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SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof

A monoclonal antibody, nucleocapsid protein technology, applied in the field of diagnosing SARS, hybridoma, specific monoclonal antibody, to achieve the effect of good repeatability, accurate detection and low cost

Inactive Publication Date: 2004-12-29
第一军医大学珠江医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently there is no commercial SARS coronavirus antigen diagnostic kit

Method used

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  • SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof
  • SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof
  • SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the preparation method of SARS-CoV nucleocapsid protein (hereinafter referred to as N protein) monoclonal antibody.

[0050] 1. Preparation of immune antigen

[0051] The immunogen used to prepare the monoclonal antibody in the present invention is a genetically recombined SARS-CoV N protein and an inactivated natural virus antigen, and the genetically recombined SARS-CoV N egg is an engineering strain carrying a SARS-CoV N protein gene. Escherichia coli strains were prepared. It was confirmed by sequencing that the nucleocapsid gene sequence of SARS coronavirus carried by the strain was consistent with the nucleocapsid gene sequence of SARS coronavirus HKU-39849 strain (or other strains) published by PUBMED. The preparation of recombinant SARS-CoV N protein is carried out according to conventional methods, and the N fusion protein antigen is obtained by purifying with nickel-nitrilotriacetic acid metal affinity chromatography. For detailed preparation me...

Embodiment 2

[0058] Embodiment 2: screening the hybridoma that secretes SARS-CoV nucleocapsid protein monoclonal antibody

[0059]In order to detect the existence of antibody-producing clones, the method for measuring the titer of immune serum in Example 1, that is, the indirect ELISA method, is used to detect the cell culture supernatant. The brief description is as follows: the hybridoma culture supernatant is added to the coated well, 0.1 ml / Wells were kept at 37°C for 30 minutes. After washing the plate five times with washing solution, horseradish peroxidase-labeled goat anti-mouse IgG (ZYMED LABORATORIES, INC., USA) diluted 1:2000 was added, 0.1 ml / well at 37°C for 30 minutes. After washing the plate as above, add the substrate TMB, 0.1 ml / well, protect from light at room temperature for 10 minutes, add 0.1 ml / well 2M H 2 SO 2 Terminate the reaction and measure the absorbance at 450 nm. The strong positive clone hybridoma cells were selected for formal cloning, and the limiting di...

Embodiment 3

[0060] Example 3: SARS-CoV Nucleocapsid Protein Monoclonal Antibody Subclass Detection

[0061] The 4 clones obtained in this example were tested by the indirect ELISA method described in Example 1 to determine the antibody subclass they produced. Add hybridoma culture supernatant to coated wells, 0.1 ml / well at 37°C for 30 minutes, wash the plate five times with washing solution, add 1:1000 dilution of horseradish peroxidase-labeled rabbit anti-mouse different Subclass-specific immunoglobulins These antibodies include rabbit anti-mouse IgG1 (ZYMED LABORATORIES, INC, USA, Cat. No. 61-0120), rabbit anti-mouse IgG2a (supra, Cat. No. 61-0220), rabbit anti-mouse IgG2b ( Same as above, catalog number 61-0320), rabbit anti-mouse IgG3 (same as above, catalog number 61-0420), rabbit anti-mouse IgM (same as above, cat. After the plate, add substrate TMB, 0.1 ml / well, protect from light at room temperature for 10 minutes, add 0.1 ml / well 2M H 2 SO 2 Terminate the reaction and measure...

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Abstract

The present invention discloses the specific monocloneal antibody of SARS-CoV nuclear capsid protein, hybrid tumor producing the antibody, reagent containing the monocloneal antibody and reagent kit therewith. The monocloneal antibody is secreted and produced with the cell line including hybrid tumor 1E8A11 of preservation number of CCTCC-C200401, hybrid tumor 1E8A17 of preservation number of CCTCC-C200402, hybrid tumor 10E4A4 of preservation number of CCTCC-C200403, and hybrid tumor 14A3A3A19 of preservation number of CCTCC-C200404. The reagent kit established with the monocloneal antibody may be used in early diagnosis of SARS-CoV infection and has the features of simplicity, convenience, fastness, high sensitivity, powerful specificity, etc.

Description

technical field [0001] The present invention relates to the monoclonal antibody that produces with hybridoma technology, particularly relate to the specific monoclonal antibody that produces SARS coronavirus nucleocapsid (N) protein, the hybridoma that produces this antibody and the application of this monoclonal antibody in the diagnosis of SARS . Background technique [0002] Infectious atypical pneumonia, also known as severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS), is a newly emerging infectious disease. The causative agent of SARS was identified as a new coronavirus by the World Health Organization on April 10, 2003, named SARS-CoV. Since it first occurred in Guangdong in my country in November 2002, it has spread in more than 30 countries and regions, and the mortality rate of the disease is 6.4-16.5%. According to the available data, the disease is mainly transmitted at close range through respiratory droplets, body fluids and dirt, and ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/44C12N5/12G01N33/577
Inventor 车小燕丘立文温坤郝卫王亚娣潘玉先廖志勇
Owner 第一军医大学珠江医院
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