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Kit box used for detecting influenza virus in sample and detection method and application thereof

A technology for detecting influenza virus and samples, applied in chemical instruments and methods, measuring devices, instruments, etc., can solve the problems of low sensitivity and achieve the effects of high detection efficiency, convenient use, and simple results

Active Publication Date: 2014-09-24
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to realize the rapid, simple and accurate detection of type A influenza virus and the problem of low sensitivity in detection, the present invention provides two kinds of anti-type A influenza virus nucleoprotein monoclonal antibodies and the enzyme chromatography detection kit prepared therefrom, The kit not only has higher sensitivity than conventional colloidal gold rapid detection test strips, but also has fast detection and easy operation, and is suitable for rapid detection of type A influenza virus

Method used

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  • Kit box used for detecting influenza virus in sample and detection method and application thereof
  • Kit box used for detecting influenza virus in sample and detection method and application thereof
  • Kit box used for detecting influenza virus in sample and detection method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1 Preparation, Purification, Identification and Testing of Anti-Influenza Virus Nucleoprotein Monoclonal Antibody

[0051] 1.1 Preparation and purification of anti-influenza virus nucleoprotein monoclonal antibody

[0052] The preparation and purification of anti-influenza virus nucleoprotein monoclonal antibody comprises the following steps:

[0053] 1.1.1 Animal immunization: Add 1 mL of inactivated virus of type A influenza virus A / California / 04 / 2009 (H1N1) strain and add the same amount of complete Freund's adjuvant and fully emulsify it, and immunize with the homologous myeloma cells used 6-8 weeks old BALB / c healthy mice, each multi-point injected with 500 μL of emulsified influenza A virus A / California / 04 / 2009 (H1N1) strain, boosted once every 2 weeks; its antiserum was measured by indirect ELISA :

[0054] After the completion of the process, the antiserum is measured by indirect ELISA, which consists of the following steps:

[0055] a. Coating:

[0...

Embodiment 2

[0100] Example 2 Pairing of Monoclonal Antibodies

[0101] 2.1 Selection of monoclonal antibody pairing mode

[0102] When screening monoclonal antibody pairing combinations, the following factors are mainly considered: first, the activity of the monoclonal antibody; second, whether there is a non-specific reaction between the monoclonal antibody and non-type A influenza virus; third, the color background.

[0103] 2.2 Detection of Monoclonal Antibody Activity

[0104] Select type A avian influenza virus strain A / Ck / HK / Yu22 / 02, dilute to 0.01HA, and test different matching modes. The results are shown in Table 2:

[0105] Table 2 Monoclonal antibody matching and activity detection

[0106]

[0107] Note: + means positive, - means negative.

[0108] The results showed that except for the Yu22 virus dilution of 0.01HA of monoclonal antibody 1 immobilized and monoclonal antibody 2 enzyme-labeled detection was negative, the other combinations were all positive results, but m...

Embodiment 3

[0117] The structure and use of embodiment 3 type A influenza virus enzyme chromatography detection kit

[0118] 3.1 Immobilization of monoclonal antibodies

[0119] The monoclonal antibody 2 prepared in Example 1 was immobilized on the nitrocellulose membrane with a BioDot XYZ3050 three-dimensional spraying platform.

[0120] 3.2 Horseradish peroxidase (HRP) labeling of monoclonal antibodies

[0121] According to the monoclonal antibody 1 prepared in Example 1, according to Tijssen P et al (Tijssen P, Kurstak E. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase antibody conjugates for enzymeimmunoassays [J]. -457) literature of anti-influenza A virus monoclonal antibody was labeled with horseradish peroxidase (HRP).

[0122] 3.3 Composition of the detection kit

[0123] The detection kit includes an enzyme chromatography detection test strip, a sample processing solution, a sample processing tube, and a sample preservation solution. ...

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Abstract

The invention provides a kit box used for detecting an influenza virus in a sample. The kit box comprises a buffer solution supply unit and a detection test strip. The detection test strip sequentially comprises a substrate supply area, a sample supply area and a detection area in the longitudinal direction. The substrate supply area comprises a substrate pad (3), a dry enzyme substrate is adsorbed to the substrate pad, and the substrate pad (3) makes contact with a nitrocellulose membrane (1). The sample supply area comprises an enzyme mark pad (2), a rat resistance avian influenza virus nucleoprotein antibody marking monoclonal antibody 1 is adsorbed to the enzyme mark pad (2), and the enzyme substrate can product a chromogenic reaction with enzymes marked on the marking monoclonal antibody 1. The detection area is provided with a rat resistance avian influenza virus nucleoprotein antibody fixed monoclonal antibody 2 in an immobilization mode. The invention further provides a method for detecting the influenza virus in the sample through the kit box. The kit box and the detection method of the kit box have the advantages of being rapid and accurate in detection, high in specificity, sensitive and good in stability.

Description

technical field [0001] The invention belongs to the field of biotechnology. Background technique [0002] Type A influenza viruses include avian influenza virus (Avian Influenza Vitus, AIV), swine influenza virus (Swine Influenza Vitus, SIV), equine influenza virus (Equine InfluenzaVitus, EIV) and canine influenza virus (Canine Influenza Vitus, CIV), etc. The Animal Health Organization (Office of International Education, OIE) lists the diseases caused by type A influenza virus as a class A animal disease, and my country classifies it as a class I animal disease. [0003] Among them, avian influenza virus can cause avian influenza, which is a highly contagious disease. According to its pathogenicity, it can be divided into highly pathogenic avian influenza and low pathogenic avian influenza, especially highly pathogenic avian influenza Influenza has brought a devastating blow to the poultry industry; swine flu virus can cause swine flu, and even lead to death of pigs. A loss...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
CPCC07K16/1018C07K2317/56G01N33/558G01N33/56983G01N33/577G01N2333/11
Inventor 张许科孙进忠李凡田克恭
Owner LUOYANG PULIKE WANTAI BIOTECH
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