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

Active Publication Date: 2021-06-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, cell culture and RT-PCR technology are time-consuming and labor-intensive, the expressed N protein and G protein fluorescent antibody technology is unstable due to the immunogenicity of the expressed protein, and it is easy to cross-react with other viruses in the rhabdovirus, farm experimental equipment Inability to meet the detection requirements and other factors lead to false detection, missed detection, delayed detection, low detection rate, etc.
At present, there is no report on the immunofluorescence detection method of rainbow trout IHNV, and there is no report on the immunofluorescence detection method of the specific monoclonal antibody of salmon trout IHNV

Method used

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  • Salmon trout ihnv monoclonal antibody and its detection kit and application
  • Salmon trout ihnv monoclonal antibody and its detection kit and application
  • Salmon trout ihnv monoclonal antibody and its detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a salmon trout IHNV monoclonal antibody, which is obtained by secreting a monoclonal hybridoma cell line specific to IHNV N protein produced by immunizing animals with the infectious hematopoietic organ necrosis virus IHNV-BJLL strain. The invention also provides a detection kit and application based on the monoclonal antibody. Among the IHNV monoclonal antibodies obtained in the present invention, the monoclonal antibody secreted by a hybridoma cell line has strong specificity, can detect the infection of domestic U-type and J-type salmon trout IHNV at the same time, and can avoid the occurrence of other rhabdoviruses The cross-reaction overcomes the misdetection and missed detection of IHNV-infected juvenile fish by current molecular biology. The monoclonal antibody secreted by another strain is more specific and can detect U-type salmon trout IHNV infection, but it has no reaction with J-type salmon trout IHNV, so it can be used to identify the genotype of salmon trout IHNV in China. The detection method of the invention can directly observe the result through a fluorescence microscope, and this operation can be completed in a common laboratory, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic microorganisms of animal infectious diseases, and relates to a salmon trout IHNV monoclonal antibody and a detection kit, which are used for rapid and accurate detection of rainbow trout infectious hematopoietic necrosis virus (IHNV) infection. [0002] technical background [0003] Infectious hematopoietic necrosis is an acute infectious disease that can cause salmon, trout and other salmonid fish. It is caused by infectious hematopoietic necrosis virus (IHNV), a member of the Rhabdoviridae family. The disease is mainly characterized by necrosis of hematopoietic organs such as the kidney and spleen, and the main route of transmission is the import and export of diseased fish or infected fish eggs. The disease first became prevalent in the Pacific Northwest of the United States and later spread throughout North America, Asia, and Europe. In 1985, the IHNV virus entered Northeast my country,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K16/10C12N5/20G01N33/577G01N33/569C12R1/90C12R1/93
CPCC07K16/10C12N7/00C12N2760/20021G01N33/56983G01N33/577G01N2333/145
Inventor 孙惠玲徐福洲李芳兵
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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