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

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...

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Benefits of technology

The Main Advantages of this Invention:

[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.

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 conformation, and when labeled by a “flexible” label with high molecular weight such as peroxidase horseradish or alkaline phosphatase it varied greatly in conformation, and its active epitopes are difficult to expose.

So the activity of CORE antigen is decreased after labelling, and as a result the sensitivity of the kits produced as the above-mentioned are even less than those of indirect methods, leading to the mis-diagnosis seriously.

But the mention sandwich method just for using NS3 antigen in the three patents, and have not research the effect of the activity of CORE antigen by adding sulfhydryl reagent.

In America patent U.S. Pat. No. 6,630,298 introduce method, secondary antigen and SOD antibody of enzymatic label secondary antigen exogenous protein are as mixture to add in reaction in one step, their disadvantages are: 1, the combination of HCV antigen and high molecular weight enzymatic antibody will result to certain conformation destroy and epitope reveal not easy, and cause the activity loss of secondary antigen, and then the detecting sensitivity is lower.

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