A double-antibody sandwich ELISA detection kit for detecting goat parainfluenza virus type 3 and its application
A double-antibody sandwich and detection kit technology is applied in antiviral immunoglobulins, measuring devices, instruments, etc., to achieve the effects of simplicity, rapidity, technical difficulty, high sensitivity, and easy operation.
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Embodiment 1
[0097] The establishment of embodiment 1 monoclonal antibody hybridoma cell line
[0098] 1. Antigen preparation: CPIV3 virus JS2013 strain (the preservation number of this strain is: CCTCC NO: V201832. The preservation date is: June 26, 2018, and the preservation address is: China, Wuhan, Wuhan University, Chinese typical culture preservation Center) to infect MDBK cells, harvest the virus after 4 days of inoculation, and centrifuge at 10,000rpm for 30min to remove cell debris; supernatant is ultracentrifuged at 40,000×g for 2h, discard the supernatant, and dissolve the precipitate with an appropriate amount of PBS overnight.
[0099] 2. Immunization of Balb / c mice: immunize 6-8 week-old female BALB / c mice (purchased from the Comparative Medicine Center of Yangzhou University) by subcutaneous multi-point injection of super-isolated virus, and immunize 3 times in total, with an interval of 2 weeks between each immunization 100 μl ultra-isolated virus and the same amount of com...
Embodiment 2
[0107] Example 2 Purification and peroxidase labeling of monoclonal antibodies
[0108] 1. Monoclonal antibody purification: Centrifuge the ascitic fluid at 4000rpm and room temperature for 15min, and take the supernatant. Slowly add saturated ammonium sulfate solution drop by drop to the supernatant of ascites under stirring at 4°C until the saturation of ammonium sulfate in the system reaches 50% to precipitate immunoglobulins, continue stirring for 30 minutes, and then centrifuge at 13,000 rpm and 4°C for 30 minutes , discard the supernatant, take the precipitate and dissolve it in PBS buffer solution (0.01M, pH7.4), slowly add saturated ammonium sulfate solution drop by drop under stirring conditions at 4°C until the saturation of ammonium sulfate in the system is 33%, using different concentrations Ammonium sulfate fractionated immunoglobulin, continued to stir for 30 min, centrifuged at 13000 rpm, 4 °C for 30 min, discarded the supernatant, took the precipitate and disso...
Embodiment 3
[0120] Example 3 Assembly of Goat Parainfluenza Virus Type 3 Double Antibody Sandwich ELISA Detection Kit
[0121] Select the anti-CPIV3 monoclonal antibody secreted by the hybridoma cell line 1F8 and the peroxidase-labeled 2E6 antibody preparation kit, and test different ELISA conditions: coating antibody concentration, detection antibody concentration, reaction time, etc., and finally determine the kit Ingredients and testing methods.
[0122] 1. Kit composition
[0123] Kit: including pre-coated microtiter plate, enzyme-labeled antibody working solution, sample diluent, chromogenic reagent, stop solution, and washing solution.
[0124] (1) Preparation of reagents
[0125] Coating buffer (0.1 M, CB buffer with pH value of 9.6): 3.2 g of sodium carbonate, 5.86 g of sodium bicarbonate, dilute to 1 L with pure water.
[0126] Sample diluent: 8g sodium chloride, 3.35g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, 10g ...
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