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Avianinflu virus H5 subtype emulsion agglutination kit and its use

A bird flu virus and kit technology, applied in the field of poultry immunology, can solve problems such as difficulty in product development, short shelf life of latex reagents, and difficulty in binding to latex surfaces, etc., to achieve a wide range of detection samples, short detection time, and easy operation Effect

Active Publication Date: 2007-06-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using latex agglutination technology to establish pathogenic detection methods, it is generally to sensitize specific antibodies to the surface of latex particles. The combination of ordinary polystyrene latex and antibodies is non-selective physical electrostatic adsorption. Due to the small molecular weight of antibodies, it is very difficult to Difficult to bind to the latex surface, even the sensitized antibody is easy to fall off from the latex particles or denatured and inactivated, resulting in a short shelf life of the sensitized latex reagent, which is difficult to meet the needs of product development

Method used

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  • Avianinflu virus H5 subtype emulsion agglutination kit and its use
  • Avianinflu virus H5 subtype emulsion agglutination kit and its use

Examples

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Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation and identification of anti-H5 subtype avian influenza virus hemagglutinin protein monoclonal antibody

[0038] 1. Preparation of monoclonal antibodies

[0039] With reference to Xue Qingshan, "Principles and Techniques of In Vitro Culture", Science Press, the method in the 2001 edition: use the avian influenza H5 subtype oil emulsion inactivated vaccine (purchased from China Harbin Veken Biotechnology Development Co., Ltd. to produce) as antigen immunization Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences), the immunization program is to select 4 BALB / c mice for immunization, the dose is 200ul / only, and each mouse is injected subcutaneously at multiple points on the back of the neck. Two weeks later, the same dose was used to boost the immunization once, and the dose was 400ul / mouse, and the mice were injected with 400ul antigen intraperitoneally after four weeks. Three days later, the splenocytes w...

Embodiment 2

[0052] Example 2 Establishment of avian influenza virus H5 subtype latex agglutination detection method

[0053] Take 125 μL of 10% carboxylated latex into EP tube, wash 3 times with pH 9.6, 0.1M carbonic acid buffer, then select 3 times with pH 4.5, 0.01M phosphate buffer, then add 0.5% water-soluble carbonic acid buffer Imine was reacted in phosphate buffer at room temperature (25°C) for 4 hours, then washed 3 times with pH 8.5, 0.01M boric acid buffer, suspended the latex in 1ml boric acid buffer, added an appropriate amount of IgG for sensitization for a certain period of time, and then terminated the addition agent to terminate the reaction.

[0054] The specific steps are as follows:

[0055] 1. Determination of the optimal amount of conjugated IgG

[0056] Add different amounts of IgG to 1 mL of carboxylated latex suspended in boric acid buffer, increasing from 200 μg, 400 μg, 600 μg to 1800 μg, collect the supernatant, measure the remaining protein in the supernatant...

Embodiment 3

[0070] Example 3 Preparation of Positive Control of Avian Influenza Virus H5 Subtype Latex Agglutination Detection Kit

[0071] The H5 subtype avian influenza virus hemagglutinin protein expressed in insect cell SF9 was selected as the positive control sample of the kit. The hemagglutinin gene of the H5 subtype avian influenza virus was cloned into the Pfastbac plasmid in the baculovirus BAC TO BAC expression system, and the Pfastbac-HA intermediate transfer plasmid was constructed. In Escherichia coli DH10BAC, homologous recombination occurred between Pfastbac-HA and the modified californica nuclear polyhedrosis virus (ACNPY) genome (BACMID) to obtain a recombinant shuttle vector containing the complete HA gene, and transfected The recombinant baculovirus ACNPY-HA was obtained by transfecting SF9 cells, and a band with a size of about 70kd was seen by SDS-PAGE electrophoresis and Western-blotting analysis, indicating that the hemagglutinin gene was expressed in insect cells. ...

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Abstract

This invention discloses a latex agglutination kit to quickly detect H5 subtype of avian influenza virus. The anti monoclonal antibodies of H5 subtype of avian influenza virus hemagglutinin protein were coupled to the surface of carboxyl polystyrene latex particles using water-soluble carbodiimide, the methods about detection of H5 subtype of avian influenza virus were established, the latex agglutination detection kits of H5 subtype of avian influenza virus were prepared supplemented by other matching reagents. This kit was made up of box body and the body latex diagnostic reagents in the box, sample handling liquid A, sample handling liquid B, the positive control samples and negative control samples. This invention kit can directly detect the H5 subtype avian flu virus, with high specificity, high sensitivity, simple and rapid diagnosis, and other significant advantages.

Description

technical field [0001] The invention belongs to the technical field of poultry immunology. Specifically, the invention relates to the development of a latex agglutination detection kit suitable for H5 subtype of avian influenza virus and its application in the detection of H5 subtype avian influenza virus. Background technique [0002] As we all know, H5N1 subtype highly pathogenic avian influenza is a severe infectious disease that seriously endangers the poultry industry and endangers human health. The "Hong Kong bird flu" incident in 1997 was the first direct cross-species transmission of bird flu to humans, and the Southeast Asian bird flu in 2004 aroused great concern around the world on the cross-species spread of bird flu. In my country in 2004, there were also 50 epidemic spots in 16 provinces and cities where bird flu outbreaks broke out on a large scale, which caused great losses to the poultry industry in my country. Since July 2005, Vietnam, Thailand, Cambodia, ...

Claims

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Application Information

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IPC IPC(8): C12N5/12C07K16/08G01N33/53G01N33/577
Inventor 金梅林陈剑锋但汉并吴斌方六荣陈焕春
Owner HUAZHONG AGRI UNIV
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