An anti-enterovirus ev71 IgA monoclonal antibody and its application

A monoclonal antibody, EV71 technology, applied in the field of biotechnology and immunology, can solve the problem of inability to induce immune protection, and achieve the effect of improving specificity and sensitivity

Active Publication Date: 2019-10-25
WUHAN AIBO TAIKE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, previous studies have shown that EV71 inactivated vaccines cannot induce immune protection against CA16 (Jia et al.The cross-reactivity of the enterovirus 71 to human braintissue and identification of the cross-reactivity related fragments.VirolJ.2010; 7:47)

Method used

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  • An anti-enterovirus ev71 IgA monoclonal antibody and its application
  • An anti-enterovirus ev71 IgA monoclonal antibody and its application
  • An anti-enterovirus ev71 IgA monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Obtaining of hybridoma cells secreting anti-enterovirus EV71IgA monoclonal antibody:

[0038] 1.1. Antigen preparation

[0039] The complete VP1 gene of EV71BrCr strain was cloned into the vector pET28a. Briefly, Escherichia coli BL21(DE3) containing recombinant 3D expression vectors were grown, induced to produce these recombinant proteins, and purified by affinity chromatography on Ni-NTA columns (Qiagen).

[0040] 1.2. Immunization and preparation of EV71VP1-specific IgG monoclonal antibody

[0041] Methods for preparing EV71VP1-specific monoclonal antibodies such as (Li YM, Liu F, Han C and YanHM. Monoclonal antibody that blocks the Toll-like receptor 5 binding region of flagellin. Hybridoma (Larchmt). 2012 Feb; 31 (1): 60-62). Briefly, 5-week-old female SPFBALB / c mice were immunized subcutaneously with 100 μg of VP1 at 2-week intervals. Four weeks after the last boost and 3 days before cell fusion, mice were boosted with 200 μg VP1 intraperitoneally. Three day...

Embodiment 2

[0054] Obtaining EV71 VP1-specific IgA monoclonal antibody:

[0055] 1. Preparation of mouse ascites

[0056] a.Prime: Each BALB / c mouse was intraperitoneally injected with 500 μl of liquid paraffin.

[0057] b. After feeding for 15 days, each mouse was intraperitoneally injected with 3-5×10 5 hybridoma cells in an injection volume of 1 ml.

[0058] c. Closely observe the physiological conditions of the mice. After the abdominal volume of the mice increases, the movement is inconvenient, and the vitality decreases (usually after 8-14 days), the mice are killed by cervical dislocation, the chest cavity is opened, and the glass straw is inserted from the diaphragm. Absorb ascites. The titer of ascites is 2 million.

[0059] 2. Silica degreasing

[0060] a. Centrifuge the freshly collected ascites at 2000r / min for 15 minutes to remove cell components and tissue debris, take the upper clear ascites, and add an equal volume of PBS with pH 7.2 to dilute;

[0061] b. Add 150 mg...

Embodiment 3

[0077] Application of IgA monoclonal antibody in the preparation of drugs for inhibiting enterovirus 71:

[0078] 1. Extracellular neutralization of 11G12-IgA

[0079] Will 1×10 4 PFU EV71 (BrCr strain) was added to serial dilutions of 200 μl monoclonal antibody 11G12-IgA (16CF7-IgA IgA antibody specific to measles virus matrix protein M served as an irrelevant antibody control). After 1 hour of incubation, the mixture was used to infect Caco-2 cells. EV71-infected Caco-2 cells were harvested and virus titers in these cell samples were determined by plaque assay. Such as image 3 As shown, at the concentration of 0.01-1 μg / well, the infection of the virus can be significantly reduced. 1ug / well (200ul) can inhibit 90% virus, 0.1ug / well can inhibit 70% virus infection, and 0.01ug / well can inhibit 50% virus infection.

[0080] 2. EV71 VP1-specific 11G12-IgA inhibits the replication of EV71 in cells

[0081] Spread Transwell with Vero C1008-pIgR cells, 2×10 5 One per hole, ...

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Abstract

The present invention discloses an anti-enterovirus EV71 lgA monoclonal antibody and application, and the anti-enterovirus EV71 lgA monoclonal antibody provided herein is secreted by hybridoma cell strain EV7_VP1_11G12_lgA, which is collected under CCTCC NO: C2016132. The monoclonal antibody lgA provided herein has good specificity to bind with virus VP1, can inhibit viral invasion at cellular level, can neutralize the virus in cells, and can protect in vivo a suckling mouse from the attack of virus EV71. Virus EV72 ELISA (enzyme-linked immuno sorbent assay) test kit prepared by using the lgA monoclonal antibody provided herein is better than other kits in terms of both specificity and sensitivity and has a promising prospect.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology; more specifically, the invention relates to an anti-enterovirus EV71IgA monoclonal antibody and its application. [0002] technical background [0003] Hand-foot-mouth disease (Hand-foot and mouth disease) is an increasingly serious threat to public health, especially for infants and young children. Severe infection can lead to aseptic meningitis, encephalitis, myocarditis, acute paralysis, pulmonary edema, and even life-threatening (Wang and Liu.Enterovirus 71:epidemiology,pathogenesis and management.Expert Rev Anti Infect Ther.2009;7:735 -742). The outbreak often occurs in Asian countries, including China, Japan, Malaysia, and Singapore (Chua and Kasri. Hand foot and mouth disease due to enterovirus 71 in Malaysia. Virol Sin 2011; 26, 221–228). There has been an outbreak trend in China in recent years. For example, there were 488,955, 1,155,525, and 1,774,669 clinically confirmed c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569
CPCA61K2039/505C07K16/1009G01N33/56983
Inventor 赵骞张福城李子龙蔡召平
Owner WUHAN AIBO TAIKE BIOTECH CO LTD
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