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Monoclonal antibody against Nipah virus envelope glycoprotein, and application thereof

A technology of monoclonal antibody and envelope glycoprotein, which is applied in the field of polypeptide to achieve the effect of excellent antigen binding activity

Active Publication Date: 2019-07-19
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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  • Monoclonal antibody against Nipah virus envelope glycoprotein, and application thereof
  • Monoclonal antibody against Nipah virus envelope glycoprotein, and application thereof
  • Monoclonal antibody against Nipah virus envelope glycoprotein, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Antibody Preparation

[0025] 1. Immunization of animals

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

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

[0028] 3) Two weeks after the third immunization, the tail vein blood of the mice was taken to measure the immune titer, which was 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 extract the spleen of the mouse, prepare a spleen cell suspension, fuse with Sp2 / 0 myeloma cells according to conventional methods, and culture with HAT selective medi...

Embodiment 2

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

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

[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 let stand at room temperature for 1 hour.

[0054] 4) Wash 3 times in a plate washer.

[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 50 μL from the first well and add to the second well, and so on, dilute the solution in a 1:3 gradient to a final volume of 100 μL in each well. Let stand at room temperature for 1 hour.

[0056] 6) Wash the plates 5 times in a plate washer.

[0057] 7) Dilute the HPR-labeled goat anti-mouse IgG secondary antibody a...

Embodiment 3

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

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

[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 let stand at room temperature for 1 hour.

[0066] 4) Wash 3 times in a plate washer.

[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 50 μL from the first well and add to the second well, and so on, dilute the solution in a 1:3 gradient to a final volume of 100 μL in each well. Let stand at room temperature for 1 hour.

[0068] 6) Wash the plates 5 times in a plate washer.

[0069] 7) Dilute the HPR-label...

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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 and belongs to the technical field of polypeptides. Background technique [0002] Nipah virus (NiV) is a highly pathogenic emerging infectious disease pathogen that has emerged in South Asia in recent years. It is related to Cedar virus (CedPV), Hendra virus (HeV) ) belong to the genus Henipavirus (genus Henipavirus) Paramyxoviridae (family Paramyxoviridae). From 2015 to 2018, for four consecutive years, the World Health Organization listed it, together with Ebola virus, Marburg virus and other pathogens, as the most likely to cause severe outbreaks and difficult to deal with. Nipah virus disease is highly contagious, has a high mortality rate, and has a wide distribution of natural hosts, seriously affecting global public health and threatening human life and health. [0003] In 1998, a novel infectious disease manifesting as encephalitis and respiratory symptoms occurred among pig farmers in Malaysia and Singapor...

Claims

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Application Information

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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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