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Method for the diagnosis or the screening of an arbovirus infection, reagents useful in said method and their applications

A technology of arthropods and mediators, applied in the field of flavivirus infection, diagnosis or screening of arthropod mediator virus infection, optimization of Flaviviridae infection, and reagents, it can solve the problems of antigen quantity change, masking, etc., and achieve the effect of accelerated diagnostic testing

Inactive Publication Date: 2010-07-21
INST PASTEUR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of monoclonal antibodies against common epitopes of different viruses or virus serotypes may raise the following problems: (i) antigen binding to human sera may mask epitopes of tracer monoclonal antibodies
As a result, the relationship between the output signal of the test and the amount of antigen captured may vary for different antigens

Method used

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  • Method for the diagnosis or the screening of an arbovirus infection, reagents useful in said method and their applications
  • Method for the diagnosis or the screening of an arbovirus infection, reagents useful in said method and their applications
  • Method for the diagnosis or the screening of an arbovirus infection, reagents useful in said method and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1: Materials and methods

[0143] - Media, buffers and kits

[0144] The media LB (Sambrook and Russell, 2001) and SB (Plückthun, 1996) have been described. Ampicillin was used at a concentration of 200 μg / mL and kanamycin was 50 μg / mL. LB medium containing ampicillin was used for all gene constructs. Plasmid DNA was prepared with Qiaprep Spin Miniprep kit (purchased from Qiagen), DNA was extracted from agar gel with Gel Extraction kit (purchased from Qiagen), DNA was ligated with Quick Ligation kit (Roche), and NuPAGE Novex system was used to (Invitrogen) for polyacrylamide gel electrophoresis. Enzyme-linked immunosorbent assay (ELISA) was performed using 96-well microtiter plates (Maxisorb, Nunc). PBS buffer (phosphate buffered saline) was purchased from Invitrogen or Sigma-Aldrich; bovine serum albumin was purchased from Roche; low-fat milk powder was purchased from Regilait; Tween 20, 4-nitrophenyl phosphate (pNPP) and 5-bromo - 4-Chloro-3-iodophosphate...

Embodiment 2

[0150] Example 2: Construction of the intermediate vector pEBL1, which was deposited at CNCM (French National Collection of Cultures and Microorganisms, 28 rue du Docteur Roux, 75015, Paris) with the deposit number I-3747 on April 23, 2007.

[0151] The restriction sites located in the cloning region of plasmid pLIP5GN-H6 are very close, and it is difficult to perform double restriction operations in this region. The kanamycin resistance cassette was therefore inserted into the Sail site in this region. Plasmid pUC-4 was digested with SalI enzyme, and the DNA fragment containing the aph gene was purified by agarose gel electrophoresis. pLIP5GN-H6 was also digested with SalI. The purified fragment and the linearized vector are recombined by ligation. Recombinant plasmid pEBL1 (SEQ ID NO: 13) was recovered by transforming the ligated mixture into XL1-Blue competent cells and selecting transformed cells on LB medium containing ampicillin and kanamycin.

[0152] more specifical...

Embodiment 3

[0159] Embodiment 3: Construction of ED3-PhoA hybrid gene

[0160] method

[0161] The ED3-phoA hybrid gene encoding the hybrid protein between the flavivirus ED3 domain and PhoA is constructed as follows: first digest the plasmid pEBL1 (see Example 2) with the restriction endonuclease SmaI, and verify the digestibility by electrophoresis. was completed, and the digested DNA was desalted by size exclusion chromatography on a Microspin G25 column (Amersham-Biosciences). The linear pEBL was then digested with SalI enzyme, and the restriction cleavage was monitored by electrophoresis and the appearance of a DNA fragment (1252 bp) corresponding to the kanamycin resistance cassette. The ED3 gene was amplified by PCR using two oligonucleotide primers and the high-fidelity polymerase Pfu-Turbo (Stratagene). Primers that hybridized at the 5' end of the ED3 gene introduced a SalI site, and primers that hybridized at the 3' end introduced ScaI and SpeI sites. The ScaI site (AGT-ACT) ...

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Abstract

Method for the diagnosis or the screening of an arbovirus infection and preferably a flaviviridae infection and more preferably a flavivirus infection, reagents useful in said method and their applications. Said method comprises: (i) contacting a sample from the subject or animal with a solid support sensitized with an Ig binding protein which is directed against a specific class of Ig molecules of the subject or animal species under consideration and (ii) incubating the immunocomplex formed in (i) with a detector molecule consisting of a hybrid protein comprising at least an arboviral ED3 domain and an alkaline phosphatase (PhoA), the detection of said immunocomplex being the sign of the presence of an arbovirus in said sample.

Description

technical field [0001] The present invention relates to a method for diagnosing or screening arbovirus infection, preferably flaviviridae infection, more preferably flavivirus infection, in said method and their use Reagents used. Background technique [0002] Arthropoviruses (arthropod-borne viruses) are viruses that circulate in nature on blood-sucking arthropod vectors and disease-susceptible vertebrate hosts. The present invention includes all Arthroviruses that contain an envelope protein, although the specification focuses primarily on the flavivirus species. [0003] Many Arthroviruses and especially many Flaviviruses cause severe disease in humans or animals, especially Yellow Fever Virus (YFV), Dengue Virus (DENV), West Nile Virus (WNV) and others. [0004] Flavivirus infection can now be detected by several methods, including virus isolation, detection of viral RNA by RT-PCR, and immunochemical tests targeting viral proteins or antiviral immunoglobulin molecules....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18G01N33/535G01N33/543C12N15/62
CPCG01N2469/20G01N33/581C12N2770/24011C07K2319/61G01N33/56983G01N2333/185Y02A50/30
Inventor 于格·贝杜埃勒埃洛蒂·布里安-利茨勒菲利普·迪萨尔菲利普·德普雷斯利蒂希娅·布勒芒
Owner INST PASTEUR
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