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Monoclonal antibody 5e1 against the gp2 subunit of ebov with a unique binding site

A monoclonal antibody, antibody light chain technology, applied in the field of peptides

Active Publication Date: 2021-07-30
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 against the gp2 subunit of ebov with a unique binding site
  • Monoclonal antibody 5e1 against the gp2 subunit of ebov with a unique binding site
  • Monoclonal antibody 5e1 against the gp2 subunit of ebov with a unique binding site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Antibody Preparation

[0031] 1. Blood Sample Collection

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

[0033] 2. FITC-labeled protein GPdM

[0034] Specific memory B cells need to be sorted by fluorescently labeled antigens. The method of FITC-labeled truncated antigen protein GPdM is as follows:

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

[0036] 2) Take 100 μL of GPdM (3.3 mg / mL), add buffer (pH9.6 carbonate buffer) to 400 μL.

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

[0038] 4) Use a 50kD centrifugal concentrator tube to replace the solution with PBS until the filtrate is transparent and colorless. Wrap the labeled protein in tinfoil and store at 4°C until use.

[0039] 3....

Embodiment 2

[0119] Example 2.ELISA detection of antibody binding activity

[0120] 1) The day before the experiment, 96-well enzyme-linked plates were coated with 1 μg / mL of EBOV GP, BDBV GP, SUDV GP and RESTV GP, 100 μL per well. Put the coated ELISA plate in a humid box overnight at 4°C.

[0121] 2) Wash 5 times with a plate washer on the day of the experiment.

[0122] 3) Add 100 μL of blocking solution to each well and let stand at room temperature for 1 hour.

[0123] 4) Wash the plates 5 times in a plate washer.

[0124] 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 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.

[0125] 6) Wash the plates 5 times in a plate washer.

[0126] 7) Dilute the HPR-labeled goat anti-human IgG secondary antibod...

Embodiment 3

[0131] Embodiment 3. Pseudovirus neutralization experiment evaluates 5E1 neutralization activity

[0132] EBOV and BDBV pseudoviruses packaged with the HIV backbone were evaluated for neutralizing activity of 5E1 in vitro. The evaluation method is as follows:

[0133] 1) Dilute the 5E1 monoclonal antibody with DMEM medium, add 75 μL of antibody diluent with a concentration of 100 μg / mL to the first well of a 96-well cell culture plate, and add 50 μL of DMEM medium to the remaining wells.

[0134] 2) Pipette 25 μL of liquid from the first well into the second well, mix well, and so on, dilute at a ratio of 1:3, and the final volume of each well is 50 μL.

[0135] 3) Dilute the pseudovirus 1:5 with DMEM medium (control the fluorescein reading in the control well between 20,000 and 100,000), add to each antibody well, 50 μL per well. Mix well and incubate at 37°C for 1h.

[0136] 4) 293T cell count, 2×10 5 cells / mL, add 100 μL to each well.

[0137] 5) Put the 96-well cell cul...

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Abstract

The invention discloses a monoclonal antibody 5E1 against Ebola virus glycoprotein GP2 subunit. The antibody has a unique CDR region and has specific binding activity with the glycan cap region of the EBOV GP1 subunit. 5E1 monoclonal antibody has good binding activity to EBOV GP, EC 50 The value was 0.027 μg / mL. Compared with the control antibody, 5E1 can effectively neutralize EBOV and BDBV pseudoviruses in vitro. The neutralizing activity of 5E1 is enhanced with the increase of antibody concentration, and 50% protection can be achieved for cells infected with EBOV and BDBV pseudoviruses at a concentration of 1 μg / mL. 5E1 also did not compete with the neutralizing antibodies MIL77‑1 and MIL77‑2 binding to the GP2 subunit, indicating that 5E1 binds to a different epitope from these antibodies. 5E1 has good neutralizing activity against EBOV and BDBV in vitro, and can be used as a candidate therapeutic drug for Ebola virus disease. Its binding epitope is different from other neutralizing antibodies, and it has the potential to form a cocktail combination therapy with other neutralizing antibodies.

Description

technical field [0001] The invention discloses an antibody and 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, with a fatality rate as high as 90%. It can be transmitted directly through contact , highly contagious and lethal. The glycoprotein (Glycoprotein, GP) spike on the surface of the Ebola virus envelope almost mediates all steps of the virus entering the cell, so Ebola GP is an important target of virus neutralizing antibodies. The GP gene of Ebola virus is processed into two proteins, one is secreted non-structural GP (secreted glycoprotein, sGP); the other is structural GP. Ebola GP is first synthesized as a polypeptide, and then Furin enzyme digests into GP1 (amino acids 1-501) and GP2 (amino acids 502-676) subunits. The two subunits are connected by disulfide bonds, and t...

Claims

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Application Information

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Patent Type & Authority Patents(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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