Salmons IHNV monoclonal antibody and detection kit

A technology of monoclonal antibody and salmon trout, applied in immunoassay, measuring device, antiviral immunoglobulin, etc., can solve the problems of delayed inspection, no report, low detection rate, etc., achieve obvious negative control, avoid Cross reaction, low cost effect

Inactive Publication Date: 2017-05-31
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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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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  • Salmons IHNV monoclonal antibody and detection kit
  • Salmons IHNV monoclonal antibody and detection kit
  • Salmons IHNV monoclonal antibody and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 monoclonal antibody

[0033] 1. Virus acquisition

[0034] 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.

[0035] 2. Virus preparation

[0036] Carp epithelial tumor cel...

Embodiment 2

[0055] The establishment of embodiment 2 immunofluorescence detection

[0056] 1. Preparation of test samples

[0057] 1.1 EPC cells inoculated with IHNV

[0058] Cultivate EPC cells at 25°C. When the cells grow into a single layer and 90% of the cells are full, the ratio of IHNV virus to cell fluid inoculation is 1:50-1:70 times, and the inoculation is optimal. When the cytopathy reaches about 50%. The cells can then be fixed for further detection. At the same time, a negative control was set up. 1.2 Determination of fixative and fixation time

[0059] 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% acetone fixation time over 5min ...

Embodiment 3

[0069] Composition and use of embodiment 3 test kit

[0070] 3.1 The components of the IHNV immunofluorescence detection kit are as follows:

[0071] cell culture plate;

[0072] IHNV N protein specific monoclonal antibody;

[0073] Enzyme-labeled secondary antibody: FITC-labeled rabbit anti-mouse IgG;

[0074] Washing solution: PBST

[0075] Positive standard: carp epithelial tumor cells (Epithelioma Papulosum Cyprini, EPC) inoculated with IHNV (fixed by 70% acetone);

[0076] Negative standard: carp epithelial tumor cells (Epithelioma Papulosum Cyprini, EPC) not inoculated with IHNV (fixed with 70% acetone).

[0077] 3.2 How to use the IHNV immunofluorescence detection kit:

[0078] (1) Take out the cell culture plate and culture carp epithelial tumor cells (EPC); culture EPC cells at 25°C, when the cells grow into a monolayer and grow to 90%, dilute the aseptically treated cells according to the ratio of 1:50-1:70 For the sample to be tested, when the cytopathic effect ...

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Abstract

The invention provides a monoclonal antibody. The monoclonal antibody is prepared from salmons IHNV (infectious hematopoietic necrosis virus) immune animals, and has specificity on IHNV N protein. The invention also provides a salmons IHNV detection kit and a method, and application of the monoclonal antibody in preparing a product for diagnosing the salmons IHNV. The prepared IHNV monoclonal antibody has the advantages that the specificity is strong, and the infection of domestic J type salmons IHNV is detected, except U type; the crossing reaction with other rhabdovirus can be avoided; the detection fault and missing of the IHNV infected juvenile fishes by the existing molecular biology can be overcome; the results can be directly observed by a fluorescent microscope, the operation is simple, convenient and quick, the cost is low, the operation can be completed via the common laboratory, and the good application prospect is realized on the IHNV immune detection.

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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IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569C12R1/91
CPCC07K16/10G01N33/56983G01N33/577G01N2333/145G01N2469/10
Inventor 孙惠玲罗琳马志宏姜娜李铁梁邢薇张莉步卫东王宏俊温书香
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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