Patent Description:
Hepatitis C is a blood-borne disease caused by hepatitis C virus (HCV). Chronic HCV infection may lead to chronic liver inflammation and necrosis and liver fibrosis, and some patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC), which is extremely harmful to the health and life of patients and has become a serious social and public health problem. Hepatitis C is a global epidemic and is the leading cause of end-stage liver diseases in Europe, United States, Japan, etc. In the United States, the number of HCV-infected people is four times that of HIV-infected people. By <NUM>, more people died from HCV infection than from HIV infection. According to the World Health Organization, the global infection rate of HCV is about <NUM>%, and it is estimated that about <NUM> million people are infected with HCV, and about <NUM>,<NUM> new hepatitis C cases are diagnosed each year.

At present, there is no effective vaccine against hepatitis C, so the control of hepatitis C can only begin with the control of the source of infection and the transmission route. Therefore, early and accurate diagnosis and detection of HCV infection is the most effective means to prevent and control the source of hepatitis C infection and block the transmission route, and immunoassay is one of the main methods for the diagnosis and management of hepatitis C virus.

HCV immunoassays include enzyme-linked immunosorbent assay, chemiluminescence immunoassay, gold-labelled antigen detection method, fluorescence immunoassay, etc. Among them, enzyme immunoassay techniques such as enzyme-linked immunosorbent assay and enzymatic chemiluminescence immunoassay are common HCV immunoassay methods using enzymes for labelling as reporter molecules. In reaction mode of the double-antigen sandwich assay, in the enzyme immunoassay, conjugate of enzyme for labelling and antigen substance, antibody to be detected in sample and antigen substance coated on solid phase support form a sandwich structure, and a qualitative or quantitative detection result of HCV is obtained by analyzing the enzyme for labelling in the sandwich structure. In reaction mode of the immunocapture assay, in the enzyme immunoassay, conjugate of enzyme for labelling and antigen substance, antibody to be detected in sample and anti-human IgG and anti-human IgM antibodies coated on solid phase support form a complex. , a qualitative or quantitative detection result of HCV is obtained by analyzing the enzyme for labelling in the complex.

Generally, in the HCV enzyme immunoassay based on the double-antigen sandwich assay or the immunocapture assay, chemically activated cross-linking method is used to link the enzyme for labelling with the antigen substance to form a crosslinked compound or a conjugate. However, this link process has the disadvantages of being complicated to operate and difficult to control; in addition, such linking manner produce a non-uniform molecular molar ratio of the enzyme to the antigen, resulting in the product being a mixture of enzyme-antigen conjugates with different molecular molar ratio. Therefore, the application of the mixture of enzyme-antigen conjugates prepared by the chemical cross-linking method in the detection of hepatitis C virus antibody leads to problems such as difficult production control of hepatitis C detection kits and large batch-to-batch variation of test results.

<NPL>) discloses an ELISA system based on a sandwich method, comprising use of genetic engineering to express fusion protein of HCV chimerical antigens in E.

<CIT> discloses a kit for detection of hepatitis c virus antibody and a method for the detection, using HCV fused antigens.

<CIT> discloses a fusion protein of chimeric HCV antigens HCV-C, NS3 and NS4 chemically connected with horseradish peroxidase.

<CIT> discloses a kit comprising HCV antigen core peptides or NS3 peptide coupled by SMASA functional group to horseradish peroxidase.

In order to solve the problems above, embodiments of the present application provide an immunoassay kit for HCV antibody, including a fusion protein of an enzyme for labelling and an HCV antigenic protein.

As used in the embodiments of the present application, "antigenic protein" or "antigen substance" refers to a protein that is immunoreactive and can be used in HCV immunological detection; it can be one HCV antigen or a fragment thereof, or a fusion antigen of two or more HCV antigens or fragments thereof (or a chimeric protein of two or more HCV antigens or fragments thereof).

In the embodiments of this application, the antigenic protein contains one or more of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen, or the antigenic protein contains a fusion antigen of two or more of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen. Core, NS3, NS4, and NS5 are the structural and membrane proteins of hepatitis C virus. A large number of studies have shown that Core, NS3, NS4, and NS5 not only have high immunogenicity, but also can be used as hepatitis C virus diagnostic antigens.

As used in the embodiments of the present application, "HCV antigen" refers to a substance that has immunoreactivity and can be used in HCV immunoassay, and is selected from HCV conserved protein or a fragment thereof. Exemplary HCV antigen can be HCV core antigen, HCV NS3 antigen, HCV NS4 antigen or HCV NS5 antigen. In the embodiments of the present application, the HCV antigen may exist in the form of one or more copies.

