Salmon trout ihnv monoclonal antibody and its detection kit and application
A monoclonal antibody and detection reagent technology, applied in the field of salmon trout IHNV monoclonal antibodies and detection kits, can solve the problems of unreported, low detection rate, time-consuming and labor-intensive, etc. specific effect
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Embodiment 1
[0034] Embodiment 1 Preparation of Monoclonal Antibody 1
[0035] 1. Virus acquisition
[0036]The virus used in the present invention is U-type rainbow trout infectious hematopoietic necrosis virus (IHNV), which was isolated from a fishery in Beijing in 2012. The virus strain was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on February 6, 2017, and it was classified and named as Infectious haematopoietic necrosis virus (IHNV) IHNV-BJLL strain, preservation number: CGMCC No .13594 (Depository Unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Zip code 100101.). This genotype IHNV virus is the first case in China, and it is reported that domestic IHNV isolates are all J-type.
[0037] 2. Virus preparation
[0038] Carp epithelial tumor cells ...
Embodiment 2
[0058] The preparation of embodiment 2 monoclonal antibody 2
[0059] Another batch of the present invention has carried out the monoclonal antibody preparation, and the method is the same as that of Example 1, and will not be repeated here. After several times of cloning and screening, four monoclonal antibody-secreting hybridomas were obtained, namely 3A5, 4A9, 1E8 and 4D6. The OD450nm values measured in the cell supernatant after continuous passage and cryopreservation were all significantly higher than the SP2 / 0 value. . The ELISA titers of the hybridoma ascitic fluid antibody secreting monoclonal antibody were 1:10 3 , 1:10 6 , 1:10 4 , 1:10 4 . Use hybridoma cell strain cell culture supernatant as primary antibody and carry out Western blot detection with purified U-type IHNV virus, the result shows, all can occur reaction band ( image 3 ), which is consistent with the size of the IHNV virus N protein, indicating that the prepared monoclonal antibody is a specif...
Embodiment 3
[0062] Example 3 Establishment of Immunofluorescence Detection
[0063] 1. Preparation of test samples
[0064] 1.1 EPC cells inoculated with IHNV
[0065] Cultivate EPC cells at 25°C. When the cells grow into a single layer and 90% of the cells are full, the ratio of U-type IHNV virus to cell fluid inoculation is 1:50—1:70 times, and the inoculation is optimal. When the cell pathology reaches 50% Cells can be fixed when left or right for the next step of detection. At the same time, a negative control was set up.
[0066] 1.2 Determination of fixative and fixation time
[0067] Use 50%, 60%, 70%, 80% acetone and 60%, 70%, 80%, 90%, 100% ethanol as the fixative solution respectively, and use 5min, 10min, 15min, 20min, 25min, 30min as the Fix the time and observe the effect of cell fixation. The optimal fixation conditions are determined by the presence or absence of cell detachment and the intensity and clarity of specific fluorescence. The results showed that 80% aceton...
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