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Human enterovirus specific antibodies and their uses in diagnostics

An enterovirus, specific technology, used in disease diagnosis, antibodies, antiviral agents, etc.

Inactive Publication Date: 2014-06-04
TEMASEK LIFE SCIENCES LABORATORY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, real-time PCR requires well-maintained laboratories and expensive equipment, while detection of serum antibodies is only possible several weeks after infection, a disadvantage when severe disease from EV71 has rapid onset and progression

Method used

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  • Human enterovirus specific antibodies and their uses in diagnostics
  • Human enterovirus specific antibodies and their uses in diagnostics
  • Human enterovirus specific antibodies and their uses in diagnostics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Monoclonal antibodies 1C6 and 7C7 were prepared and purified from intact EV71 virus.

[0092] Hybridomas secreting specific monoclonal antibodies were obtained from BALB / c mice that had been treated with purified EV71-B5 strain in 0.1 ml PBS (with an equal volume of adjuvant (SEPPIC, France) emulsified) two immunizations by intramuscular injection. Three days before the fusion of splenocytes with SP / 2.0 myeloma cells purchased from ATCC, they were given an intraperitoneal booster of the virus at the same dose. The hybridomas identified to produce specific antibodies were cloned by limiting dilution, and grown in 75cm 2 Expand in the flask. One week later, the hybridoma suspension was harvested and centrifuged at 400g for 10 minutes to pellet cell debris, then the supernatant was collected and stored at -20°C. The concentration of monoclonal antibodies was determined with a spectrophotometer (Nanodrop, DE, USA).

[0093] Antigen capture ELISA.

[0094] Use 100 μl of...

Embodiment 2

[0100] Preparation and purification of recombinant 3CD from EV71C4 subtype

[0101] The nonstructural protein 3CD from the EV71C4 subtype genome was expressed and used as an antigen to generate monoclonal antibodies. This 3CD DNA was amplified by a synthetic gene designed based on the published sequence of the EV71C4 subtype genome (Fuyang-08). This sequence was assigned accession number EU703813.1 by NCBI and cloned into pGex-4T-1 vector and transformed into E. coli. Total lysates of induced and uninduced cells were analyzed by SDS-PAGE, where expressed 3CD-GST fusion protein could be detected at 98.5 kDa. The fusion protein can be enriched by extraction with 8M urea and is GST positive by Western blotting. The enriched protein was then purified by gel cleavage and the concentration of recombinant protein was determined by BCA assay.

[0102] Production of monoclonal antibody against EV713CD

[0103] injected protein

mouse number

first injection

first...

Embodiment 3

[0108] epitope Figure 7 C7

[0109] To characterize the identity of the 7C7 epitope, the VP2 protein was fragmented into GST-tagged contiguous overlapping peptides and cloned into the pGex-4T-1 vector, expressed in E. coli BL21 cells and western blotted the protein using monoclonal antibody 7C7 detected in the blot. The gradual reduction in fragment length resulted in the recognition of the 7C7 linear epitope mapped to amino acid positions 142-147 of the VP2 protein or amino acid positions 211-216 of the EV71 open reading frame. like Figure 4 As shown, the minimal epitope consists of 6 amino acids EDSHPP (B5) (SEQ ID NO: 1) or ENSHPP (A, CA16) (SEQ ID NO: 2).

[0110] Epitope mapping of monoclonal antibody 4B12

[0111] To characterize the identity of the 4B12 epitope, the 3CD protein was fragmented into GST-tagged contiguous overlapping peptides and cloned into the pGex-4T-1 vector and expressed in E. coli BL21 cells. like Figure 5 As shown in C, a gradual decrease i...

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Abstract

There is provided at least one isolated antibody or fragment thereof, wherein the antibody or fragment thereof is capable of specifically binding to at least one EV71 derived peptide.

Description

technical field [0001] The present invention mainly relates to the field of detection and / or treatment of enterovirus type 71 (EV71) and immune-mediated therapy of diseases related to EV71, and methods of preparing such treatment. In particular, immune-mediated therapy may be in the form of EV71-specific monoclonal antibodies. Background technique [0002] Enteroviruses are a heterogeneous group of pathogens that cause a broad spectrum of human and non-human diseases. Enteroviruses belong to a large genus within the picornaviridae family, which includes other genera including rhinoviruses, hepatoviruses, cardioviruses, and foot-and-mouth disease viruses. The Enterovirus genus includes polioviruses, coxsackieviruses A (CAV), coxsackieviruses B (CBV), echoviruses, and enteroviruses 68-71, as well as some species from humans and other primates. Atypical enterovirus isolated from . Like other picornaviruses, enterovirions consist of a non-enveloped icosahedral capsid surround...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/42A61P31/14C07K16/10
CPCG01N2333/085G01N2800/28C07K2317/34G01N33/56983C07K16/1009C07K2317/33A61P31/14
Inventor 贾强林晓芳塔尼娅·基纳况慧星何放
Owner TEMASEK LIFE SCIENCES LABORATORY
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