In some embodiments, the HCV antigenic protein of the present application contains HCV core antigen. For example, the HCV antigenic protein of the present application contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen.

In some embodiments, the antigenic protein is a chimeric protein or a fusion antigen containing HCV core antigen and one or more additional HCV antigens. Wherein, the additional HCV antigen may be a highly immunoreactive HCV antigen, for example, one or more of HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen.

In an exemplary embodiment, the HCV antigenic protein of the present application is a chimeric protein containing HCV core antigen and HCV NS3, or a chimeric protein containing HCV core antigen, HCV NS3 antigen and HCV NS4, or a chimeric protein containing HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen.

When the antigenic protein contains two or more HCV antigens, these HCV antigens may be present in any order. In an exemplary embodiment, the antigenic protein of the present application may contain HCV core antigen and HCV NS3 antigen in the order from N-terminus to C-terminus; alternatively, the antigenic protein of the present application may contain HCV NS3 antigen and HCV core antigen in the order from N-terminus to C-terminus.

In a specific embodiment, the HCV antigenic protein of the present application contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen in the order from N-terminus to C-terminus.

In a specific embodiment, the antigenic protein of the present application contains HCV NS3 antigen, HCV NS4 antigen, HCV core antigen and HCV NS5 antigen in the order from N-terminus to C-terminus.

In a specific embodiment, the antigenic protein of the present application contains HCV NS3 antigen, HCV core antigen, HCV NS4 antigen and HCV NS5 antigen in the order from N-terminus to C-terminus.

In the embodiments of the present application, the HCV antigens may be directly linked with each other or may be linked through a Linker with each other, as long as the HCV antigens are linked while ensuring that their respective structures and activities are not affected. In the embodiments of the present application, a flexible linker may be used, such as flexible (Gly<NUM>Ser)n, GGGS, GGSGGGSG, and the like.

In the embodiments of the present application, "enzyme" and "enzyme for labelling" may be used interchangeably, and refer to an enzyme used in enzyme immunoassays. For example, it may be an enzyme used in enzyme-linked immunosorbent assay and enzymatic chemiluminescence immunoassay.

In the present invention, the enzyme is alkaline phosphatase (EC <NUM>. <NUM>), which, for example, can catalyze the hydrolysis of phosphate group-containing chromogenic substrates and chemiluminescent substrates such as nitrophenyl phosphate (PNP), sodium β-glycerophosphate, naphthyl phosphate, <NUM>-(<NUM>-spiroadamantane)-<NUM>-methoxy-<NUM>-(<NUM>-phosphoryloxy)-phenyl-<NUM>,<NUM>-dioxetane (AMPPD).

The alkaline phosphatase in the embodiments of the present application may be naturally occurring, artificially synthesized or produced by genetic engineering. In addition, the alkaline phosphatase in the embodiments of the present application may be a modified, such as surface glycosylated or deglycosylated alkaline phosphatase.

The source of alkaline phosphatase is not particularly limited in the embodiments of the present application, as long as the enzyme immunoassay can be realized. Exemplary alkaline phosphatase can be derived from but not limited to bacteria, such as E. coli; mammals such as cattle (for example Genebank: AF052227. <NUM> (https://www. gov/)) or human (for example Genebank: M12551. <NUM>); and shrimp.

In another embodiment of the invention, the enzyme is horseradish peroxidase (EC <NUM>. <NUM>), which uses iron porphyrin as a prosthetic group and can catalyze the polymerization of phenol, aniline and their substitutes in the presence of hydrogen peroxide, and is widely distributed in the plant kingdom, with the highest content in horseradish.

The horseradish peroxidase in the embodiments of the present application may be naturally occurring, artificially synthesized or produced by genetic engineering. In addition, the horseradish peroxidase in the embodiments of the present application may be modified.

In some embodiments, the enzyme of the present application also includes mutants thereof. Compared with wild type, the mutants of the enzymes in the embodiments of the present application have more than <NUM>%, optionally more than <NUM>%, more than <NUM>%, more than <NUM>%, more than <NUM>% or more than <NUM>% sequence homology. An exemplary alkaline phosphatase mutant may be GeneBank: M29670. <NUM> (https://www. gov/), but the embodiments of the present application are not limited thereto. An exemplary horseradish peroxidase mutant may be GeneBank: XM_018585035. <NUM>, but the embodiments of the present application are not limited thereto.

Sequence homology is determined by comparing a test sequence to a reference sequence by using a wild-type sequence as the reference sequence. The sequence homology of the test sequence relative to the reference sequence is then calculated using a sequence comparison algorithm. Two examples of algorithms suitable for determining sequence homology are the BLAST and BLAST <NUM> algorithms, described in <NPL>, and <NPL>, respectively. Software for performing BLAST analysis is publicly available through NCBI.

