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A high-throughput method for rapid detection of Hantaan virus neutralizing antibody titer

A technology for antibody titer and Hantaan virus, which is applied in the field of high-throughput rapid detection of Hantaan virus neutralizing antibody titer, can solve the problems of multiple transmission routes, difficult prevention and control tasks, and lack of specific and effective therapeutic drugs, etc. Good repeatability, easy promotion, and standardized methods

Active Publication Date: 2019-05-03
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because HTNV has a wide range of hosts and multiple transmission routes, and there is still a lack of specific and effective therapeutic drugs in clinical practice, the task of its prevention and treatment is very arduous.

Method used

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  • A high-throughput method for rapid detection of Hantaan virus neutralizing antibody titer
  • A high-throughput method for rapid detection of Hantaan virus neutralizing antibody titer
  • A high-throughput method for rapid detection of Hantaan virus neutralizing antibody titer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1.ICW detects the selection of the optimal time of HTNV infection cell ( figure 1 ).

[0105] After A549 cells were infected with HTNV (MOI=1), the expression of viral protein NP in cells at different infection time points was detected by ICW. The left picture is the scanning result, and the right picture is the software analysis result. Through the Student t test analysis, it is found that compared with the uninfected group, the expression of viral protein can be detected on the second day after infection (2dpi). (*P<0.05,**P<0.01,***P<0.001)

Embodiment 2

[0106] Embodiment 2.ICW detects the selection of the optimal virus titer of HTNV infected cell (attachment figure 2 ).

[0107] After A549 cells were infected with HTNV according to different MOI, the expression of viral protein NP in cells was detected by ICW at 2dpi. The left picture is the scan result, and the right picture is the software analysis result. Through the Student t test analysis, it is found that compared with the uninfected group, the expression of viral protein can be clearly detected when the infection dose is MOI=0.1. (*P<0.05,**P<0.01,***P<0.001)

Embodiment 3

[0108] Example 3. Screening of antiviral protein antibodies used by ICW ( image 3 ).

[0109] Anti-HTNV NP mouse monoclonal antibody 1A8 and anti-HTNV GP mouse monoclonal antibody 3D8 and 3G1 were prepared. The antibody was prepared according to the standard monoclonal antibody preparation method; the antibody purification method was the ammonium sulfate precipitation method, and the specific steps were as follows:

[0110] 1. Prepare saturated ammonium sulfate solution (SAS)

[0111] Slowly add 767g (NH4)2SO4 to 1L of distilled water while stirring. Use ammonia or sulfuric acid to adjust the pH to 7.0. This is ammonium sulfate solution (4.1mol / L, 25°C) with 100% saturation.

[0112] 2. Precipitation

[0113] (1) Centrifuge the sample (such as ascites) at 20,000 g for 30 min to remove cell debris;

[0114] (2) keep the supernatant and measure the volume;

[0115] (3) Slowly add an equal volume of SAS to the supernatant while stirring, the final concentration is 1:1;

...

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Abstract

The invention discloses a high-flux method for quickly detecting hantaan virusneutralizing antibody titer. The method is characterized by utilizing an In-cell Western (ICW) method for simply, conveniently and intuitively detecting cells infected by HTNV (Hantaan Virus). The method is high in detection sensitivity (capable of detecting 5g of target protein), short in period (72 hours), objective in result interpretation, good in repetitiveness, high in flux, standardized in method, and convenient to inter-laboratory popularize, and can be particularly used for detecting the infectivity of acellular cytopathic effect virus. The invention also discloses application of the method in HTNV infectivity titration (TCID50), HTNV neutralizing antibody titration, anti-HTNV molecular screening and identification, human HTNV vaccine immunity protection evaluation and the like.

Description

technical field [0001] The invention belongs to the technical field of detection of Hantaan virus, and relates to a method for rapidly detecting the titer of neutralizing antibodies of Hantaan virus with high throughput. Background technique [0002] Hantaan virus (HTNV) belongs to the Bunyaviridae (Bunyaviridae) Hantavirus genus (Hantavirus), is a single-stranded negative-sense RNA virus, the genome consists of three segments, L, M, and S, respectively encoding the virus RNA-dependent RNA polymerase (RdRp), envelope glycoprotein (GP) and nucleocapsid protein (NP). The host animal and source of infection of the virus are rodents, and it is mainly transmitted to humans through the respiratory tract, digestive tract or damaged skin through animal excrement contamination, causing a type of natural focal disease, namely hemorrhagic fever with renal syndrome (Hemorrhagic fever with renal syndrome, HFRS). The clinical manifestations of the disease are fever, hemorrhage, and acut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/569
CPCG01N21/64G01N21/6428G01N33/56983G01N2021/6421G01N2021/6443G01N2333/175
Inventor 张芳琳马宏炜雷迎峰张亮程林峰陈何嵩石静琦韩佩君叶伟吴兴安徐志凯
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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