Method for pretreating sample and method for immunoassay of hcv

An immunoassay and sample technology, applied in the field of HCV immunoassay, can solve the problems of high sensitivity, variation, detection, etc.

Inactive Publication Date: 2011-06-01
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009]However, HCV, which is known as the pathogen of hepatitis C, is an RNA virus that causes a high degree of mutation
For example, when the amino acid at position 49 of the core region protein of HCV genotype 1b shown in SEQ ID NO: 2 is mutated, even if HCV is detected by the above-mentioned immune complex transfer assay method using the above-mentioned known antibody, it may not be possible. Cases detected with high sensitivity (Journal Of Clinical Microbiology, Sept. 2000, p.3450-3452)

Method used

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  • Method for pretreating sample and method for immunoassay of hcv
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  • Method for pretreating sample and method for immunoassay of hcv

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Experimental program
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Embodiment approach

[0062] Preferred embodiments of the present invention will be described below with reference to the accompanying drawings.

[0063] The pretreatment method of samples for HCV core protein detection in this embodiment is a pretreatment method for samples used in the detection of HCV core protein using the immunoassay method using particles, including: using samples suspected of containing hepatitis C virus with The first treatment step of the reagent treatment of alkaline substances, the sample obtained in the first treatment step is treated with the reagent containing acidic substances, the second treatment step, at least one of the reagents used in the first treatment step and the second treatment step Contains a reducing agent.

[0064] In this embodiment, at least one of the reagents used in the first treatment step and the second treatment step contains a reducing agent. When at least one of the reagents used in the first treatment step and the second treatment step conta...

Embodiment 1

[0176]

[0177] (A) Construction of expression plasmid

[0178] The following plasmids were obtained by merging the DNA encoding the amino acid sequence at positions 1 to 160 of the HCV core region represented by SEQ ID NO: 2, 3, or 4.

[0179] pUC·HCV-J1b: plasmid of the core region protein (HCV-J1b) of HCV genotype 1b shown in SEQ ID NO: 2

[0180] pUC·HCV-J1bT49P: The plasmid of the core region protein (HCV-J1bT49P) of HCV genotype 1b in which the 49th amino acid threonine shown in SEQ ID NO: 3 is mutated to proline

[0181] pUC·HCV-3bNE137: plasmid of core region protein (HCV-3bNE137) of HCV genotype 3bNE137 strain shown in SEQ ID NO: 4

[0182] SEQ ID NO: 2

[0183] MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDPRRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLED

[0184] SEQ ID NO: 3

[0185] MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRAPRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDP...

Embodiment 2

[0206]

[0207] Using the HCV-J1b and HCV-J1bT49P obtained in Example 1 and the synthetic peptide CDP-2 shown in Table 1, CDP-2-1, CDP-3, and CDP-3-1 were analyzed as anti-HCV core protein single Cloning the epitope of the antibody HCF4-801 antibody and HE25 antibody. It should be noted that CDP-2, CDP-2-1, CDP-3, and CDP-3-1 are synthetic peptides conjugated with keyhole limpet hemocyanin, respectively, which were produced by Operon Biotechnology Co., Ltd. externally.

[0208] Table 1: List of amino acid sequences of peptides derived from HCV core

[0209]

Peptide name

amino acid sequence

AA configuration

SEQ ID NO: 5

CDP-2

DVKFPGGGQIVGGVYLLPRR

21～40

SEQ ID NO: 6

CDP-2-1

DVKFPGGGQI

21～30

SEQ ID NO: 7

CDP-3

GPRLGVRATRKTSKRSQPRG

41～60

SEQ ID NO: 8

CDP-3-1

GPRLGVRATR

41～50

[0210] (A) Confirmation of reactivity between CDP-2 and HCF4-801 antibody

[0211] To contain ...

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Abstract

There are provided: a method for pretreating a sample for HCV core protein detection by an immunoassay using particles, which includes treating a sample suspected of containing hepatitis C virus (HCV) with an alkaline material-containing reagent and neutralizing the sample with an acid material-containing reagent, wherein at least one of the reagents contains a reducing agent; a reagent kit for HCV core protein detection; a method for determining the presence or absence of hepatitis C virus in a sample; and a method for immunoassay of HCV.

Description

【Technical field】 [0001] The invention relates to a sample pretreatment method for HCV core protein detection, a test kit for HCV core protein detection, a method for judging whether there is hepatitis C virus in a sample, and an HCV immunoassay method. 【Background technique】 [0002] Hepatitis C is a contagious disease caused by the hepatitis C virus (HCV). Not only has the preventive method of hepatitis C not yet been established, but most of the infected persons (carriers) are presumed to have metastasized to liver cirrhosis or liver cancer after 10 to 30 years. Early detection and early treatment of HCV infection are very important. [0003] HCV, which is the causative agent of hepatitis C, is an RNA virus with a diameter of about 60 nm and has an envelope. The RNA of HCV is a single-stranded positive strand consisting of about 9500 bases. The gene structure is similar to that of flaviviruses, with untranslated regions (UTR) at the 5' end and 3' end, and a translated r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N33/68G01N33/576C12N5/18
CPCG01N33/5767G01N2469/10
Inventor 山垣内孝博武田和彦高马卓也
Owner SYSMEX CORP
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