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Hybridoma cells and monoclonal antibody capable of secreting II type dengue virus NS1 monoclonal antibody and application

A technology of monoclonal antibody and hybridoma cells, applied in the field of immunoassay, can solve the problems that cannot meet the needs of clinical diagnosis well, and achieve the effect of high secretion yield

Active Publication Date: 2015-02-18
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the common antigenic determinants among flavivirus structural proteins, there is cross-reactivity, and the diagnostic reagents using dengue virus structural proteins as antigens cannot well meet the needs of clinical diagnosis.

Method used

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  • Hybridoma cells and monoclonal antibody capable of secreting II type dengue virus NS1 monoclonal antibody and application
  • Hybridoma cells and monoclonal antibody capable of secreting II type dengue virus NS1 monoclonal antibody and application
  • Hybridoma cells and monoclonal antibody capable of secreting II type dengue virus NS1 monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Establishment of hybridoma cells and preparation of anti-type II dengue virus NS1 monoclonal antibody.

[0033] 1. Preparation of immunogen.

[0034] Recombinant type II dengue virus NS1 protein expressed by insect cells (sf9 cells and High Five cells in this example) was used as an immunogen. The NS1 gene of type II dengue virus (Shanghai Sangon Bioengineering Co., Ltd.) and the genome of baculovirus (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) were subjected to homologous recombination to construct a dengue virus carrying NS1 gene of type II. Baculovirus, and then by transfecting sf9 cells (purchased from Shanghai Yingjun Biotechnology Co., Ltd., preserved by the company), a large amount of recombinant baculovirus was amplified, and the amplified recombinant baculovirus was infected with High Five cells ( Purchased from Shanghai Yingjun Biotechnology Co., Ltd. and preserved by our company), the target NS1 protein is efficiently secreted and expressed by ...

Embodiment 2

[0075] Preparation of colloidal gold test paper for detection of type II dengue virus NS1 antigen.

[0076] 1. Preparation of nitrocellulose membrane

[0077] Preparation of coating buffer: 6% methanol, 0.01M pH7.2 PBS buffer as coating buffer, filtered through 0.22 μm membrane, set at 4°C for later use, valid for one week. 1000ml 6% methanol 0.01M pH 7.2PBS buffer formulation: 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.

[0078] Preparation of nitrocellulose membrane: Dilute the monoclonal antibody DN2-9 prepared in Example 1 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 pad end of the gold label is about 5mm away from the pad end of the gold label; dilute the goat anti-mouse IgG antibody (manufactured by Shenzhen Feipeng Biological Co., Ltd., product number BA-PAB-MU0001) to 1-5mg / ml with coating buffer, Adjust the ...

Embodiment 3

[0103] A kit for detecting the NS1 protein of dengue virus type II.

[0104] 1. The kit for rapid detection of type II dengue virus NS1 protein comprises: the detection test paper and sample diluent prepared in Example 2.

[0105] The sample diluent was 8% NaCl solution. Preparation method: 80gNaCl, add distilled water to make up to 1000ml.

[0106] 2. Detection of type II dengue virus NS1 protein by colloidal gold method.

[0107] (1) Directly pipette 120 μl of collected human serum or plasma into the sample hole of the test paper card, and wait for 15 minutes to observe the results.

[0108] (2) Result judgment: When the test strip has a red quality control line visible to the naked eye, but no red detection line visible to the naked eye, the result is judged as negative; when the test strip shows a red quality control line visible to the naked eye, it also appears Visible red test line, the result is judged as positive. The darker the detection line, the higher the leve...

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Abstract

The invention relates to an II type dengue virus test paper which is high in sensitivity and specificity. The test paper uses the II type dengue virus NS1 monoclonal antibody secreted by the hybridoma cells for indirectly marking when markers are large-sized nano particles, so as to reduce the usage amount of labelled antigen, and improve the sensitivity while improving the specificity. The invention, in addition, further relates to hybridoma cells and a monoclonal antibody which are capable of secreting an II type dengue virus NS1 monoclonal antibody and the application. The two hybridoma cells are preserved in the preservation center of Wuhan University, Luojiashan street, Wuchang district, Wuhan City, Hubei province, and the preservation numbers are respectively CCTCC No: C2014183 and CCTCC No: C2014182.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a hybridoma cell capable of secreting an anti-type II dengue virus NS1 monoclonal antibody, a monoclonal antibody and an application thereof. Background technique [0002] Dengue virus belongs to the Flaviviridae family and is mainly transmitted by Aedes aegypti. Dengue virus includes 4 serotypes (types I-IV). Any type of dengue virus infection can cause a series of clinical symptoms. Manifested as invisible infection, fever, dengue fever or more severe dengue hemorrhagic fever and dengue shock syndrome. Dengue virus is mainly prevalent in tropical and subtropical regions. At present, more than 100 countries in the world have reported dengue fever epidemics, 2.5 billion people live in high-risk areas of infection, and 50 million to 100 million cases are reported every year, of which 200,000 ~500,000 dengue infections become dengue hemorrhagic fever. In dengue-endemic areas, 4 ty...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569
CPCY02A50/30
Inventor 杨耿周朱碧银龚春喜霍永庭冯琼李可国范凌云彭亮韩日才刘德荣
Owner FAPON BIOTECH INC
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