Anti-rabies virus monoclonal antibody and preparation method and application

A technology of rabies virus and monoclonal antibody, applied in the field of biomedicine, can solve the problems of cumbersome operation of detection methods and the like

Inactive Publication Date: 2009-10-21
NANJING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of anti-rabies virus monoclonal antibody and its preparation method and application

Method used

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  • Anti-rabies virus monoclonal antibody and preparation method and application
  • Anti-rabies virus monoclonal antibody and preparation method and application
  • Anti-rabies virus monoclonal antibody and preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Immunogen preparation and immunization

[0043] The immunogen was prepared by using inactivated rabies virus CTN strain to purify the virus (a purity of more than 99%, donated by Zhang Aihua, Wuhan Institute of Virology).

[0044] Animal immunization: Purify the above-mentioned inactivated rabies virus CTN strain virus, measure the protein concentration to be 0.67 mg / mL, add Freund's complete adjuvant to mix at 1:1, oscillate and emulsify for half an hour, and subcutaneously immunize BALB / c mice with 100 μg; Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant, and the same method was used to immunize again; two weeks after the second immunization, 200 μg of purified antigen was injected intraperitoneally for booster immunization, and fusion was performed three days later.

Embodiment 2

[0045] The fusion of embodiment 2 hybridoma cells

[0046] Fusion of hybridoma cells is performed according to conventional methods. Positive blood was obtained by removing the eyeball before fusion. Spleen cells were fused with SP2 / 0 cells in logarithmic growth phase with PEG-2000. At the same time, blood was collected from BALB / c immunized mice and ICR mice provided with feeder cells as positive and negative sera for control when screening monoclonal antibodies. The fused cells were suspended in HAT medium, distributed into 96-well plates, and placed at 37°C, 5% CO 2 Cultivate in the incubator, add fresh HAT for 5-7 days, observe frequently, change the medium and detect in due course.

Embodiment 3

[0047] The detection of embodiment 3 hybridoma cells

[0048] Enzyme plate coated on the plate, do square array test, that is: use the purified virus of inactivated rabies virus CTN strain as antigen, coat ELISA plate with PBS doubling dilution horizontally, use positive serum as primary antibody, Dilute longitudinally by 100 times, 200 times, 400 times, 800 times, and select the optimal coating concentration (5 μg / mL) according to the reaction results. The coating of the microtiter plate is briefly described as follows: Take the purified antigen protein and mix it with carbonate buffer solution, that is, 0.2mol / L Na 2 CO 3 8mL, 0.2mol / L NaHCO 3 17mL, add deionized water to 100mL, pH9.6, dilute to the coating concentration, and set a negative control at the same time; add 100μL / well to the ELISA plate, 37℃, 3 hours; PBST, 0.01mol / L, pH7.3 , PBS+0.5% Tween-20, wash 3 times, 5 minutes / time; add PBS / FCS, namely 0.01mol / L, pH7.3, PBS+10%FCS or 1%BSA, 200μL / well, 37℃ to block ...

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Abstract

The invention discloses an anti-rabies virus monoclonal antibody and a preparation method and an application, belonging to the field of biomedicine and particularly relating to the preparation of a monoclonal antibody capable of identifying rabies virus and the application. The monoclonal antibody of the invention is screened by indirect Enzyme-linked immunosorbent assay (ELISA), and specificity and affinity thereof combined with antigen are identified by methods such as polyacrylamide gel electrophoresis analysis, speckle ELISA, immunoblot analysis and the like. The anti-rabies virus monoclonal antibody of the invention can be applied in multiple testing methods of antigen of rabies virus and can be also applied in the preparation of rabies virus detecting kit. The anti-rabies virus monoclonal antibody is secreted by anti-rabies virus monoclonal antibody hybridoma cell strain 2C5 with the preservation number being CGMCC No.3014.

Description

technical field [0001] The anti-rabies virus monoclonal antibody and its preparation method and application of the present invention relate to the field of biomedicine, in particular to the preparation and application of a monoclonal antibody capable of recognizing rabies virus. Background technique [0002] Rabies is a worldwide zoonotic disease caused by Rabies virus. Mortality rate is as high as 100% in humans and animals. About 55,000 people die from rabies every year in the world, mainly in developing countries in Asia, Africa and Latin America. Human patients are mostly infected by the bites of sick animals, and have central nervous system infection diseases mainly characterized by hydrophobia, photophobia, dysphagia, and mania. The disease is widespread and has many hosts. Almost all warm-blooded animals can be infected. It takes various mammals as the main host. Infectious diseases are also one of the diseases that humans have not been able to effectively control....

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/06C12P21/08G01N33/577C12N5/20
Inventor 刘新建朱进冯振卿管晓虹黄剑飞唐奇李琛
Owner NANJING MEDICAL UNIV
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