Human anti-H7N9 avian influenza virus neutralizing antibody 1F7L and its use

A bird flu virus and antibody technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of weakening the clinical efficacy of drugs and not being able to mediate cytotoxic killing

Inactive Publication Date: 2017-10-03
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing H7N9 subtype-specific monoclonal antibodies have certain neutralizing activity in vitro, and can protect animals from infection or reduce symptoms, but these antibodies have not been reported or have no ability to mediate cell-dependent cytotoxicity (ADCC ) ability, which will weaken the clinical efficacy of drugs with such antibodies as effector molecules

Method used

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  • Human anti-H7N9 avian influenza virus neutralizing antibody 1F7L and its use
  • Human anti-H7N9 avian influenza virus neutralizing antibody 1F7L and its use
  • Human anti-H7N9 avian influenza virus neutralizing antibody 1F7L and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Human-derived anti-H7N9 avian influenza virus neutralizing antibody 1F7L and its preparation method

[0041] 1. Antigen labeling

[0042] Purchase HA from Acrobiosystems (HA9-V5227) 7 protein, diluted to 0.5-2mg / ml with PBS, and then using Thermal company's biotin (EZ-LinkTMSulfo-NHS-LC-Biotin, 21335) according to its kit process to HA 7 Protein labeling (the ratio of the number of molecules is protein:biotin=1:20-100), incubate at room temperature in the dark for 0.5-2h, and then use a 10KD centrifugal semi-permeable column (MerckMillipore, UFC501096) to centrifuge 4-6 times at 8000g, and use Supplement with sterile PBS, remove excess biotin molecules, and mark HA 7 Protein molecules will be used to screen HA 7 specific memory B cells.

[0043] 2. Antigen-specific memory B cell sorting and reverse transcription

[0044] The convalescent peripheral blood mononuclear cells were isolated from patients with H7N9 infection, washed once with PBS, and then resu...

Embodiment 2

[0111] Embodiment 2 monoclonal antibody 1F7L is aimed at HA 7 protein EC 50 determination

[0112] Dilute HA with ELISA coating solution 7 The protein was adjusted to 1 μg / ml, and then 50 μl per well was coated on an ELISA plate (Corning, 3690) at 4° C. overnight. Plates were washed with PBST and blocked with PBS containing 5% skimmed milk for more than 2 hours. Starting from the initial concentration of 50 μg / ml, the monoclonal antibody was diluted 4-fold with PBS containing 5% skimmed milk, and then added to the blocked ELISA plate. A total of 10 gradients were set up, with two repetitions. The wells without antibodies were used as negative controls, and IgG9114L (ie CR9114, Dreyfus C, Laursen NS, Kwaks T, Zuijdgeest D, Khayat R, Ekiert DC, et al. Highlyconserved protective epitopes on influenza B viruses. Science 2012 Sep 14; 337(6100): 1343-8) is an antibody control. Goat anti-human IgG Fc-HRP (1:10000, Jackson immunolab, 109-036-098) as secondary antibody, 100μl TMB ...

Embodiment 3

[0113] Example 3 Determination of neutralizing activity of monoclonal antibody 1F7L against H7N9 (A / Shenzhen / SP17 / 2014 (H7N9))

[0114] Inoculate MDCK cells in a 96-well cell culture plate, culture until the cell density is about 70%-90%, wash twice with PBS and set aside; dilute the monoclonal antibody to be detected 3 times on a 96-well microtiter plate (starting at 100μg / ml), each antibody to be detected is repeated in 4 wells, 8 gradients; according to the TCID of the virus 50 Titer dilute the virus so that the diluted virus titer is 200TCID 50 / 100μl; mix 60μl of the diluted virus solution with 60μl of the diluted monoclonal antibody sample, and incubate in a 37°C incubator for 2 hours to fully act on the antigen and antibody; then take 100μl of the virus plasma mixture and put it into the washed 96-well MDCK cells, Infect in an incubator at ℃ for 1 hour, replace the virus liquid with 150 μl of MEM medium supplemented with TPCK trypsin, and store at 37℃ CO 2 Cultivate i...

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Abstract

The invention discloses a human anti-H7N9 avian influenza virus neutralizing antibody 1F7L screened by a single cell sorting technology. The amino acid sequences in the light and heavy chain variable regions are shown in the formulas of SEQ ID No. 2 and SEQ ID No. 5. The antibody has the ability to neutralize the H7N9 influenza viruses in vitro and mediates the killing (ADCC) of the H7N9 influenza virus-infected cells with effector cells mainly comprising NK cells. The antibody can be used as a treatment drug for highly pathogenic avian influenza infection and can also be used for the development of H7N9 influenza virus antigen detection reagents.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering, single cell sorting technology and antibody library display, in particular to a human neutralizing antibody 1F7L against H7N9 avian influenza virus and its application. Background technique [0002] Since February 2013, cases of human infection caused by H7N9 avian influenza virus have been found in southeast China, gradually spreading from Shanghai and Anhui to other provinces and cities across the country. As of February 2017, a total of 1079 cases of H7N9 influenza infection were reported nationwide, with relatively high death cases. At present, there is no specific drug for the treatment of highly pathogenic avian influenza infection, and the curative effect of the existing non-specific neuraminidase inhibitor-oseltamivir is limited to the early stage of infection. Since the early symptoms of highly pathogenic H7N9 infection are not significantly different from common influenza,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16G01N33/569
CPCC07K16/1018C07K2317/24C07K2317/565C07K2317/622C07K2317/732C07K2317/76C07K2317/92
Inventor 杨争陈心春周伯平
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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