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Monoclonal antibody 5E1 with unique binding site and resisting EBOV GP2 subunit

A monoclonal antibody and subunit technology, applied in the field of peptides

Active Publication Date: 2020-05-12
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the first two ways have been confirmed by literature, and the third way is the speculation of some literature, and there is no direct evidence yet.

Method used

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  • Monoclonal antibody 5E1 with unique binding site and resisting EBOV GP2 subunit
  • Monoclonal antibody 5E1 with unique binding site and resisting EBOV GP2 subunit
  • Monoclonal antibody 5E1 with unique binding site and resisting EBOV GP2 subunit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Antibody preparation

[0031] 1. Collection of blood samples

[0032] After obtaining the informed consent, 5 mL of blood samples were collected from subjects in clinical trials of recombinant Ebola vaccine 28 days after the second immunization for subsequent experiments.

[0033] 2. FITC labeled protein GPdM

[0034] Fluorescence-labeled antigens are needed to sort specific memory B cells. The method of FITC labeling truncated antigen protein GPdM is as follows:

[0035] 1) Fluorescein Isothiocyanate_FITC (SIGMA, F4274) is dissolved in DMSO at a concentration of 20 mg / mL.

[0036] 2) Take 100 μL of GPdM (3.3 mg / mL) and add buffer (pH 9.6 carbonate buffer) to 400 μL.

[0037] 3) Add 8μL FITC to GPdM solution and incubate for 3h at 4°C in the dark.

[0038] 4) Change the solution with PBS with a 50kD centrifugal concentration tube until the filtrate is transparent and colorless. Wrap the labeled protein in tin foil and store it at 4°C until use.

[0039] 3. Flow cytometric...

Embodiment 2

[0120] Example 2. ELISA to detect antibody binding activity

[0121] 1) One day before the experiment, a 96-well ELISA plate was coated with 1μg / mL EBOV GP, BDBV GP, SUDV GP and RESTV GP, and each well was coated with 100μL. Put the coated ELISA plate into a humid box at 4°C overnight.

[0122] 2) Wash 5 times with a plate washer on the day of the experiment.

[0123] 3) Add 100 μL of blocking solution to each well, and place it at room temperature for 1 hour.

[0124] 4) Machine wash the plate 5 times.

[0125] 5) Add 150 μL of 5E1 monoclonal antibody at a concentration of 10 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.

[0126] 6) Machine wash the plate 5 times.

[0127] 7) Dilute the HPR-labeled goat anti-human IgG secondary antibody at 1:10000 with the diluent, add 100 μL ...

Embodiment 3

[0132] Example 3. Pseudovirus neutralization experiment to evaluate 5E1 neutralization activity

[0133] The EBOV and BDBV pseudoviruses that package the HIV backbone were evaluated for the neutralizing activity of 5E1 in vitro. The evaluation method is as follows:

[0134] 1) Dilute 5E1 monoclonal antibody with DMEM medium, add 75 μL of antibody diluent with a concentration of 100 μg / mL to the first well of 96-well cell culture plate, and add 50 μL of DMEM medium to the remaining wells.

[0135] 2) Take 25μL of liquid from the first hole and add it to the second well, mix well, and so on, dilute by a ratio of 1:3, and the final volume of each well is 50μL.

[0136] 3) Dilute the pseudovirus with DMEM medium in a ratio of 1:5 (control the fluorescein reading value of the control well to be between 20,000 and 100,000), and add it to each antibody well, 50 μL per well. Mix well and incubate at 37°C for 1h.

[0137] 4) Count of 293T cells, 2×10 5 cells / mL, add 100μL to each well.

[0138]...

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Abstract

The invention discloses a monoclonal antibody 5E1 for resisting an Ebola virus glycoprotein GP2 subunit, and the antibody has a unique CDR region and has specific binding activity with a glycan cap region of an EBOV GP1 subunit. The 5E1 monoclonal antibody has good binding activity with EBOV GP, and the EC50 value of the 5E1 monoclonal antibody is 0.027 [mu] g / mL. 5E1 is effective in neutralizingEBOV and BDBV pseudotype viruses vitro compared to a control antibody. The neutralization activity of 5E1 is enhanced along with the increase of the concentration of antibody, and 50% protection can be realized on EBOV and BDBV pseudo-virus infected cells at the concentration of 1 [mu] g / mL. 5E1 does not compete with neutralizing antibodies MIL77-1 and MIL77-2 which are combined with the GP2 subunit, so that the binding epitopes of 5E1 and the antibodies are different. The 5E1 has good in-vitro neutralizing activity on EBOV and BDBV, can be used as a candidate treatment drug for Ebola virus diseases, has different binding epitopes from other neutralizing antibodies, and has the potential of forming cocktail combination therapy with other neutralizing antibodies.

Description

Technical field [0001] The invention discloses an antibody, which belongs to the technical field of polypeptides. Background technique [0002] Ebola virus (EBOV) can cause acute severe hemorrhagic fever in humans and non-human primates. It is one of the viruses with the highest fatality rate found so far. The fatality rate is as high as 90%, and it can be transmitted directly through contact. , Has a very strong infectiousness and fatality rate. Glycoprotein (GP) spikes on the surface of the Ebola virus envelope mediate almost all aspects of the virus entering the cell, so Ebola GP is an important target of virus neutralizing antibodies. The Ebola virus GP gene is processed into two proteins, one is the secreted non-structural GP (secreted glycoprotein, sGP); the other is the structural GP. Ebola GP is first synthesized as a polypeptide and then Furin enzyme cleaves into GP1 (amino acids 1-501) and GP2 (amino acids 502-676). The two subunits are connected by disulfide bonds, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N5/10A61K39/12A61P31/14
CPCA61P31/14C07K16/10C07K2317/565C07K2317/76C07K2317/92C12N5/0636C12N2510/00
Inventor 陈薇迟象阳于长明范鹏飞张冠英侯利华房婷吴诗坡陈旖陈郑珊王美荣刘渝娇
Owner ACADEMY OF MILITARY MEDICAL SCI
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