8w / w% sodium chloride, 0.2w / w% potassium chloride, 2.9w / w% disodium hydrogen phosphate and 0.2w / w% potassium dihydrogen phosphate aqueous solution, pH7.4;
[0131] (3) 10× sample diluent: the 10× sample diluent contains 5w / w% bovine ser...
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