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Anti-influenza A virus nucleoprotein monoclonal antibody, its preparation and application

A type of influenza A virus, monoclonal antibody technology, applied in antiviral immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of lack of good domestic kits, high price, etc. The detection rate advantage, the effect of high affinity

Active Publication Date: 2015-07-01
GUANGDONG WESAIL BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, NP is not completely conserved, so it is necessary to ensure that the prepared monoclonal antibody is directed against its conserved antigenic site
At present, in the field of rapid detection of influenza, there are no good domestic kits at present, and the price of foreign kits is relatively expensive, so it is of great significance to research and develop antibodies suitable for rapid detection

Method used

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  • Anti-influenza A virus nucleoprotein monoclonal antibody, its preparation and application

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Experimental program
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Effect test

Embodiment 1

[0030] The establishment of embodiment 1 hybridoma cell line and the preparation of anti-influenza virus nucleoprotein monoclonal antibody

[0031] 1. Antigen Immunization

[0032] An equal amount of recombinant influenza A nucleoprotein antigen (manufactured by Shenzhen Feipeng Biological Co., Ltd., product number BA-IAV0004) was mixed with complete Freund's adjuvant to obtain an oily emulsion. The emulsion was subcutaneously administered to the back site of BALB / c mice (Experimental Animal Center of Guangzhou Province, 6-week-old female, 5) with a dose of 0.2 ml, and the abdominal cavity was boosted immunization (equal amount of antigen and Ephrine) 14 days after the first immunization. Mixed with incomplete adjuvant), after boosting immunization to four injections, the tail blood was collected for titer detection, and the titer reached the fusion requirement.

[0033] Three days before fusion, the same dose of antigen was injected intraperitoneally for booster immunization...

Embodiment 2

[0053] The preparation of embodiment 2 influenza A virus colloidal gold rapid detection test paper

[0054] 1. Preparation of Nitrocellulose Membrane

[0055] Coating buffer preparation: 0.01M PH7.2 PBS buffer containing 6% methanol was used as the coating buffer, filtered through a 0.22μ membrane, set at 4°C for later use, and was valid for one week. 1000ml 6% Methanol 0.01M PH 7.2 PBS Buffer Recipe: NaCL 8g, KCL 0.2g, NaCl 2 HPO 4 .12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60ml, distilled deionized water to 1000ml.

[0056] Preparation of nitrocellulose membrane: Dilute the anti-influenza A virus nucleoprotein rabbit polyclonal antibody (produced by Shenzhen Feipeng Biological Co., Ltd., product number BA-PAB-NP0003) to 1-5 mg / ml with coating buffer, Adjust the machine, draw the T line, which is the detection line, and the T line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad; the goat anti-mouse IgG antibody (Shenzhen ...

Embodiment 3

[0080] Example 3 The kit for rapid detection of influenza A virus NP protein

[0081] 1. The kit for rapid detection of influenza A virus NP protein includes:

[0082] ①A pack of test strips (10 strips / pack)

[0083] ② One bottle of sample diluent (10ml / bottle)

[0084] Preparation of relevant solutions

[0085]Sample diluent: The sample diluent is 8% NaCl solution. Preparation method: 80gNaCL, add distilled water to make up to 1000ml.

[0086] 2. Detection of Influenza A Virus NP Protein by Colloidal Gold Method

[0087] (1) Place the collected nasopharyngeal swab directly into a plastic test tube containing 300 μl of diluent, squeeze it fully to completely dissolve the nasal secretion in the diluent, take 120 μl of the dissolved sample and add it to the test paper card for sample addition Hole, wait for 15 minutes to observe the results.

[0088] (2) Result judgment: when the test strip shows a purple-red quality control line visible to the naked eye, but there is no v...

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Abstract

The invention discloses an anti-influenza A virus nucleoprotein monoclonal antibody with high affinity and high specificity, its preparation and application. The anti-influenza A virus nucleoprotein monoclonal antibody is secreted by a hybridoma cell strain H1N1-2F10, and the preservation number of the cell strain is CCTCC C201119. The invention also provides a kit prepared with monoclonal antibody for rapid detection of influenza A. The kit has the characteristics of simple operation, strong specificity and high sensitivity, etc.

Description

technical field [0001] The invention relates to the field of influenza immune detection, in particular to an anti-influenza A virus nucleoprotein monoclonal antibody and its preparation and application. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and are RNA viruses. The core inside the virus is composed of single-stranded ribonucleic acid and nucleoprotein (NP). According to the antigenicity of the nucleoprotein, it can be divided into three types: A (A), B (B), and C (C). Each type can be further divided into three types: different subtypes. Type A mutates quickly and can occur once every 2-3 years, while Type B mutates slowly. When the antigen changes greatly, it is completely different from the previous epidemic strain, which is a qualitative change of the antigen, called antigen strain change, and a new subtype is produced at this time. Because the population lacks antibodies to new subtypes, it often causes large epidemics. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569
Inventor 崔鹏李泓彦何志强胡鹏范凌云彭亮
Owner GUANGDONG WESAIL BIOTECH CO LTD
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