In the present invention, the enzyme is fused to any position of the antigenic protein. The enzyme may be fused to the N-terminus of the HCV antigenic protein; alternatively, the enzyme may be fused to the C-terminus of the antigenic protein; alternatively, the enzyme may be fused between any two adjacent antigens in the antigenic protein.

In some embodiments, the fusion protein of the HCV antigenic protein and the enzyme contains enzyme, HCV core antigen, HCV NS3 antigen, and HCV NS4 antigen in the order from N-terminus to C-terminus.

In some embodiments, the fusion protein of the HCV antigenic protein and the enzyme contains enzyme, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen in the order from N-terminus to C-terminus.

In some embodiments, the fusion protein of the HCV antigenic protein and the enzyme contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and enzyme in the order from N-terminus to C-terminus.

In some embodiments, the fusion protein of the HCV antigenic protein and the enzyme contains HCV core antigen, HCV NS3 antigen, enzyme, HCV NS4 antigen, and HCV NS5 antigen in the order from N-terminus to C-terminus.

In a specific embodiment, the fusion protein of the present application contains, in the order from N-terminus to C-terminus, alkaline phosphatase, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen; or the fusion protein of the present application contains, in the order from N-terminus to C-terminus, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and alkaline phosphatase; alternatively, the fusion protein of the present application contains, in the order from N-terminus to C-terminus, HCV core antigen, HCV NS3 antigen, alkaline phosphatase, HCV NS4 antigen and HCV NS5 antigen; alternatively, the fusion protein of the present application contains, in the order from N-terminus to C-terminus, horseradish peroxidase, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen; alternatively, the fusion protein of the present application contains, in the order from N-terminus to C-terminus, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and horseradish peroxidase; alternatively, the fusion protein of the present application contains, in the order from N-terminus to C-terminus, HCV core antigen, HCV NS3 antigen, horseradish peroxidase, HCV NS4 antigen and HCV NS5 antigen.

In a specific embodiment, the immunoassay kit may include at least one fusion protein of: a fusion protein of alkaline phosphatase and HCV core antigen, a fusion protein of alkaline phosphatase and HCV NS3 antigen, a fusion protein of alkaline phosphatase and HCV NS4 antigen, and a fusion protein of alkaline phosphatase and HCV NS5 antigen; or the immunoassay kit may include at least one fusion protein of: a fusion protein of horseradish peroxidase and HCV core antigen, a fusion protein of horseradish peroxidase and HCV NS3 antigen, a fusion protein of horseradish peroxidase and HCV NS4 antigen, and a fusion protein of horseradish peroxidase and HCV NS5 antigen.

In the embodiments of the present application, the enzyme may be directly linked to the antigenic protein or may be linked to the antigenic protein through a Linker, as long as the enzyme and the antigenic protein are linked while ensuring that their respective structures and activities are not affected. In the embodiments of the present application, a flexible linker is used, such as flexible (Gly<NUM>Ser)n, GGGS, or GGSGGGSG.

A "fusion protein" refers to a fusion protein of an enzyme and an antigenic protein. For example, it may be expressed as enzyme + core + NS3, in this case, it refers to a fusion protein of enzyme, HCV core antigen and HCV NS3 antigen in the order from N-terminus to C-terminus.

The fusion protein of the enzyme and the HCV antigenic protein may be prepared by conventional recombinant expression technology. In the embodiments of the present application, the recombinant expression technology may be prokaryotic expression technology, such as E. coli expression technology, etc.; and eukaryotic expression technology, such as yeast expression technology, insect cell expression technology, etc..

Those skilled in the art would understand that the kit in the embodiments of the present application may further include other reagents or components for the detection of HCV antibody based on double antigen sandwich assay or immunocapture assay, for example, include a solid phase support coated with an HCV antigenic protein (the HCV antigenic protein coated on the solid phase support and the HCV antigenic protein in the fusion protein can bind to a same HCV antibody in a sample) or a solid phase support coated with anti-human IgM antibody and anti-human IgG antibody; a calibrator for drawing a standard curve; a quality control for controlling quality; a substrate solution for chemiluminescence reaction; and/or a wash buffer and a sample diluent, and the like.

In some embodiments, the kit includes a solid phase support coated with a fusion antigen containing HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen; alternatively, the kit includes a solid phase support coated with HCV core antigen, a solid phase support coated with HCV NS3 antigen, a solid phase support coated with HCV NS4 antigen, and a solid phase support coated with HCV NS5 antigen.

