H1-subtype influenza A virus double-antibody sandwich ELISA kit and application

A double-antibody sandwich and influenza virus technology, applied in the direction of virus/bacteriophage, antiviral immunoglobulin, biochemical equipment and methods, etc., can solve the problems of human and animal health impact, economic loss, etc., and achieve a wide range of detection samples, Long shelf life and good stability

Active Publication Date: 2012-08-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Influenza A (H1N1) has a huge impact on human and animal health, and has caused huge economic losses

Method used

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  • H1-subtype influenza A virus double-antibody sandwich ELISA kit and application
  • H1-subtype influenza A virus double-antibody sandwich ELISA kit and application
  • H1-subtype influenza A virus double-antibody sandwich ELISA kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation Example

[0035] 1. Isolation of Influenza A H1 Subtype Virus

[0036] 1. Separation process: collect throat samples from sick pigs with sterile throat swabs, place them in sterile phosphate buffered saline (PBS for short, formula: Na 2 CO 3 1.59g, NaHCO 3 2.93g, with ddH 2 (0 was fixed to 1000ml), and then 0.2ml of throat swab sample liquid was inoculated into 9-day-old specific pathogen-free (Specific pathogen Free, SPF) chicken embryos (purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd.), and placed at 35 Incubate at ℃ for 72 hours, receive the allantoic fluid and do the hemagglutination qualitative test, and the samples that are positive in the hemagglutination test will be finalized by RT-PCR test.

[0037] 2. Identification basis: RT-PCR detected positive samples, amplified HA, sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing, and compared the obtained DNA sequence with the sequence published on t...

Embodiment 2

[0053] Example 2 Preparation of Anti-Influenza A H1 Subtype Influenza Hemagglutinin Protein Monoclonal Antibody

[0054] 1. Inactivated and purified H1 subtype influenza virus A-Influ / JML-F9 original strain (preserved in the Chinese Type Culture Collection Center in Wuhan University, Wuhan City, Hubei Province, the preservation number is CCTCC NO: V201105 ) as antigen, emulsified with Freund's adjuvant (freund's complete adjuvant for the first immunization, and incomplete freund's adjuvant for the second and third immunization) to immunize 5-8 week-old BALB / c mice (purchased from Experimental Animals of Hubei Province Research center). The first immunization dose is 20ug / mouse, each mouse is injected subcutaneously at multiple points on the back of the neck. Two weeks later, a booster immunization was given with the same dose, and a booster immunization was given again two weeks later, with a dose of 40ug / mouse. After two weeks, a small amount of mouse blood was collected by ...

Embodiment 3

[0081] Example 3 Identification of Anti-Influenza A H1 Subtype Influenza Hemagglutinin Protein Monoclonal Antibody (Deposit Number CCTCC NO: C201106)

[0082] 1. Identification of HI potency

[0083] The H1 subtype influenza virus isolated by the present invention (preservation number: CCTCCNO: V201105) was used as an antigen to measure the titer of hybridoma cell culture supernatant and mouse ascites by hemagglutination inhibition method. The results are shown in Table 1.

[0084] Table 1: Determination of titers of hybridoma cell culture supernatant and mouse ascites by hemagglutination inhibition method (HI)

[0085]

[0086] 2. Identification of the Ig subclass (type) of the monoclonal antibody (the deposit number is CCTCC NO: C201106)

[0087] The monoclonal antibody obtained in the present invention is identified with a mouse monoclonal antibody subtype identification kit (Mouse Mab Isotyping Test Kit, purchased from ROCKLAND Company), and the monoclonal antibody an...

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Abstract

The invention belongs to the fields of detection technology of animal virology and epizootiology. The invention particularly relates to an H1-subtype influenza A virus double-antibody sandwich ELISA kit and an application. The kit of the invention comprises an enzyme label plate which contains anti-H1-subtype influenza A virus hemagglutinin, has a preservation number of CCTCC NO: C201106, and is coated by a monoclonal antibody, and a horseradish peroxidase labeled H1-subtype influenza A virus hemagglutinin monoclonal antibody is used as a second antibody. The invention discloses separation, amplification, inactivation, and purification methods of the H1-subtype influenza A virus, and preparation and purification methods of the H1-subtype influenza A virus hemagglutinin monoclonal antibody. The invention also discloses an H1-subtype influenza A virus double-antibody sandwich ELISA detection method.

Description

technical field [0001] The invention relates to the technical field of animal virology and animal infectious disease detection. Specifically, the present invention relates to a type A H1 subtype influenza virus double-antibody sandwich ELISA detection kit and its application in the detection of type A H1 subtype influenza virus antigen in swine. Background technique [0002] In 1975, Kohler G and Milstein C published the cell fusion method to establish hybridoma technology in Nature magazine, creating an epoch-making hybridoma monoclonal antibody technology. After cloning, high-purity antibodies with identical structures and various characteristics are produced, which are called monoclonal antibodies (Monoclonal Antibody, McAb), or monoclonal antibodies for short. The discovery and use of monoclonal antibody technology has greatly promoted the development of modern life science research and has become an important aspect in the field of biotechnology. So far, the applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N5/20C12N7/00G01N33/577C12R1/91C12R1/93
Inventor 金梅林赵刚但汉并王灿郭学波周洪波张安定
Owner HUAZHONG AGRI UNIV
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