Method for determination of SARS virus nucleocapsid protein, reagent kit for the determination, test device, monoclonal antibody directed against SARS virus nucleocapsid protein, and hybridoma capable

A technology of SARS virus and nucleocapsid protein, applied in the field of hybridoma, can solve the problems of low sensitivity and insufficient sensitivity, and achieve the effect of sensitive detection

Inactive Publication Date: 2008-10-08
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunochromatography is a measurement method with lower sensitivity than ELISA, and even if polyclonal antibodies and monoclonal antibodies described in Non-Patent Document 1 and Non-Patent Document 2 are used in immunochromatography, sufficient sensitivity may not be obtained

Method used

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  • Method for determination of SARS virus nucleocapsid protein, reagent kit for the determination, test device, monoclonal antibody directed against SARS virus nucleocapsid protein, and hybridoma capable
  • Method for determination of SARS virus nucleocapsid protein, reagent kit for the determination, test device, monoclonal antibody directed against SARS virus nucleocapsid protein, and hybridoma capable
  • Method for determination of SARS virus nucleocapsid protein, reagent kit for the determination, test device, monoclonal antibody directed against SARS virus nucleocapsid protein, and hybridoma capable

Examples

Experimental program
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Effect test

preparation example Construction

[0070] [V] Production of monoclonal antibody: The method for obtaining monoclonal antibody from hybridoma can be appropriately selected according to the required amount of monoclonal antibody and the properties of hybridoma. For example, there is a method of obtaining from the ascites in the peritoneal cavity of a mouse inoculated with the hybridoma cells, a method of obtaining from the culture supernatant by cell culture, and the like. Monoclonal antibodies at high concentrations of several mg / ml can be obtained from ascites fluid as long as they are hybridomas that can proliferate in the peritoneal cavity of mice. Hybridomas that cannot proliferate in vivo obtain monoclonal antibodies from the culture supernatant of cultured cells. The production of monoclonal antibodies obtained from cultured cells is lower than in vivo, but it has the advantages of less mixing of immunoglobulins and other impurities contained in the peritoneal cavity of mice, and it is easy to purify.

[...

Embodiment 1

[0076] Example 1: Production of Monoclonal Antibodies to SARS-NP

[0077] The monoclonal antibody of this example was produced through the following steps [I] to [V]. Specifically, [I] using genetic engineering technology to prepare antigen solution containing recombinant SARS-NP, [II] immunizing mice with this antigen solution, [III] fusing splenocytes and myeloma cells obtained from immunized mice, [IV] Select cells producing antibodies specific to SARS-NP from the obtained hybridoma cells, [V] proliferate the hybridomas in the peritoneal cavity of mice, and isolate monoclonal antibodies from the ascites. The details are as follows.

[0078] [I] Preparation of antigen solution

[0079] First, using the gene analysis software BioEdit version 7.0.0 (BioEdit Company), the base sequence of the nucleocapsid protein cDNA of the SARSTOR2 strain (published in the American Gene Database (accessionNumber; AY274119, protein id; AAP41047.1)) was expressed in Escherichia coli Convert ...

Embodiment 2

[0157] Embodiment 2: application in immunochromatography

[0158] Immunochromatography was performed using the nine kinds of monoclonal antibodies obtained in Example 1 (monoclonal antibody Nos. 1, 2, 3, 12, 13, 14, 15, 16, and 17).

[0159] (Production of test equipment for immunochromatography)

[0160] In this example, the figure 2 A test appliance of the shape shown. The substrate 1 of the test device of this example used a gasket with an adhesive surface, the absorbent layer 5 used Waterman paper WF1.5, and the carrier 4 for chromatography used a nitrocellulose membrane. The chromatography carrier 4 has a determination device 6 immobilized with any one of the above nine kinds of monoclonal antibodies. In this example, the sample application layer of the test equipment is immersed in the measurement sample prepared from the specimen, and the sample solution is diffused to the judgment device by capillary phenomenon.

[0161] (Antibody Sensitive Glue Emulsion)

[0162...

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Abstract

Disclosed is a method for determination of SARS virus nucleocapsid protein (SARS-NP) using a first antibody and a second antibody both capable of binding specifically to SARS-NP, wherein the first or second antibody can recognize an epitope present in a region lying between the 283th nucleotide and the 422th nucleotide to the N-terminus in the amino acid sequence for SARS-NP (region C).

Description

Technical field: [0001] The invention relates to an assay method for assaying SARS virus nucleocapsid protein (SARS-NP), a kit for assay and a test device. The present invention also relates to a monoclonal antibody against SARS-NP and a hybridoma producing the above monoclonal antibody. Background technique: [0002] Severe Acute Respiratory Syndrome (SARS) is an infectious disease discovered only in recent years, and it has been confirmed that the causative pathogen of SARS is a novel virus (SARS virus) classified into the Coronaviridae family. As a diagnostic method for SARS infection, immunological assays have been used to detect SARS virus in specimens. One example of this is the methods described in Non-Patent Document 1 and Non-Patent Document 2. [0003] Non-Patent Document 1 describes an assay method using an enzyme-linked immunoassay (ELISA) using a polyclonal antibody against SARS-NP. Specifically, the polyclonal antibody is immobilized on an ELISA microtiter p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C07K16/08C12N5/10C12N15/02G01N33/53G01N33/543G01N33/545G01N33/569G01N33/577
CPCG01N33/56983C07K14/005C07K16/10C12N2770/20022
Inventor 藤本幸太郎梶田忠宏武田和彦冈本尚
Owner SYSMEX CORP
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