Anti-infectious hematopoietic necrosis virus monoclonal antibody and its application

A technology of necrosis of hematopoietic organs and monoclonal antibodies, applied in the field of immunology, can solve the problems of lack of good antigens and antiserum, unsuitable for grassroots laboratories, and inability to meet rapid detection, etc., to achieve improved purity, good specificity, and potency high effect

Active Publication Date: 2015-12-09
北京市水产技术推广站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the accuracy of cell culture separation technology is high, but the detection cycle is long, and it takes at least 15 days to get the final result, which cannot meet the needs of rapid detection; molecular biology methods are based on cell separation methods and are used to detect cell culture After the virus, or further confirmation of the virus carried by the dominant infected fish; immunological detection technology based on monoclonal antibody technology is an effective way to meet the rapid and large-scale detection of IHNV in grassroots laboratories. It has been reported, or There is no good antigen and antiserum, or more expensive precision instruments are required, which is not suitable for grassroots laboratories, and has not been able to be applied to disease monitoring on a large scale

Method used

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  • Anti-infectious hematopoietic necrosis virus monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of monoclonal antibodies against infectious hematopoietic necrosis virus

[0027] 1. Antigen purification

[0028] The antigen used was IHNV-UK (UK strain of infectious hematopoietic necrosis virus) deposited by the OIE Reference Laboratory in Weymouth, UK. Inoculate EPC (carp epithelioma cells) in the exponential growth phase, and use M199 (containing Earle’s balanced salt solution, 10% fetal calf serum and 200U / mL penicillin-streptomycin, pH7.2-7.6) at 17.5℃ to increase the value. The virus was collected, centrifuged at 8000r / min for 30min, and then centrifuged at 24000r / min for 2h. The pellet was resuspended in 0.5 mL of 0.01MPBS.

[0029] 2. Animal immunity

[0030] The immunized mice were SPF-grade 5-week-old female BALB / c mice. Each mouse was immunized by intraperitoneal injection with the purified virus as the antigen. The virus was mixed with Freund’s complete adjuvant 1:1, and injected intraperitoneally; after 2 weeks, the booster immunization ...

Embodiment 2

[0035] Example 2: Determination of the biological characteristics of monoclonal antibodies against infectious hematopoietic necrosis virus

[0036] 1. Determination of potency

[0037] Methods: The ELISA plate was coated with purified infectious hematopoietic necrosis virus at a concentration of 15 μg / mL, and the ascites titer of monoclonal antibodies was identified by indirect ELISA.

[0038] Results: The ascites titer of the monoclonal antibody of the present invention is 1:2.5×10 5 , Indicating that the hybridoma cell line has the ability to secrete high-titer antibodies.

[0039] 2. Subtype identification of monoclonal antibodies

[0040] Method: Using SouthernBiotech's typing kit (SBAClonotyping TM System / HRP) to identify the Ig subtype of the monoclonal antibody of the present invention according to its instructions.

[0041] Results: The subtype of the monoclonal antibody of the present invention is IgG1 type k chain.

[0042] 3. Specificity analysis of monoclonal antibodies

[00...

Embodiment 3

[0047] Example 3 Infectious hematopoietic organ necrosis virus double sandwich ELISA detection kit

[0048] 1. The composition of the double-sandwich ELISA test kit for infectious hematopoietic necrosis virus

[0049] The composition of the Infectious Hematopoietic Organ Necrosis Virus Double Sandwich ELISA Test Kit is as follows: 1) A solid-phase carrier containing polyclonal antibodies against infectious Hematopoietic Organ Necrosis Virus; 2) Monoclonal antibodies prepared in Example 1; 3) Horseradish Peroxidase labeled goat anti-mouse antibody (purchased from sigma company); 4) enzyme substrate reaction solution; 5) positive control and negative control; 6) washing solution; 7) enzyme reaction termination solution.

[0050] 1) Preparation of solid-phase carrier containing polyclonal antibody against infectious hematopoietic organ necrosis virus: immunize goats with infectious hematopoietic necrosis virus (ATCCVR-714) purified by gradient centrifugation. The serum is prepared by a...

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Abstract

The invention discloses an infectious haematopoietic necrosis virus resisting monoclonal antibody and application thereof. The infectious haematopoietic necrosis virus resisting monoclonal antibody is generated through the secreting of a mouse hybridoma cell strain with the collection number of CGMCC No. 8556. The infectious haematopoietic necrosis virus resisting monoclonal antibody can be used for detecting an infectious haematopoietic necrosis virus. The invention further discloses an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting the infectious haematopoietic necrosis virus.

Description

Technical field [0001] The invention belongs to the field of immunology, and specifically relates to a monoclonal antibody against infectious hematopoietic organ necrosis virus, hybridoma cell strains and a sandwich ELISA method kit for detecting infectious hematopoietic organ necrosis virus. Background technique [0002] Infectious haematopoietic necrosisvirus (IHNV) belongs to the Rhabdoviridae family (Rhabdoviridae) and Novirhabdovirus in classification. It mainly infects cold-water fishes such as rainbow trout and hardhead trout. The cumulative mortality rate during acute infection is over 90-95%, and it can be transmitted vertically to offspring. It was first popular in some countries in North America and Europe. With the continuous spread of aquatic product trade, it has spread to major cold-water fish breeding areas in Northeast my country, Hebei, Shandong, Beijing, Gansu, Qinghai, etc., and outbreaks in some areas , Causing greater economic losses. IHN is listed as a dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569G01N33/543
Inventor 曹欢徐立蒲景宏丽王静波王姝王小亮
Owner 北京市水产技术推广站
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