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Detection method for agglutination test of rabies virus neutralizing antibody cells

A rabies virus and antibody technology, applied in the field of bioengineering, can solve the problems of high detection cost, complicated operation and long time.

Inactive Publication Date: 2012-07-04
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention relates to a detection method of rabies virus neutralizing antibody cell agglutination test, which solves the problems of long time, complicated operation and high detection cost of the traditional method for measuring rabies virus neutralizing antibody

Method used

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  • Detection method for agglutination test of rabies virus neutralizing antibody cells
  • Detection method for agglutination test of rabies virus neutralizing antibody cells
  • Detection method for agglutination test of rabies virus neutralizing antibody cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of Eukaryotic Expression Vector of Rabies Virus Glycoprotein Gene

[0030] Using the commercially available eukaryotic expression plasmid vector pIRES-neo ( figure 1 ) polyclonal restriction site EcoR V and Bam H I inserted the rabies virus glycoprotein gene (GenBank: AF499686.2) to construct an expression cassette expressing the glycoprotein gene.

[0031] EcoR V and Bam H I double enzyme digestion system (product of Treasure Bioengineering (Dalian) Co., Ltd.): 10 U of each of the two endonucleases, 2 μl of K buffer, 1 μg of pIRES-neo plasmid, and make up to 20 μl of distilled water. After bathing in water at 37°C for 1 hour, a 5.3bp band was recovered by 1% agarose gel electrophoresis (for the recovery step, refer to the instructions of the DNA gel recovery kit from Axygen).

[0032] DNA ligation system (product of Treasure Bioengineering (Dalian) Co., Ltd.): T4 DNA ligase 200U, reaction buffer 1 μl, rabies virus glycoprotein gene fragment...

Embodiment 2

[0036] Establishment of detection method of human rabies virus neutralizing antibody cell agglutination test

[0037] 1. Establishment of Stable Expression Cell Lines

[0038] Under the mediation of liposome 2000, the constructed eukaryotic expression vector pIRES-neo / G was transfected into human embryonic kidney cells 293. Transfection step: take 10 μl liposome 2000, mix it with 5 μg pIRES-neo / G in equal volume for 5 minutes; remove the cell culture medium, spread the mixture on the monolayer cells, 37°C 5% CO 2 Induction for 30min, add DMEM medium containing 5% calf serum, 37°C 5%CO 2 nourish. The next day, the cell culture medium was removed, and the cells were screened with DMEM containing 150 μg / mL G418 and 5% calf serum to obtain the 293 cell line stably expressing the neo gene. The obtained cells were stained with commercial FITC-labeled rabies virus antibody, and the expression of glycoprotein was identified under a fluorescent microscope ( figure 2 ).

[0039]...

Embodiment 3

[0053] Establishment of detection method of canine rabies virus neutralizing antibody cell agglutination test

[0054] 1. Establishment of stable expression cell lines

[0055] Under the mediation of liposome 2000, the constructed eukaryotic expression vector pIRES-neo / G was transfected into canine kidney cells MDCK. Transfection step: take 10 μl liposome 2000, mix it with 5 μg pIRES-neo / G in equal volume for 5 minutes; remove the cell culture medium, spread the mixture on the monolayer cells, 37°C 5% CO 2 Induction for 30min, add DMEM medium containing 5% calf serum, 37°C 5%CO 2 nourish. The next day, the cell culture medium was removed, and the cells were screened with DMEM containing 300 μg / mL G418 and 5% calf serum to obtain the MDCK cell line stably expressing the neo gene. The obtained cells were stained with commercial FITC-labeled rabies virus antibody, and the expression of glycoprotein was identified under a fluorescent microscope ( Figure 4 ).

[0056] 2. Pr...

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Abstract

The invention provides a detection method for an agglutination test of rabies virus neutralizing antibody cells. In the detection method, cells for expressing rabies virus glycoprotein are taken as antigens and carrier particles; and through agglutination reaction with human, dog and cat serum antibodies, whether the rabies virus neutralizing antibodies in the serum reach an immune protection level can be judged. By the detection method, the problems that the conventional detection method for the rabies virus neutralizing antibodies is long in detection time, complicated in operation, high in detection cost and the like can be solved.

Description

technical field [0001] The invention relates to a detection method of rabies virus neutralizing antibody cell agglutination test, that is, cells expressing rabies virus glycoprotein are used as antigens and carrier particles, and through agglutination reaction with serum antibodies of humans, dogs and cats, it can be determined Whether the neutralizing antibody containing rabies virus reaches the level of immune protection; it belongs to the technical field of bioengineering. Background technique [0002] Rabies is a deadly infectious disease caused by rabies virus infection, which can lead to 100% death once onset. The fundamental means to prevent the occurrence of rabies is pre-exposure prophylaxis for animals or emergency post-exposure treatment for humans. Both methods are to vaccinate the vaccinated animals or humans to produce a sufficient level of neutralizing antibodies to prevent the occurrence of viral infection. The rabies virus neutralizing antibody in immunize...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/566
Inventor 扈荣良刘晔张守峰张菲
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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