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Preparation method and application of EHNV (Epizootic Haematopoietic Necrosis Virus) antiserum

An anti-serum, blood sample technology, applied in the direction of serum immunoglobulin, anti-animal/human immunoglobulin, measuring device, etc., can solve the problems of unfavorable promotion and use of EHN immunological diagnosis method, high price and high cost. The effect of promoting the use of

Inactive Publication Date: 2014-10-08
LIANYUN PORT IMMIGRATION INSPECTION & QUARANTINE BUREAU PEOPLES REPUBLIC OF CHINA
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] In addition, immunological identification methods such as ELISA are also commonly used and recommended by the "Diagnostic Manual of Aquatic Animal Diseases". This method requires various EHN immunological diagnostic reagents (mainly EHNV antiserum) ), and there are no these reagents (especially EHNV antiserum) on the domestic market, mainly rely on imports, which are expensive and costly, which is not conducive to the promotion and use of EHN immunological diagnostic methods

Method used

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  • Preparation method and application of EHNV (Epizootic Haematopoietic Necrosis Virus) antiserum
  • Preparation method and application of EHNV (Epizootic Haematopoietic Necrosis Virus) antiserum
  • Preparation method and application of EHNV (Epizootic Haematopoietic Necrosis Virus) antiserum

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Embodiment 1

[0037] For the preparation method of this EHNV antiserum provided by the embodiments of the present invention, please refer to figure 1 , including the following steps:

[0038] Step 101: adding EHNV virus liquid to the newly cultured sensitive cells for a predetermined period of time, and then adding M199 medium for cultivation to obtain diseased cell liquid;

[0039] In step 101, the sensitive cells refer to cell lines established in vitro. By adding EHNV virus liquid to sensitive cells, the virus liquid will be adsorbed on the sensitive cells in large quantities, and a large amount of virus multiplication will be carried out, while the M199 medium will provide nutrients for the proliferation of sensitive cells. When the (CPE) reaches 80%, the poison collection operation can be carried out, and it can be quickly frozen in a -80°C refrigerator for later use.

[0040] Step 102: Centrifuge the diseased cell liquid at 10000×g-14000×g for 25-35 minutes to obtain the first super...

Embodiment 2

[0057] In this embodiment, the preparation method of the EHNV antiserum preparation method comprises the following steps:

[0058] S1: Add EHNV virus liquid to newly cultured sensitive cells, add M199 medium for culture after adsorption for a predetermined time, and obtain diseased cell liquid;

[0059] In the present invention, a sensitive cell refers to a cell line established by in vitro culture, specifically, a sensitive cell can be a perch gill cell line (BF-2), a fat-headed carp muscle cell line (FHM) or a carp epithelial tumor Cell line (EPC). In this example, the sensitive cells used are specifically CHSE-214. In addition, during the adsorption process, in order to achieve a large amount of virus adsorption in cells, the adsorption temperature is 12-18°C (15°C is the best) , The scheduled time is 50-70min (60min is the best). In addition, in order to provide sufficient nutrition for CHSE-214 cells, preferably, the M199 medium contains 2% fetal bovine serum by volume,...

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Abstract

The invention relates to the field of aquatic animal disease control and in particular relates to a preparation method and an application of an EHNV (Epizootic Haematopoietic Necrosis Virus) antiserum. The preparation method of the EHNV antiserum comprises the steps of preparing a high-purity antigen, purifying and carrying out immunization so as to obtain the EHNV antiserum. With the adoption of the preparation method of the EHNV antiserum, the EHNV antiserum is prepared. According to the preparation method, as a low-speed long-time centrifugation method is adopted, high-concentration EHNV (purified virus) is obtained, the immune operation is carried out on an experimental animal with the high-concentration EHNV as an antigen, and a foundation of preparing the antiserum with strong specificity and high titer is laid; the problems in China EHNV detection technology that an immunology diagnostic reagent is high dependent on import, the detection cost is high and the like are solved, and an EHNV immunology diagnostic method is beneficial to popularization and application.

Description

technical field [0001] The invention relates to the field of prevention and treatment of aquatic animal diseases, in particular to a preparation method and application of EHNV antiserum. Background technique [0002] Epidemic hematopoietic necrosis (EHN) is a notifiable fish disease announced by the World Organization for Animal Health (OIE). [0003] At present, at home and abroad, there are mainly the following diagnostic methods for EHN: virus isolation, immunoperoxidase test, antigen capture ELISA, immunoelectron microscopy and PCR. Among them, virus isolation is the most commonly used method, and the isolated virus is mainly identified by PCR technology. [0004] In addition, immunological identification methods such as ELISA are also commonly used and recommended by the "Diagnostic Manual of Aquatic Animal Diseases". This method requires various EHN immunological diagnostic reagents (mainly EHNV antiserum) ), and there are no these reagents (especially EHNV antiserum...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06G01N33/573
Inventor 周毅段宏安徐晔丁超姚燕林曹洁李金华
Owner LIANYUN PORT IMMIGRATION INSPECTION & QUARANTINE BUREAU PEOPLES REPUBLIC OF CHINA
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