On the other hand, the embodiments of the present application (but not part of the present invention) also relate to the use of a fusion protein of an enzyme and an HCV antigenic protein in the manufacture of an immunoassay kit for detecting HCV. The immunoassay kit includes:.

In a specific instance, HCV is detected based on double antigen sandwich assay or immunocapture assay.

In a variation of the disclosure, an immunoassay kit for detecting HCV is provided, and the kit includes:.

Those skilled in the art can understand that the HCV antigenic protein coated on the solid phase support in the first reagent and the HCV antigenic protein fused in the fusion protein in the second reagent are of the same type, but the source or the linking manner or sequence between multiple antigens may be the same or different. For example, the HCV antigenic protein coated on the solid phase support may be a fusion antigen of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen linked in order, and the HCV antigenic protein fused in the fusion protein in the second reagent may be a fusion antigen of HCV core antigen, HCV NS4 antigen, HCV NS5 antigen and HCV NS3 antigen linked in order.

In addition, in the embodiments of the present application, the HCV antigenic protein coated on the solid phase support may be the HCV antigenic protein described above, or may be an HCV antigenic protein linked in other ways, such as linked chemically.

In the embodiments of the present application, an HCV antibody refers to an antibody produced in a subject after HCV infection, such as anti-HCV IgG antibody and anti-HCV IgM antibody.

In another variation of the embodiments of the present application, an immunoassay kit for detecting HCV is provided, and the kit includes:.

As used in the embodiments of the present application, "solid phase support" refers to a solid surface to which an antigen or an antibody can be attached. There is no particular limitation on the solid phase support used in the embodiments of the present application, and commercial solid phase supports and any solid phase supports that can be used in immunoassay can be used in the present application. Exemplary solid phase supports may be magnetic beads (such as superparamagnetic microspheres), ELISA plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates or microchips, but the embodiments of the present application are not limited thereto.

In the embodiments of the present application, the solid phase coating may be contained in a conventional diluent containing proteins and surfactants and having buffer capacity.

In the embodiments of the present application, the enzyme label conjugate may be contained in a conventional diluent containing a protein and a surfactant and having buffer capacity.

In the embodiments of the present application, the fusion protein may be contained in the second reagent, for example, at a concentration of about <NUM> ng/mL to <NUM> ng/mL.

In a specific embodiment, the kit of the present application may further include a third reagent, which contains a blocking agent and a surfactant. For example, the blocking agent includes one or more components selected from the group consisting of: skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, a surfactant (for example: Tween-<NUM>, Tween-<NUM>, TritonX-<NUM>, etc.).

In a specific embodiment, the kit of the present application may further include a fourth reagent, which contains a reducing agent. For example, the reducing agent includes one or more components selected from the group consisting of: DTT and β-mercaptoethanol.

In the embodiments of the present application, the blocking agent and the surfactant may be dissolved in a conventional diluent with buffering capacity; the reducing agent may be dissolved in a conventional diluent with buffering capacity.

In some embodiments, the kit may further include a reaction substrate for the enzyme for labelling, preferably the reaction substrate is <NUM>-(<NUM>-spiroadamantane)-<NUM>-methoxy-<NUM>-(<NUM>-phosphoryloxy)-phenyl-<NUM>,<NUM>-dioxetane.

Unless otherwise specified, the terms "first", "second", "third" and "fourth" in the embodiments of the present application are only used to distinguish multiple similar elements, and not intended to indicate any difference in importance or order between the elements.

The present invention further relates to a method for detecting an HCV antibody produced after infection with HCV in a sample by using a fusion protein of an enzyme for labelling and a HCV antigenic protein, and the method includes the following steps:.

The first ligand is HCV antigenic protein, or anti-human IgG antibody and anti-human IgM antibody.

In the embodiments of the present application, a fusion protein of an enzyme for labelling and an HCV antigenic protein is obtained by recombinant expression, and the fusion protein can be applied to an HCV immunoassay kit based on double-antigen sandwich assay or immunocapture assay. It should be noted that:
by replacing conjugate of enzyme and antigen substance produced by chemically activated cross-linking with such a fusion protein, the fusion protein of the enzyme for labelling and the HCV antigenic protein in the kit has a uniform molar ratio of the enzyme for labelling to the HCV antigenic protein, thereby avoiding the problem of large batch-to-batch differences in kit production and test results.

On the other hand, compared with a conventional kit for double antigen sandwich assay or immunocapture assay, the kit in the embodiments of the present application shows better discrimination between negative and positive samples, and has a better sample coincidence rate.

