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Hepatitis C virus envelope antigen sandwich method kit and detecting method

A technology of hepatitis C virus and envelope antigen, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve the problems of low magnification, false positives, and high cost

Active Publication Date: 2013-03-06
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitations of bDNA technology are also obvious, that is, low magnification, low sensitivity, narrow detection range, and not suitable for low-level detection of HCV RNA
Gene chip technology is suitable for studying the epidemiology, mutation trend, and transmission route of HCV. It is of great significance in judging the condition, guiding treatment, predicting the curative effect and prognosis of hepatitis C patients, but it is costly and prone to false positives

Method used

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  • Hepatitis C virus envelope antigen sandwich method kit and detecting method
  • Hepatitis C virus envelope antigen sandwich method kit and detecting method
  • Hepatitis C virus envelope antigen sandwich method kit and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] The configuration of the hepatitis C virus envelope antigen sandwich method kit:

[0173] a. ELISA plate (CellStar, purchased from Wuhan Dafeng Biotechnology Co., Ltd.;

[0174] b, biotin-labeled 12 peptides that can specifically bind to the HCV E2 protein;

[0175] c. The polypeptide is a sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4;

[0176] d, horseradish peroxidase-labeled streptavidin;

[0177] e, horseradish peroxidase;

[0178] f, ELISA (purchased from Nanjing Huadong Electronics Group Medical Equipment Co., Ltd.);

[0179] g, 2 protein as a standard positive control.

[0180] 1. The steps for preparing E2-GST fusion protein are as follows:

[0181] A. Pick the Escherichia coli BL21DE3 (purchased from American invitrogen company) were cultured in 3 ml of LB medium containing ampicillin (50 g / ml), and the control group was inoculated with pGEX-KG (prokaryotic expression vector of GST protein, purchased from Amersham Biosciences...

Embodiment 2

[0302] The steps of surface plasmon resonance (SPR) detection of affinity between polypeptide and E2 protein are as follows:

[0303] A. Let the test instrument Biocore (USA) pass through a period of HBS buffer until the baseline is stable.

[0304] B. Inject 40 μl of amino coupling reagent (containing 0.02M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS) ), to activate the carboxyl group on the CM5 sensor chip.

[0305] C. Dilute the E2 protein to 5 mg / ml with an acetate buffer solution with a pH value of 5.0, and inject 40 μl of the solution.

[0306] D. On-machine detection, the flow rate is 20 μl / min.

[0307] E. Dilute the polypeptides PE2A, PE2B, PE2C, and PE2D to different concentrations, and react with the E2 protein on the CM5 sensor chip at 2 μl / min for 7 minutes.

[0308] F. Inject 10 μl of HCl regeneration solution at a flow rate of 2 μl / min to restore the baseline, and collect response signals in real time.

[0309] G. ...

Embodiment 3

[0313] The steps for flow cytometry to detect the combination of polypeptides and target molecules are as follows:

[0314] A. Culture human liver cancer cells Huh7.5 (see reference Zhong, et al., Robust hepatitis C virus infection in vitro, 2005, PNAS, 102:9294-9299) and DC-SIGN + - NIH3T3 (mouse fibroblasts with DC-Sign molecules on the surface) cells (see reference Wu, et al., Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGNinteractions with ICAM-3 do not promote human immunodeficiency virustype I transmission. J. virus. 2002, 76: 5905-5914). The culture conditions were all DMEM medium (purchased by Gibco) with 10% fetal bovine serum, cultured at 37° C. in an incubator containing 5% carbon dioxide.

[0315] B. Mix the peptides PE2A, PE2B, and PE2D with the E2-GST protein respectively (GST protein is added to the control tube), the total volume is 50 μl, the concentration of the polypeptide is 500 μM, and the concentration of E2-GST protein and GST pro...

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Abstract

The invention discloses a kit for detecting a hepatitis C virus envelope antigen by a sandwich method and a detection method thereof. The kit comprises an ELISA plate, three polypeptides which are capable of being in specific binding with HCV E2 protein, three polypeptides which are labeled by biotin and capable of being in specific binding with the HCV E2 protein, and streptavidin labeled by horseradish peroxidase. A method for preparing the kit for detecting the hepatitis C virus envelope antigen by the sandwich method comprises the following steps: firstly, coating the polypeptides; then adding blood serum of a patient to be detected; thirdly, adding the polypeptides labeled by the biotin; fourth, adding the streptavidin labeled by the horseradish peroxidase; and fifth, adding o-phenylenediamine for color development and reading an OD value by an ELISA reader at OD450nm. The polypeptides and labels thereof are applied to the kit for detecting the hepatitis C virus envelope antigen. The kit can be widely used in medicine-related fields and the like.

Description

technical field [0001] The invention belongs to the technical field of hepatitis C detection. More specifically, it relates to a sandwich method kit with hepatitis C virus envelope antigen. At the same time, the present invention also relates to a method for detecting hepatitis C virus with the hepatitis C virus envelope antigen sandwich method kit, as well as polypeptides and biological The application of the labeled polypeptides such as peptides as a sandwich method kit for detecting hepatitis C virus surface envelope antigen can be widely used in medicine and other related fields. Background technique [0002] Hepatitis C virus (hepatitis C for short) is an inflammatory disease of the liver caused by hepatitis C virus (Hepatitis C Virus, HCV). HCV infection has a global distribution and is mainly transmitted through blood. According to the World Health Organization, the global HCV infection rate is about 3%, an estimated 170 million people are infect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/576G01N33/543
Inventor 章晓联
Owner WUHAN UNIV