Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies

a technology of anti-hepatitis c virus and kit, which is applied in the field of virus diagnostics, can solve the problems of affecting human health, bringing heavy burden to the bur society, and not satisfying sensitivity and specificity, so as to reduce the activity of hcv antigens, deactivate enzymes, and influence the structure of enzymes

Inactive Publication Date: 2008-08-14
CUI PENG
View PDF6 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0065]B, If the secondary antigens and labels are added at the same time or previously well-mixed then added for the incubation, and if the reductant is not added into the diluting solutions, the activity of HCV antigens decreases. If adding reductant, the activity of enzyme label will be destroyed, because most enzymes contain natural disulfide bonds, thus adding the reductant destroys the natural disulfide bonds, and further influences the structure of enzymes, and deactivates the enzymes. During the present testing procedure, the method of adding the secondary antigens and labels into the reacting system then incubating together, and then the adding of sulfhydryl reagents into the secondary antigen diluting solution, solves this conflict.
[0067]B, During the procedure of successively adding sample to be tested and the secondary antigens in two separate steps into the reacting system then incubating, if reductant is not added into the sample diluting solutions, the IgM antibodies will be catched by the primary antigens in the form of pentamer; and after adding the secondary antigens, in order to increase the activity of secondary antigens the reductant is added into their diluting solutions, the disulfide bonds connecting the IgM pentamer will be broken, causing part of secondary antigens to bind dissoc...

Problems solved by technology

So HCV harms human's healthy and brings heavy burden to bur society.
The primitive Elisa-1 kits were developed by Ortho Diagnosis Company (U.K), the antigen was recombinant HCV NS4 antigen C100-3 which was supplied by Chiron Company (U.S.A), but its sensitivity and specificity are not very satisfied.
Unfortunately even Elisa-3 has many disadvantages, false positive, false negative and uncertain results still exist.
1. Poor specificity. Non-specific binding labeled second antibody (SA) was employed in the indirect method, which lead to low specificity.
Because of poor specificity, the concentration of coating antigen and labeled SA must be low, thus the sample volume has to be reduced and the dilution multiples should be higher, resulting even lower sensitivity.
Presently to conjugate the antigen with main label such as peroxidase horseradish (HRP) is the dominant enzyme labeling method, but there are some problems when this method is applied for HCV antigen.
First, the NS3 antigen as the main antigen epitope area of HCV, is the one strongly depending on its confo...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies
  • Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies
  • Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 2

Preparation of Secondary Antigen and Enzyme Label

1. The Preparation of Biotinylation Secondary Antigen

[0082]We amplify the gene encoding HCV antigen a.a.1-28 (the 1st to 84th nucleotide in SEQ ID NO.1), the forward primer thereof has BamHI site, and the reserve one has BgIII and EcoRI sites between which there is a termination codon TAA. Then we amplify segment L (SEQ ID NO.3) encoding biotin acceptor protein by PCR, sense primer thereof has BamHI site, and anti-sense has BgIII site and EcoRI site between which there is a termination codon TAA.

[0083]Gene coding a.a.1-28 cleaved by restriction endonuclease BamHI and EcoRI is cloned into P2-G expression vector cleaved by BgIII and EcoRI endonuclease, the resulting positive clone P2-G-CORE (a.a.1-28) is named P2-HDVAg2, and the antigen expressed is named as HCVAg2. Clone gene L cleaved by BgIII / EcoRI into P2-G-CORE (a.a.1-28) treated by same endonucleases, we can obtain the positive clone P2-G-CORE (a.a.1-28)-L, named P2-X.

[0084]We ext...

example 3

SELECTION OF THE CONCENTRATION OF REDUCING AGENT

[0091]First, respectively use HCVAg1 and HCVAg as coating antigen, add different final concentration of β-mercaptoethanol in coating buffer, and detect using indirect method, (data in tab 1). We get similar result by adding different final concentration of β-mercaptoethanol in blocking buffer. The results show that reducing agent can increase HCV antigenic activity to a certain extent, indicating that there have some mismatching disulfide bond in the HCV antigen. The reducing agent can break up the mismatching disulfide bond of HCV antigen, and restore the natural conformation of the HCV antigen. The β-mercaptoethanol optimization concentration range is 0.5-5‰, prefers 1‰.

TABLE 1The effect of β-mercaptoethanol in coatingbuffer to sensitivity of HCV indirect kitfinal concentration of20‰10‰5‰2‰1‰0.5‰0.2‰0.1‰0β-mercaptoethanol incoating bufferaverage OD mean5.105.625.896.316.546.195.785.125.20value / Co of thepositive serumsIndication: Cut ...

example 4

PRODUCTION AND TEST OF THE PRESENT INVENTION KIT

1. Using Biotin Labeled HCV Antigen as the Second Antigen in the Kit

[0099]Dilute the HCV coating antigen with bicarbonate buffer (50 mM, pH9.51, contains 1‰β-mercaptoethanol (Sangon, Article No. M0482)) by a certain ratio, add 100 ul to each well of Polystyrene plate (Shenzhen jincanhua industry Co., Ltd irradiation Polystyrene microliter), Tween-20, pH7.4) washing buffer to wash it twice, then we desiccate the wells by patting. Add 120 ul pH7.4, 10 mM PB blocking buffer into each well, which contains 30% fetal calf serum (Beijing Yuanhengshenma Biotechnology Research Institute), 8% sucrose, 5‰casein (America Sigma-Aldrich Inc., article no. C-8645), 1‰β-mercaptoethanol (Sangon, Article No. M0482), 150 mM NaCl, and then block for 2 hours at 37° C. We then discard liquid in wells and desiccate the wells by patting. After dried by air in room equipped with draft equipment where temperature is 20-25° C. and humidity is 55%-65%, the plate i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention provides a kit for the detection of anti-hepatitis C virus (HCV) antibodies using a double-antigen sandwich technique, said kit carries out the detection by the process ‘solid support-first antigen-HCV antibody to be detected-second antigen-enzyme linked substance-recognizable signal’, wherein one or more coupling reactions between the second antigen and the enzyme linked substance occur, and the diluent for the second antigen and that for the enzyme linked substance are respectively placed into two container, they are added in order in two steps to reaction system and incubated respectively. The present invention can solve the following problems encountered by prior art: 1) the influence of the enzyme linked substance on the activity of the second antigen; 2) the mismatch of disulfide bonds in second antigen, thereby enhance the activity of the second antigen and obviate loss of enzyme activity, therefore, the present kit highly improves the sensitivity and specificity of the detection.

Description

TECHNICAL FIELD[0001]The present invention pertains generally to virus diagnostics. In particular, this invention relates to HCV antibody diagnostic kit and its preparation method.BACKGROUND[0002]Hepatitis C is a serve hepatic disease attributed to infection with hepatic C virus (HCV). About 170 million patients are infected with HCV over the world and in China this number has exceeded 40 million. 50˜85% of HCV-infected individuals subsequently develop to chronic hepatitis and 10˜15% of them will be deteriorated to hepatocirrhosis. So HCV harms human's healthy and brings heavy burden to bur society.[0003]HCV Diagnosis got much progress since the HCV gene was cloned by Choo et al in 1989. Now there are 3 main approaches for Diagnosis of HCV: detecting the HCV RNA by polymerase chain reaction (PCR); detecting the HCV antigens using monoclonal antibody; diagnosing the HCV antibody with ELISA and Western blotting.[0004]ELISA reagents for diagnosis of HCV have developed into the third ge...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N33/00
CPCG01N33/56983
Inventor CUI, PENG
Owner CUI PENG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap