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Hepatitis C virus envelope antigen ELISA kit and detecting method

A technology of hepatitis C virus and envelope antigen, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of low magnification, false positive, high cost, etc.

Inactive Publication Date: 2009-06-17
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 ELISA kit and detecting method
  • Hepatitis C virus envelope antigen ELISA kit and detecting method
  • Hepatitis C virus envelope antigen ELISA kit and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Configuration of Hepatitis C virus envelope antigen kit:

[0182] a. 12 peptides labeled with biotin and the like that can specifically bind to the HCV E2 protein;

[0183] b. The polypeptide is a sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4;

[0184] c. Rabbit polyclonal antibody against E2;

[0185] d. horseradish peroxidase-labeled streptavidin;

[0186] E. horseradish peroxidase substrate;

[0187] f. Microplate plate (CellStar@, purchased from Wuhan Dafeng Biotechnology Co., Ltd.), microplate reader;

[0188] g. E2 protein as a standard positive well;

[0189] h. Enzyme-linked immunoassay instrument (purchased from Nanjing Huadong Electronics Group Medical Equipment Co., Ltd.).

[0190] The steps of a polypeptide PF2A, PE2B, PE2C, PE2D are as follows:

[0191] 1. The steps for preparing E2-GST fusion protein are as follows:

[0192] A. Pick the Escherichia coli containing pGEX-KG-E2 (see reference Li, et al., Engineering of N-glycosylation o...

Embodiment 2

[0317] The steps of surface plasmon resonance (SPR) detection of affinity between polypeptide and E2 protein are as follows:

[0318] A. Let the test instrument Biocore (USA) pass through a period of HBS buffer until the baseline is stable.

[0319] 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.

[0320] 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.

[0321] D. On-machine detection, the flow rate is 20 μl / min.

[0322] 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.

[0323] 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.

[0324] G. Us...

Embodiment 3

[0328] The steps for flow cytometry to detect the combination of polypeptides and target molecules are as follows:

[0329] 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 (fibroblasts from mice with DC-Sign molecules on their 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.

[0330] B. Mix the polypeptides 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 ...

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Abstract

The invention discloses an ELISA kit for a hepatitis C virus envelope antigen and a detection method thereof. The ELISA kit comprises an ELISA plate, three polypeptides (12-peptide ZA, 12-peptide ZB and 12-peptide ZD) which are labeled by biotin and capable of being in specific binding with HCV E2 protein, a rabbit anti-E2 polyclonal antibody and streptavidin labeled by horseradish peroxidase. A method of the kit for detecting the hepatitis C virus envelope antigen comprises the following steps: firstly, coating the rabbit anti-E2 polyclonal antibody; 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. Meanwhile, the polypeptides can also be used for preparing a medicine for treating or preventing the hepatitis C virus infection. The ELISA 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 detection ELISA kit for hepatitis C virus envelope antigen. Meanwhile, the present invention also relates to a method for detecting hepatitis C virus envelope antigen and virus with the hepatitis C virus envelope antigen kit. The polypeptide and the E2 antibody can be widely used in related fields such as medicine and biology as an enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) for detecting the surface envelope antigen of hepatitis C virus. 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 infected wit...

Claims

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Application Information

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IPC IPC(8): G01N33/576G01N33/543
Inventor 章晓联李冬青俞晶
Owner WUHAN UNIV
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