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HCV antigen detection board and method for detecting HCV antigen by using same

An antigen detection and detection board technology is applied in the detection of hepatitis C virus HCV antigen and the detection of virus antigens, which can solve the problems of low detection rate of hepatitis C virus core antigen, and achieve shortened detection time, high sensitivity, The effect of reducing the background

Inactive Publication Date: 2011-05-04
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of low detection rate of the existing hepatitis C virus core antigen, the present invention provides a high sensitivity and good specificity HCV antigen detection panel

Method used

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  • HCV antigen detection board and method for detecting HCV antigen by using same
  • HCV antigen detection board and method for detecting HCV antigen by using same
  • HCV antigen detection board and method for detecting HCV antigen by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Obtaining the HCV antigen detection panel:

[0049] A, preparation of anti-HCV serum

[0050] A1. Recombinant HCV antigen NS expressed in four highly conserved region genes with a concentration of 512Eu / ml 3 、NS 4 、NS 5 2 ml of the equal volume mixture of C and C, and immunize monkeys, rabbits or other suitable animals;

[0051] A2. Blood was collected at 2, 3, and 4 weeks respectively, and the HCV antibody titer was tested with a conventional hepatitis C virus antibody diagnostic kit. When the titer did not reach 1:4000, a booster immunization injection was required;

[0052] A3. When the anti-HCV serum titer is greater than 1:8000, blood is collected from the carotid artery, and serum is prepared according to a conventional method to obtain anti-HCV serum, which is stored below -60°C until use;

[0053] B. Purification of Anti-HCV Antibody

[0054] B1. Take protein A or protein A / G affinity chromatography column, equilibrate at room temperature for 30 minutes, an...

Embodiment 2

[0064] Obtaining the HCV antigen detection panel:

[0065] A, preparation of anti-HCV serum

[0066] A1. Recombinant HCV antigen NS expressed in four highly conserved region genes with a concentration of 1024Eu / ml 3 、NS 4 、NS 5 1ml of the equal volume mixture of C and C, to immunize monkeys, rabbits or other suitable animals;

[0067] A2. Blood was collected at 2, 3, and 4 weeks respectively, and the HCV antibody titer was tested with a conventional hepatitis C virus antibody diagnostic kit. When the titer did not reach 1:4000, a booster immunization injection was required;

[0068] A3. When the anti-HCV serum titer is greater than 1:8000, blood is collected from the carotid artery, and serum is prepared according to a conventional method to obtain anti-HCV serum, which is stored below -60°C until use;

[0069] B. Purification of Anti-HCV Antibody

[0070] B1. Take protein A or protein A / G affinity chromatography column, equilibrate at room temperature for 30 minutes, and...

Embodiment 3

[0089] Preparation of biotin-labeled HCV antibody use solution: After dissolving conventional long-arm biotin in water for injection as usual, mix it with conventional HCV monoclonal antibody at a molar ratio of 1:10; leave it at room temperature for 30 minutes, or After 2 hours in the refrigerator at 4°C, dialyze overnight with phosphate buffered saline PBS as usual, add an equal amount of glycerol, and store at -20°C for later use to obtain biotin-labeled HCV antibodies; use phosphate buffered saline PBS+2% volume ratio Dilute the biotin-labeled HCV antibody to a volume concentration of 1:500 to 1:2000 in the enzyme junction solution composed of bovine serum albumin to obtain a biotin-labeled HCV antibody use solution, which is stored at 4°C for future use.

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Abstract

The invention provides a hepatitis C virus (HCV) antigen detection board and a method for detecting an HCV antigen by using the same. The method comprises the following steps of: diluting a purified HCV resistant polyclonal antibody until the concentration is 5 to 50mu g / ml by the conventional method; adding 50 to 200mu l of polyclonal antibody into each hole of a polystyrene board; standing overnight at the temperature of 4 DEG C; washing the board; adding 100 to 300mu l of the conventional bovine serum albumin into each hole of the polystyrene board and sealing the detection board; standing overnight at the temperature of between 2 and 8 DEG C; washing the board and drying in the air; storing at the temperature of between 2 and 8 DEG C to obtain the HCV antigen detection board; and detecting the HCV antigen through the HCV antigen detection board by using long arm biotin labeled monoclonal (polyclonal) HCV-immunoglobulin G (IgG), horse radish peroxidase labeled streptavidin or horse radish peroxidase labeled monoclonal (polyclonal) HCV-IgG as an amplification system. Through the method, detection processes can be reduced, detection time can be shortened, a background can be lowered by lowering a cross reaction and detection sensitivity can be improved; and the method for detecting the HCV antigen has high specificity and sensitivity, and is conveniently and quickly operated; and a reliable technical path is provided for early diagnosis, large-scale quick screening, quantitative detection and the like of HCV infection.

Description

technical field [0001] The invention relates to a virus antigen detection method, in particular to a hepatitis C virus HCV antigen detection method, and belongs to the field of biotechnology. Background technique [0002] Currently, there are three main types of hepatitis C detection methods: [0003] The first type is hepatitis C antibody detection: its disadvantage is that after infection with hepatitis C virus HCV, there is a incubation period of 2 to 26 weeks. Generally, there is a longer window period until the anti-HCV antibody appears (turning positive), with an average of 70 days In some patients, the window period can be extended to 6-9 months or longer, about 1-3% of patients can continue to be negative for anti-HCV, and the anti-HCV antibody rises only after the alanine aminotransferase increases. Due to the existence of the window period, it is easy to miss the detection of the latent period and invisible infection. After the body is infected with HCV, virus par...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N21/31
Inventor 谢忠平李琦涵龙润乡李华杨蓉白惠珠董承红蒋蕊鞠易红昆崔萍芳
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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