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Fully human anti-rabies virus neutralizing antibody and application thereof

A rabies virus, all-human technology, applied in the field of cellular immunology and molecular biology, can solve the problem of no drug approval for marketing

Active Publication Date: 2018-11-09
CHANGCHUN BCHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some anti-rabies virus-specific mAbs have entered the clinical research stage, but so far no such drugs have been approved for marketing

Method used

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  • Fully human anti-rabies virus neutralizing antibody and application thereof
  • Fully human anti-rabies virus neutralizing antibody and application thereof
  • Fully human anti-rabies virus neutralizing antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Preparation of fully human anti-rabies virus monoclonal neutralizing antibody TRN073

[0071] 1. Memory B cell sorting

[0072] Two healthy volunteers were selected to be injected with rabies vaccine (Novartis, batch number: 1928). On the 7th day (28th day) after the full immunization, 100 mL of blood was drawn on an empty stomach. ELISA was used to detect positive results for rabies virus IgG. Then, the anticoagulated whole blood samples were separated with Ficoll lymphocyte separation medium to obtain human peripheral blood mononuclear cells (PBMC), and the CD3-, CD14-, CD16-, CD19+, CD27+ single B cells, the results are as follows figure 1 shown. A single B cell obtained by sorting was placed in a 96-well PCR plate containing an RT reaction system so that each well contained one memory B cell, and the PCR plate was frozen at -80°C for future use.

[0073] 2. Reverse transcription to synthesize cDNA strand

[0074] Add 0.5 μM constant region primers and ...

Embodiment 2

[0085] Example 2 TRN073 Antibody Binding Activity Detection

[0086] Use the same ELISA method mentioned above to detect the binding activity of the expressed and purified antibody: use rabies vaccine as the antigen, and coat the ELISA 96-well plate after diluting the antigen 10 times with the coating solution, and coat each well with 100 μL overnight at 4°C , and blocked with blocking solution for 2 hours at room temperature. Dilute the TRN073 antibody supernatant with an initial concentration of 100 μg / ml 10 times and then use it as the primary antibody for serial dilution. Add the primary antibody and incubate at room temperature for 2 hours. At the same time, use the untransfected cell supernatant as a negative antibody control. Goat anti-human IgG (diluted at 1:2000) was incubated as the secondary antibody at room temperature for 1 hour, and 100 μl / well of the substrate chromogenic solution was added. After standing in the dark at room temperature for 5 minutes, the react...

Embodiment 3

[0088] Example 3 TRN073 Antibody Binding Specific Detection

[0089] (1) Binding to rabies virus G protein (RVG)

[0090] Steps: It is the main protective antigen of rabies virus, which can stimulate the body to produce neutralizing antibodies, clone the gene fragment of RVG into the eukaryotic expression vector pcDNA3.1, and transfect 293T with PolyFect (Qiagen, Valencia, CA) transfection reagent cells, take 1×10 5 48-72 hours after transfection, centrifuge 293T cells at 300×g for 10 minutes, remove the supernatant, add 10 μL of commercial human anti-rabies virus serum or antibody TRN073, and use other expressed human monoclonal antibodies as negative controls, and stain at 4°C After incubation for 45 minutes, wash twice with PBS, then add FITC-labeled goat anti-human IgG antibody, stain and incubate at 4°C for 30 minutes, wash twice with PBS, and analyze with BD FACSria flow cytometer.

[0091] The results indicated that antibody TRN073 is a functional antibody specific to...

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Abstract

The invention discloses a fully human anti-rabies virus neutralizing antibody and application thereof. The neutralizing antibody disclosed by the invention is capable of being specifically combined with the anti-rabies virus and effectively neutralizing the virus activity. The invention further relates to the preparation method and the application of the neutralizing antibody. The antibody disclosed by the invention can be used for emergency prevention and / or treatment of the rabies as well rabies virus detection. In addition, the antibody can be further used for preparing a rabies virus detection reagent capable of effectively neutralizing antigenic epitopes and developing rabies virus recombinant proteins and subunit vaccines.

Description

technical field [0001] The invention belongs to the field of cellular immunology and molecular biology, relates to a fully human monoclonal antibody, in particular to a fully human monoclonal neutralizing antibody against rabies virus. In addition, the present invention also relates to the preparation method and application of the neutralizing antibody. Background technique [0002] According to the recommendations of the World Health Organization (WHO), active and passive immunotherapy should be carried out simultaneously for severely exposed persons to obtain a rapid and effective protective effect. Rabies vaccine is an attenuated rabies virus (that is, a harmless antigen) that is harmless to humans. It takes 15 to 20 days to produce enough antibodies to stimulate the body to produce active immunity against rabies virus. Passive immunization preparations are immune serum or cytokines containing specific antibodies, which can immediately and specifically neutralize rabies ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
CPCA61K2039/505C07K16/10C07K2317/21C07K2317/565C07K2317/76C07K2317/92G01N33/56983G01N33/577
Inventor 廖化新王月明袁晓辉
Owner CHANGCHUN BCHT BIOTECH
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