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A kind of anti-Nipah virus envelope glycoprotein monoclonal antibody and its application

A monoclonal antibody, envelope glycoprotein technology, applied in the field of peptides, to achieve the effect of excellent antigen binding activity

Active Publication Date: 2020-12-18
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No vaccine or antibody drug has yet entered clinical trials

Method used

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  • A kind of anti-Nipah virus envelope glycoprotein monoclonal antibody and its application
  • A kind of anti-Nipah virus envelope glycoprotein monoclonal antibody and its application
  • A kind of anti-Nipah virus envelope glycoprotein monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024]Example 1. Antibody preparation

[0025]1. Immunize animals

[0026]1) The purified NIV-GP recombinant protein was mixed with the same amount of Freund's complete adjuvant, and injected into the groin of each mouse at multiple points. The injected protein was measured at 20 μg per mouse, and 3 mice were immunized.

[0027]2) After 2 weeks and 4 weeks, the PSCA recombinant protein and Freund's incomplete adjuvant mixture were injected, and the method and dosage were the same as the first time.

[0028]3) Two weeks after the third immunization, the tail vein blood of the mice was taken to test the immunization titer, which reached more than 1:1600 to prepare for fusion, and boosted immunization (20 μg / mouse) 3 days before fusion.

[0029]2. Cell fusion and cloning

[0030]1) Aseptically harvest the mouse spleen, prepare a spleen cell suspension, fuse with Sp2 / 0 myeloma cells according to a conventional method, and culture with HAT selective medium.

[0031]2) On the 4th day after fusion, replace the...

Embodiment 2

[0050]Example 2. ELISA experiment to detect the binding activity of 14F8 monoclonal antibody and Nipah virus G protein

[0051]1) One day before the experiment, a 96-well ELISA plate was coated with 1μg / mL NIV-GP, and each well was coated with 200μL. Put the coated ELISA plate into a humid box at 4°C overnight.

[0052]2) Wash 5 times with a plate washer on the day of the experiment.

[0053]3) Add 100 μL of blocking solution to each well, and place it at room temperature for 1 hour.

[0054]4) Machine wash the plate 3 times.

[0055]5) Add 150 μL of 14F8 monoclonal antibody at a concentration of 20 μg / mL to the first well, and add 100 μL of diluent to the remaining wells. Aspirate 50μL from the first well and add it to the second well, and so on, with a 1:3 gradient, and the final volume of each well is 100μL. Let stand at room temperature for 1 hour.

[0056]6) Machine wash the plate 5 times.

[0057]7) The HPR-labeled goat anti-mouse IgG secondary antibody was diluted 1:10000 with the diluent, 100 μL...

Embodiment 3

[0062]Example 3. ELISA experiment to detect the binding activity of 14F8 monoclonal antibody to Hendra virus envelope glycoprotein

[0063]1) One day before the experiment, a 96-well ELISA plate was coated with 1μg / mL Hendra virus G protein HEV-GP, and each well was coated with 200μL. Put the coated ELISA plate into a humid box at 4°C overnight.

[0064]2) Wash 5 times with a plate washer on the day of the experiment.

[0065]3) Add 100 μL of blocking solution to each well, and place it at room temperature for 1 hour.

[0066]4) Machine wash the plate 3 times.

[0067]5) Add 150 μL of 14F8 monoclonal antibody at a concentration of 20 μg / mL to the first well, and add 100 μL of diluent to the remaining wells. Aspirate 50μL from the first well and add it to the second well, and so on, with a 1:3 gradient, and the final volume of each well is 100μL. Let stand at room temperature for 1 hour.

[0068]6) Machine wash the plate 5 times.

[0069]7) The HPR-labeled goat anti-mouse IgG secondary antibody was dilut...

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Abstract

The present invention discloses a monoclonal antibody 14F8 against Nipah virus envelope glycoprotein. The antibody has a unique CDR region and has a binding titer of 0.47 ng / mL with the Nipah virus envelope glycoprotein, and when an antibody concentration is greater than 9.14 ng / mL, an ELISA OD value is greater than 2.0, indicating excellent antigen binding activity. The antibody has a binding titer of 129.66 ng / mL with Hendra virus envelope glycoprotein, and when the antibody concentration is 20,000 ng / mL, the OD value is only 0.29, indicating the 14F8 can be used for detection of the Nipah virus envelope glycoprotein and can effectively distinguish the Nipah virus envelope glycoprotein and the Hendra virus envelope glycoprotein. The present invention also discloses an application of the14F8 monoclonal antibody in preparation of drugs for treating Nipah virus diseases, the antibody can effectively inhibit binding of the Nipah virus envelope glycoprotein to a cellular receptor EFNB2,an IC50 value is 50 ng / mL, besides, neutralizing activity is enhanced with increase of the antibody concentration, and when the antibody concentration exceeds 1 [mu]g / ml, an inhibition rate tends to reach 100%, indicating a prospect of the 14F8 monoclonal antibody as a candidate therapeutic antibody for the Nipah virus diseases.

Description

Technical field[0001]The invention discloses an antibody, which belongs to the technical field of polypeptides.Background technique[0002]Nipah virus (NiV) is a highly pathogenic new infectious disease pathogen that has emerged in South Asia in recent years. It is closely related to Cedar virus (Cedar virus, CedPV) and Hendra virus (HeV). ) Belong to the genus Henipavirus (genus Henipavirus) Paramyxoviridae (family Paramyxoviridae). From 2015 to 2018, the World Health Organization listed it together with pathogens such as Ebola virus and Marburg virus as the most potent pathogens that are most likely to cause severe epidemics and are difficult to respond to. Nipah virus disease is highly contagious, has a high mortality rate, and has a wide distribution of natural hosts. It seriously affects global public health and threatens human life and health.[0003]In 1998, a new type of infectious disease occurred in pig farmers in Malaysia and Singapore, manifested as encephalitis and respirat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/1027C07K2317/35C07K2317/56C07K2317/565C07K2317/76
Inventor 陈薇徐俊杰李耀辉殷瑛宰晓东张军刘树玲李汭桦
Owner ACADEMY OF MILITARY MEDICAL SCI
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