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Method for detecting hepatitis C virus antibody

A hepatitis C virus and antibody technology, applied in measurement devices, material analysis, instruments and other directions by observing the impact on chemical indicators, can solve the problems of inability to use blood donors for screening, low sensitivity, etc., and achieve high sensitivity, Reproducible and specific effects

Inactive Publication Date: 2006-04-05
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity is low and cannot be used for the screening of blood donors

Method used

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  • Method for detecting hepatitis C virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of Hepatitis C Virus Multi-epitope Chimeric Antigen and Enzyme-labeled Tether

[0049] 1. Materials:

[0050] pIL1 vector and pIL1a-NS 4 CNS 3 Plasmid: Chinese patent has been applied for and authorized, patent number: ZL00100695.9, patent name: "expression vector PBVIL1 and its construction method and use", and has been preserved, preservation number: CGMCC No: 0437.

[0051] pIL1-HCV / C plasmid:

[0052] E. coli HB101:

[0053] Q-Sepharose-FF anion exchange column:

[0054] S-Sepharose-FF cation exchange column:

[0055] Horseradish Peroxidase (HRP):

[0056] SMPB:

[0057] Dithiothreitol (DTT):

[0058] HCV-Ag:

[0059] Iodoacetamide:

[0060] Enzyme Linker:

[0061] EcoR I, BamH I enzymes:

[0062] 2. Method results:

[0063] 1. Development of chimeric antigens with multiple epitopes of hepatitis C virus:

[0064] The HCV antigen epitopes were analyzed with Goldkey computer-aided software. Determined the amino acid positions of th...

Embodiment 2

[0081] Example 2 Preparation of Hepatitis C Virus Multi-epitope Chimeric Antigen Labeled Horseradish Peroxidase

[0082] One, material: with embodiment one.

[0083] 2. Method results:

[0084] (1) HRP activation: 1ml (10mg HRP+1mlPBS pH7.6) horseradish peroxidase (HRP), add 100μl SMPB, stir gently at 18-20°C for 20 minutes.

[0085] (2) Antigen activation: Activation of hepatitis C virus multi-epitope chimeric antigen (HCVAg) by DTT: 5 mg HCVAg plus 115 μl (30.9 mg / ml) dithiothreitol (DTT) at 18-20° C. and gently stirred for 20 minutes.

[0086] (3) Desalting: Use PD-10 desalting column to desalt the above-mentioned activated antigen and HRP respectively, and collect 1.5ml of each volume.

[0087] (4) Binding: the desalted antigen is combined with HRP and gently stirred at 18-20°C for 45 minutes.

[0088] (5) Termination: Add 40 μl of 0.1 M iodoacetamide and stir gently at 37° C. for 30 minutes.

[0089] (6) Storage: aliquot 100 μl / tube, and store at -20°C.

Embodiment 3

[0090] Example 3 Double Antigen Sandwich Method for Detecting Hepatitis C Virus Antibody

[0091] One, material: with embodiment one.

[0092] 2. Method results:

[0093] 1. Dilute the purified hepatitis C virus multi-epitope fusion antigen with 0.05M pH9.6 carbonate buffer to 0.33 μg / ml, coat 100 μl / well on the enzyme-linked assay plate, and wash the plate with buffer 2 The second time, 3% BSA was blocked for 2 hours, and dried at room temperature for later use. Enzyme-labeled HCV multi-epitope fusion antigens of 4 different tethers were used, and the labeled antigens were diluted to 1:2000 times with a special diluent for enzyme-labeled antigens at 4°C for later use. The 4 HCV antibody positive and 4 negative sera were tested separately.

[0094] 2. The research procedure of the method for detecting hepatitis C virus antibody: see the attached figure 1 .

[0095] 3. Kit composition:

[0096] (1) 1 HCV antigen-coated enzyme-linked plate

[0097] (2) HCV antibody positi...

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Abstract

The invention discloses a method for adopting enzyme immune technology to test hepatitis C virus antibody, it also discloses the hepatitis C virus multiple bit table jogging antibody and the enzyme label connecting arm. The testing method comprises: preparing the hepatitis C virus multiple bit table jogging antibody, preparing the jogging antibody and the label connecting arm, preparing the jogging antibody label horse radish peroxides compound, using the jogging antibody to uterus the enzyme checker board, adding tested antibody and enzyme label antibody compound and so on.

Description

technical field [0001] The invention relates to a method for detecting hepatitis C virus antibody, in particular to a method for detecting hepatitis C virus antibody by using enzyme-linked immunosorbent technology, and also relates to the multi-epitope embedding method of hepatitis C virus used in the method. Synthesize antigen and enzyme-labeled tether. Background technique [0002] Hepatitis C virus (HCV) is the main pathogen that causes viral non-A non-B hepatitis after blood transfusion. It is mainly transmitted through blood transfusion and intravenous drug use, and can also be transmitted through close contact and mother-to-child transmission. There are currently about 170 million HCV-infected people in the world. About half of those infected develop chronic hepatitis, and some develop cirrhosis and hepatocellular carcinoma. It is extremely harmful as there is currently no proven treatment and vaccine to prevent its further spread. According to recent reports, there...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/535G01N21/78
Inventor 张贺秋宋晓国陈坤王国华凌世淦
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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