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Sample pretreatment solution and kit for combined detection of hepatitis B virus core-related antigens

A technology of hepatitis B virus and pretreatment solution, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high human body infringement, high price, and complicated operation, and achieve improved detection sensitivity, wide application, and high clinical application value effect

Active Publication Date: 2021-03-19
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Blood HBV DNA detection as a common method to detect HBV load in liver tissue has the following problems: (1) HBV DNA detection is usually expensive and the operation is too complicated; (2) Drugs inhibit the synthesis and release of HBV-DNA, HBV DNA detection Although gene amplification methods such as PCR (Polymerase Chain Reaction) and TMA (Transcription-Mediated Amplification) are used, when antiviral drugs such as lamivudine and adefovir dipivoxil are taken, DNA synthesis will be inhibited, and the The release of viral particles of HBV DNA, that is, HBV DNA testing using the gene amplification method described above may be negative even if HBV is present in the liver tissue
In addition, although the method of detecting cccDNA (covalently closed circular DNA) through biopsy (collecting liver tissue) can effectively understand the amount of HBV in liver tissue, it is not easy to implement because it is too invasive to the human body

Method used

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  • Sample pretreatment solution and kit for combined detection of hepatitis B virus core-related antigens
  • Sample pretreatment solution and kit for combined detection of hepatitis B virus core-related antigens
  • Sample pretreatment solution and kit for combined detection of hepatitis B virus core-related antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Hepatitis B virus core-associated antigen detection kit

[0045] 1. Preparation method

[0046] (1), preparation of magnetic bead coatings coupled with anti-HBeAg, HBcAg and p22cr antibodies

[0047] The magnetic bead coating is prepared by linking the specific antibody to the magnetic beads by means of covalent coupling. The amino group or carboxyl group on the surface of the antibody reacts with the chemical group on the surface of the magnetic bead under the action of a chemical cross-linking agent to form a covalent conjugate of the antibody-magnetic bead. The reacted antibodies, as well as the blocked non-specific binding sites, are finally prepared into the magnetic bead coating of the kit.

[0048] The operation process is as follows:

[0049] 1. Take a certain functional group (selected from -COOH, -NH 2 , -HS and other functional groups) of paramagnetic nanoparticle solution 500 μL, wash with coupling buffer (MES buffer, pH 5.0~6.0), and replace t...

Embodiment 2

[0075] Preparation of Example 2 Sample Treatment Solution

[0076] The function of the sample treatment solution is to release HBcAg and p22cr from Dane virus particles, HBcAg, p22cr, and HBeAg are all denatured into linear antigens, and at the same time, anti-HBe and anti-HBc in the sample become inactive antibodies. Dane particles are infectious and complete HBV particles, spherical in shape, 42nm in diameter, and have a double-layer capsid. Its outer capsid is equivalent to the envelope of a general virus. It is composed of lipid bilayer and protein. After the virus outer capsid can be removed by detergent, the 20-hedron core structure with a diameter of 27nm can be exposed. The surface of the core is the inner shell of the virus. , the inner capsid protein is HBcAg. The sample treatment solution should not only fully lyse the virus particles but also be mild enough to affect the activity of the coated antibody on the solid phase carrier in the subsequent kit.

[0077] 1....

Embodiment 3

[0098] Example 3 Correlation between hepatitis B virus core-associated antigen and PCR quantitative detection

[0099] 1. Kit preparation

[0100] The HBcrAg content of the sample was detected with the hepatitis B virus core-associated antigen detection kit in Example 1, and the HBV-DNA content of the sample was detected with the hepatitis B virus nucleic acid PCR quantitative detection kit purchased from Daan Gene Co., Ltd.

[0101] 2. Test specimens

[0102] The samples were obtained from 39 cases of HBV-DNA positive serum samples in Da'an Clinical Laboratory Center, and were frozen at -20°C.

[0103] 3. Test items and result analysis

[0104] Detect the HBV-DNA content and HBcrAg content of the sample, and analyze the concentration correlation between the two. For the correlation results, see image 3 . According to statistics, the r value of the correlation between the two is 0.482, and the 95% CI is 0.1963-0.6922, indicating that there is an obvious parallel relations...

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Abstract

The invention relates to a sample pretreatment solution and a kit for combined detection of hepatitis B virus core related antigens. The sample pretreatment solution is a buffer solution containing achelating agent, high-concentration salt ions, a protein denaturing agent, a reducing agent and a surfactant. The sample pretreatment solution is utilized to construct the kit for combined detection of the hepatitis B virus core related antigens. The kit can effectively break virus shells to release three related antigens, greatly improves the detection sensitivity of samples, has extremely high clinical application value, and can be widely applied to inspection of various types of samples.

Description

technical field [0001] The invention relates to the technical field of hepatitis B virus antigen detection, and more specifically, to a sample pretreatment solution and a kit for combined detection of hepatitis B virus core-associated antigen. Background technique [0002] Hepatitis B is an infectious disease that seriously endangers human health. Hepatitis B virus (HBV) is prevalent in the world. There are 2 billion people in the world who have been infected with HBV, of which about 350 million people are chronically infected with HBV. About 1 million people die of HBV-related liver failure or primary liver cancer every year. Chronic hepatitis B virus infection is a global health problem. [0003] Blood HBV DNA detection as a common method to detect HBV load in liver tissue has the following problems: (1) HBV DNA detection is usually expensive and the operation is too complicated; (2) Drugs inhibit the synthesis and release of HBV-DNA, HBV DNA detection Although gene ampl...

Claims

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Application Information

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IPC IPC(8): G01N33/576G01N33/543G01N33/531
CPCG01N33/5762G01N33/54326G01N33/54346G01N33/531Y02A50/30
Inventor 吴英松李志雄王红翠刘天才董志宁
Owner GUANGZHOU DARUI BIOTECH
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