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Monoclonal antibody for detecting coxsackie virus a16 type virus solid particles and use thereof

A monoclonal antibody, Coxsackie virus technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulin, etc.

Active Publication Date: 2018-09-11
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports on the detection method of CA16 solid particle antigen
There is no report on the detection method of CA16 solid particle antigen

Method used

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  • Monoclonal antibody for detecting coxsackie virus a16 type virus solid particles and use thereof
  • Monoclonal antibody for detecting coxsackie virus a16 type virus solid particles and use thereof
  • Monoclonal antibody for detecting coxsackie virus a16 type virus solid particles and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of CA16 virus solid particles

[0039] The cultivation of virus: the CA16 strain used in the present invention is 2007-00190 (GenBank No. JF420555), and the cell used for cultivating virus is human rhabdoid cell RD ( CCL-136 TM ). First, RD cells were cultured in a 10 cm culture dish (NEST), and the medium was MEM medium (GIBCO) containing 10% fetal bovine serum (PAA). When the cell confluency reaches 80%, replace it with serum-free MEM medium, and inoculate the virus according to the amount of MOI=0.1. Culture at 37°C, and after 3 days, the virus was harvested after the cells were completely damaged. The virus harvesting method is as follows: the cells were scraped off and then frozen and thawed 3 times, the cell debris was removed by centrifugation, the cell lysed supernatant was collected, filtered through a 0.22 μm filter membrane to obtain the virus stock solution, and stored at -80°C for later use.

[0040] Sucrose density gradient cent...

Embodiment 2

[0042] Embodiment 2: Preparation of monoclonal antibody

[0043] The CA16 virus stock solution prepared in Example 1 was mixed and emulsified evenly with Freund's complete adjuvant, and multi-point injections were performed on 6-8 week-old BALB / c female mice, including back subcutaneous injection, groin subcutaneous injection, foot pad injection, limb muscle injection The immunogen was injected by injection and other ways, and the injection dose was 500 μL / time. After that, boost once every 2 weeks, and boost immunization in the same way. The immunogen is the mixture of the CA16 virus stock solution prepared in Example 1 and Freund's incomplete adjuvant. Before each immunization, 20 μL of tail vein blood or 200 μL of ocular vein blood were collected for titer determination. The serum titer was measured by indirect ELISA. When the serum titer of the mice reached a plateau, the mice were stopped from immunization and rested for two months before fusion. 72 hours before the fus...

Embodiment 3

[0045] Example 3: Screening of antibodies that can specifically bind CA16 virus solid particle antigens

[0046] Indirect ELISA experiment: Dilute the different CA16 virus particle antigens prepared in Example 1 100 times with 20mM PB7.4, coat a 96-well microtiter plate, 100 μL / well, coat at 37°C for 2 hours, wash the plate once with PBST , with relevant blocking solution (20mM PB7.4 containing 150mM NaCL, 0.5% casein, 0.002% gelatin) to block non-specific binding sites, 200 μL / well, 4°C overnight. Dilute the monoclonal antibody sample to be tested (1 mg / mL) prepared in Example 2 by 200 times (the formula of the diluent is the same as that of the blocking solution), add 100 μL to the microtiter plate, and incubate at 37°C for 1 h. Wash the plate 5 times with PBST, add GAM-HRP (horseradish peroxidase-labeled goat anti-mouse antibody, purchased from bio-rad company in the United States, the diluent formula is the same as the blocking solution), and incubate at 37°C for 30min. Was...

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Abstract

The invention relates to a monoclonal antibody of solid particles of specific binding coxsackievirus A16 (CA16), and a conservative variant or an active fragment of the monoclonal antibody. The invention further relates to a method for detecting a CA16 solid particle antigen by using the monoclonal antibody and use of the monoclonal antibody for preparing drugs for preventing or detecting or treating CA16.

Description

technical field [0001] The present invention relates to a monoclonal antibody specifically binding to the solid particle of Coxsackievirus A16 type virus, or its conservative variant or active fragment and its coding nucleotide sequence, or its degenerate sequence. The invention also relates to a method for detecting the solid particle antigen of Coxsackievirus A16 virus. The present invention also relates to the use of the monoclonal antibody, or its active fragment or conservative variant, for the preparation of medicaments for diagnosing, preventing and / or treating Coxsackievirus A16 infection. Background technique [0002] Coxsackievirus type A16 (hereinafter referred to as CA16), belonging to the Picornaviridae Enterovirus genus, is one of the main pathogens causing hand-foot-mouth disease (hereinafter referred to as HFMD) (Rabenau et al., 2010, Medical Microbiology and Immunology Med. Microbiol. Immunol. 199:45-51; Wang et al., 2011, Epidemiology 22:781-792). The gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
Inventor 程通杨立生徐龙发何德磊陈毅歆张军夏宁邵
Owner XIAMEN UNIV
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