Monoclonal antibody hybridoma cell of HIV P24 and application

A hybridoma cell and monoclonal antibody technology, applied in the field of HIV detection, can solve the problems of expensive imported reagents, unfavorable scientific research work, and difficulty in meeting the needs of AIDS research, achieving good sensitivity and specificity, and simple operation. Effect

Inactive Publication Date: 2010-03-17
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high cost of imported reagents, the widespread use of more domestic laboratories is limited, which is not conducive to the development of relevant scientific research in China
In addition, the P24 monoclon...

Method used

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  • Monoclonal antibody hybridoma cell of HIV P24 and application
  • Monoclonal antibody hybridoma cell of HIV P24 and application
  • Monoclonal antibody hybridoma cell of HIV P24 and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Establishment of a monoclonal antibody against the P24 protein of HIV strains prevalent in China.

[0036] 1. Construct the expression plasmid pET32a-p24 of the domestic popular HIV strain P24 protein:

[0037] 1. Material acquisition and PBMCs separation:

[0038] The blood of HIV-1 infected patients was collected through the Wuhan Center for Disease Control and Prevention in Hubei Province, and the blood of HIV-1 (-) healthy people was provided by the central blood station in Wuhan, Hubei Province. Isolate PBMCs as follows:

[0039] 1) Pour the anticoagulated whole blood into a conical centrifuge tube, and centrifuge at 2,000 rpm / min for 30 min at room temperature (20-25°C, the same below).

[0040] 2) Take the intermediate leukocyte-enriched layer, put it into a centrifuge tube, and add 4 times its volume of PBS buffer solution containing 1% bovine serum (volume ratio, the same below).

[0041] 3) Take another centrifuge tube with conical bottom, add ly...

Embodiment 2

[0249] Embodiment 2, establishment of double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method

[0250] 1. Preparation of P24 polyantizyme conjugate:

[0251] Use the obtained purified recombinant antigen as immunogen, immunize 5 rabbits, take the rabbit serum with the highest titer, purify it with DEAE52 ion exchange, and then use periodate oxidation method to label the rabbit polyclonal antibody with HRP.

[0252] 1) Dissolve 5mg of HRP in newly prepared 1ml of 0.30Mol / LpH8.1 sodium bicarbonate, add 0.1ml of 1% (volume ratio, the same below) fluorodinitrobenzene (FDNB) absolute ethanol solution, and mix at room temperature ;

[0253] 2) Add 1ml 0.04Mol / L~0.08Mol / L sodium periodate (NaIO 4 ), put it at room temperature (the same as below 20-25°C) and gently stir for 30 minutes. When the solution is yellow-green, add 1ml of 0.16Mol / L ethylene glycol solution, and place it at room temperature for 1 hour to stop the oxidation reaction;

[0254] 3) Dialyze again...

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PUM

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Abstract

The invention discloses a monoclonal antibody hybridoma cell expressing an HIV P24 and an application, wherein, through separating HIV virus in an HIV positive infector, the P24 protein gene of the separated virus is enlarged; P24 protein can be obtained through external expression of carriers and purification; after using the purified P24 protein to immunize the mice, the spleen B cells of the immunized mice are separated out, and an hybridoma cell efficiently expressing a P24 monoclonal antibody (whiov-P24C-MoAb, CCTCC NO:C200818) can be obtained by adopting a hybridoma technology; and the P24 monoclonal antibody can be in specific binding with P24 protein of HIV virus for detecting the expression of P24 antigen. The P24 monoclonal antibody can be in specific binding with P24 protein ofHIV virus, interferences of other bad reactions can be reduced when being used for specific detection of P24 protein.

Description

technical field [0001] The present invention relates to the technical field of HIV (HIV) detection, more specifically to a hybridoma cell expressing HIV P24 protein monoclonal antibody (abbreviated as monoclonal antibody), and also to a kind of HIV-1 P24 monoclonal antibody in Application in the ELISA detection method for detecting P24 protein (enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, ELIS A) method for detecting P24 protein antigen) Background technique [0002] Acquired immunodeficiency syndrome, referred to as AIDS (AIDS), is a serious infectious disease caused by human immunodeficiency virus (humanimmunodeficiency virus, HIV) infection. Since the first AIDS patient was discovered in China in 1985, the epidemic has been accelerating, and the epidemic is spreading from high-risk groups to the general population. Since there is no effective preventive vaccine or cure for AIDS, early detection and early treatment can minimize the harm to society...

Claims

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Application Information

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IPC IPC(8): C12N5/20G01N33/577
Inventor 杨荣阁严兵童骁刘萍萍
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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