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A method and detection kit for preprocessing hepatitis C antigen

A detection kit and pretreatment agent technology, applied in the field of in vitro diagnostic medical testing, can solve the problems of poor specificity, low sensitivity, unfavorable commercialization, etc.

Active Publication Date: 2019-12-24
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Due to the unique protein space structure of hepatitis C antigen, the main detection site is placed inside the protein structure, making it difficult to be detected
Traditional HCV mostly uses polypeptides for cross-linking, but this will cause disadvantages such as poor specificity; in recent years, expressed genetically engineered antigens have been used to replace HCV polypeptides, but the processing process is difficult to control, and the stability of the processed antigens is poor, so it is not easy to Good for commercialization, difficult to control batch-to-batch variation, and low sensitivity

Method used

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  • A method and detection kit for preprocessing hepatitis C antigen
  • A method and detection kit for preprocessing hepatitis C antigen
  • A method and detection kit for preprocessing hepatitis C antigen

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preparation example Construction

[0053] Correspondingly, the present invention also provides a method for preparing the biotinylated hepatitis C antigen pretreated above, the method comprising the following steps:

[0054] 1) Add a pretreatment agent to the hepatitis C antigen and react for a period of time;

[0055] 2) using biotin to cross-link the treated hepatitis C antigen;

[0056] 3) Preserving the cross-linked biotinylated hepatitis C antigen in a diluent containing a pretreatment agent.

[0057] In a specific embodiment, the following process parameters are used in the preparation process of the biotinylated pretreated hepatitis C antigen of the present invention with excellent detection sensitivity and stability of hepatitis C virus antibody.

[0058] In a specific embodiment, the hepatitis C antigen pretreatment agent is 4-nitrothiophenol or sodium 2-mercaptoethanesulfonate; preferably sodium 2-mercaptoethanesulfonate.

[0059] In another preferred embodiment, the hepatitis C antigen pretreatment...

Embodiment 1

[0100] Example 1. Preparation of biotinylated pretreated hepatitis C antigen

[0101] 1) Dilute hepatitis C antigen and sodium 2-mercaptoethanesulfonate to 1 mg / mL and 1% (w / v) with pH 7.4, 20 mM PBS, respectively;

[0102] 2) Add sodium 2-mercaptoethanesulfonate solution to the hepatitis C antigen at a ratio of 1:10, mix well, place at 2-8°C, and react for 30 minutes;

[0103] 3) Dilute the pretreated hepatitis C antigen to 1mg / mL with pH7.4, 20mM PBS;

[0104] 4) Dissolving Biotin containing a sulfhydryl functional group in purified water to prepare a 2 mg / mL Biotin solution;

[0105] 5) According to the ratio of 1:15, add Biotin solution containing sulfhydryl functional group to the pretreated hepatitis C antigen;

[0106] 6) Mix well, place at room temperature, and react for 30 minutes;

[0107] 7) Dialyze overnight in 20mM PBS solution at pH 7.4;

[0108] 8) Add glycerol at a volume ratio of 1:1, mix well, and store at -20°C to prepare biotinylated pretreated hepatiti...

Embodiment 2

[0109] Example 2. Preparation of Hepatitis C Virus Antibody ELISA Detection Kit

[0110] 1. Preparation of Biotinylated HCV Antigen Reagent

[0111] Biotinylated Hepatitis C Antigen Reagent Diluent Formula:

[0112]

[0113] Dilute the biotinylated hepatitis C antigen with diluent to 0.2 μg / mL, and mix thoroughly at room temperature for at least 30 minutes to prepare the biotinylated hepatitis C antigen reagent.

[0114] 2. Preparation of Streptavidin Enzyme Reagent

[0115] Streptavidin Enzyme Reagent Diluent Recipe:

[0116]

[0117] Dilute horseradish peroxidase-labeled streptavidin with enzyme diluent to 1:1000-1:5000, and mix thoroughly at room temperature for at least 30 minutes to prepare streptavidin enzyme reagent.

[0118] 3. Preparation of Washing Solution

[0119] Washing liquid formula:

[0120]

[0121]

[0122] Prepare the washing solution according to the formula, and mix thoroughly at room temperature for at least 30 minutes to obtain the wash...

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Abstract

The invention discloses a hepatitis C antigen pretreatment method, a preparation method of a biotinylated hepatitis C antigen, and a hepatitis C virus antibody detection kit comprising the biotinylated hepatitis C antigen. The hepatitis C antigen is pretreated to change the protein conformation, and the pretreated hepatitis C antigen reacts with biotin to produce the biotinylated hepatitis C antigen. The hepatitis C virus antibody detection kit adopts the biotinylated hepatitis C antigen prepared through a reaction of the pretreated hepatitis C antigen and biotin, so the detection ability of the hepatitis C antibody is greatly improved, the specificity is improved, and the kit also has the advantages of good stability and easiness in production control, so the kit is massively suitable for the assisted diagnosis and blood screening of the hepatitis C virus.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic medical testing. Specifically, the present invention relates to a method for pretreatment of hepatitis C antigen, the hepatitis C antigen obtained by the pretreatment method, a kit for implementing the pretreatment method on the hepatitis C antigen, and the hepatitis C antigen pretreated by the method. Antigen detection kits and their use in the detection of hepatitis C. Background technique [0002] HCV is a blood-borne virus with worldwide distribution. According to the statistics of the World Health Organization, the global hepatitis C virus (HCV) infection rate is about 3%, and there are about 3.15 million new HCV cases every year. It is mainly transmitted through blood transfusion or blood products, hemodialysis, plasmapheresis, kidney transplantation, intravenous drug injection, sexual transmission, and mother-to-child transmission. [0003] Hepatitis C virus (HCV) is a single-stranded ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/553G01N33/535G01N21/76
CPCG01N21/76G01N33/535G01N33/553G01N33/56983
Inventor 陈超夏泽张巍佳罗紫臣彭珊姚姣妮
Owner SHANGHAI KEHUA BIO ENG
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