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Avian influenza virus latex agglutination assay kit and its use

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

Active Publication Date: 2007-08-29
WUHAN KEQIAN BIOLOGY CO LTD
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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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  • Avian influenza virus latex agglutination assay kit and its use
  • Avian influenza virus latex agglutination assay kit and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Isolation and identification of embodiment 1 bird flu strain

[0036] The number used in the present invention is the QLG-001 avian influenza virus strain, which was isolated from ducks sent for inspection in a certain place in Hubei Province on April 5, 2003. It was identified as the H9N2 subtype by the China Influenza Center, and was observed by electron microscopy and serology. The results of the test, pathogenicity and animal regression test confirmed that the virus isolate was a typical type A influenza virus. EID 50 And pathogenicity tests (IVPI and ICPI) show that the virus is a low pathogenicity strain (LPAIV). The avian influenza virus strain QLG-001 was deposited in CCTCC on September 2, 2005, and the preservation number is CCTCC-V200512. The preservation method of the virus strain is to freeze-dry and preserve it under the condition of -80°C.

[0037] The specific separation and identification steps are as follows:

[0038] (1) Incidence and necropsy lesi...

Embodiment 2

[0086] Preparation and inspection of rabbit anti-avian influenza virus nucleoprotein immune serum of embodiment 2

[0087] 1 Preparation of immune antigen

[0088] (1) Proliferation of virus

[0089] Get QLG-1 virus strain, make 10 with sterilized normal saline -4 Dilute and inoculate specific pathogenic microorganism-free (SPF) chicken embryos into the allantoic cavity, 0.2 mL per embryo, seal the pinhole after inoculation, and continue to incubate at 37°C. Eggs were illuminated once every 12 hours, and the chicken embryos that died 24 hours ago were discarded, and the chicken embryos that died 24 hours to 72 hours were cooled at 4°C overnight, and the allantoic fluid of the chicken embryos was harvested, and the HA titer ≥ 9log2 was qualified. .

[0090] (2) Inactivation of virus

[0091] After freezing and thawing the qualified chicken embryo allantoic fluid for 3 times, add formaldehyde with a final concentration of 0.2%, mix well and replace it into another sterile co...

Embodiment 3

[0102] Embodiment 3 Establishment of avian influenza virus latex agglutination detection method

[0103] Take 125 μL of 10% carboxylated latex into EP tube, wash 3 times with pH9.6, 0.1M carbonic acid buffer, then select 3 times with pH4.5, 0.01M phosphate buffer, then add 0.5% water-soluble carbodioxide The amine was reacted in phosphate buffer at room temperature for 4 hours, and then washed 3 times with pH 8.5, 0.01M boric acid buffer, the latex was suspended in 1ml boric acid buffer, and an appropriate amount of IgG was added to sensitize for a certain period of time, and then a terminator was added to terminate the reaction. .

[0104] 1 Determination of the amount of optimally coupled IgG

[0105] Add different amounts of IgG to 1 mL of carboxylated latex suspended in boric acid buffer, increasing from 200 μg, 400 μg, and 600 μg to 1800 μg. Protein amount, calculate the coupling efficiency of IgG, the formula is as follows:

[0106] Coupling efficiency of IgG (%)=(Ini...

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Abstract

The invention discloses a reagent box of latex agglutination which is used for quickly detecting the birds flu virus. Putting the hare birds flu anti-virus nuclein IgG couple to the surface of carboxylation polystyrene latex particles, founding the method to test the latex agglutination of birds flu virus, then supplemented by other match reagents prepared the reagent box of detect the bird flu virus latex agglutination. the reagent box of detect the bird flu virus latex agglutination is composed of box body and latex diagnostic reagents, sample handling liquid of A which is located in box, sample handling liquid of B, positive contrast sample and negative contrast sample. The reagent box is referred directly detect the birds flu virus, its notable advantages are that strong specificity, high sensitivity, simple operation and rapid diagnosis.

Description

technical field [0001] The invention belongs to the technical field of poultry immunology, and relates to related fields such as immunology and applied chemistry. Specifically, the present invention relates to a latex agglutination detection kit of avian influenza virus and the application of the kit in detection of avian influenza virus. Background technique [0002] As we all know, 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. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
Inventor 金梅林陈剑锋喻正军张安定李红超陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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