On the other hand, the kit including the fusion protein in the embodiments of the present application avoids the destruction of the activity of the antigen or the activity of the enzyme caused by chemically activated cross-linking method.

On the other hand, the embodiments of the present application omit the step of chemical cross-linking reaction, which makes operation more convenient and faster.

The technical solutions of the embodiments of the present application will be described below clearly and comprehensively.

As mentioned above, the fusion protein of the enzyme and the HCV antigenic protein in the embodiments of the present application may be produced by recombinant expression technology.

An exemplary recombinant expression method may include the following steps:.

In the steps above, the nucleic acid molecule encodes the fusion protein above; the expression vector is used to prepare the fusion protein of the enzyme and the HCV antigenic protein by recombinant expression, the expression vector includes a nucleic acid molecule encoding the fusion protein above and operably linked to an expression control sequence, and additionally includes genetic elements for maintenance and propagation in the respective host cells, such as origins of replication and/or selectable marker genes.

Production of the fusion protein using an E. coli expression system:.

Nucleic acid sequence information of HCV antigen: Core, GeneBank; AJ222676. <NUM>; NS3, GeneBank: EU84764. <NUM>; NS4, GeneBank: Z84363. <NUM>; NS5, GeneBank: S70341. <NUM> (https://www.

<NUM> of "magnetic beads coated with Core+NS3+NS4+NS5 chimeric protein" was measured off using a pipette or graduated cylinder and added to a magnetic bead-coated tube to replace a supernatant, that is, after magnetic separation was completed, the supernatant suctioned off and then an equal volume (<NUM>) of a magnetic bead coating diluent was added and mixed evenly; the magnetic beads were mixed evenly and then added to a liquid preparation bottle containing <NUM> of the magnetic bead coating diluent; and the magnetic bead suspension was stirred until completely mixed to prepare the first reagent Ra, where the concentration of the magnetic beads coated with the Core+NS3+NS4+NS5 chimeric protein is <NUM>/mL; and the magnetic bead coating diluent is a conventional diluent with buffering capacity and contains a protein and a surfactant.

<NUM> of a tracer diluent is measured off using a suitable graduated cylinder and added to a liquid preparation bottle, <NUM> of "a fusion protein of an enzyme and an HCV antigenic protein" or "a conjugate of an enzyme and an HCV antigenic protein linked chemically" or "a conjugate of anti-human IgG and anti-human IgM linked chemically" was pipetted as an enzyme label conjugate using a pipettor and added into the tracer diluent; the solution was stirred with a stirrer to fully dissolve and mix evenly; then the prepared solution was filtered using a suitable filter with a pore size of <NUM>, and the filtrate was collected, to prepare the tracer Rb, where the concentration of the above enzyme label conjugate is <NUM> ng/mL; and the tracer diluent is a conventional diluent with buffering capacity and contains a protein and a surfactant.

Third reagent Rc:
a diluent with buffering capacity and containing BSA, Tween-<NUM> and skimmed milk powder.

Fourth reagent Rd:
a diluent with buffering capacity and containing DDT.

Step <NUM>: the sample, the fourth reagent and the first reagent were added to a reaction tube and incubated at <NUM> for <NUM> minutes, so that the HCV antigen coated on the magnetic bead solid phase were fully bound to anti-HCV IgG and anti-HCV IgM antibodies in the sample; and after incubation was complete, the magnetic bead solid phase was placed in a magnetic field and attracted, the substances bound to the magnetic bead solid phase were retained, and other unbound substances were washed off.

Step <NUM>: the third reagent and the second reagent were added to the reaction tube and mixed evenly; After incubation at <NUM> for <NUM> minutes, the HCV antigen on the enzyme label conjugate was bound to the anti-HCV IgG and anti-HCV IgM antibodies captured on the magnetic beads to form a sandwich complex. After incubation in the reaction tube was complete, the complex was attracted by the magnetic field, and other unbound substances were washed off.

Step <NUM>: a chemiluminescent substrate was added to the reaction tube to generate chemiluminescence; and the number of photons produced by the reaction was then measured by a photomultiplier to obtain a chemiluminescence signal value of the sample.

In the embodiments of the present application, COI (Cutoff index) is the ratio of the chemiluminescence signal value (RLU) of the measured sample to a threshold value (Cutoff value), where COI ≥ <NUM> indicates that the measured sample is a positive sample, and COI < <NUM> indicates that the measured sample is a negative sample. For qualitative detection methods, the threshold value (cutoff value) is a cut-off value for judging whether the test result is positive or negative.

In the embodiments of the present application, the negative coincidence rate refers to the ratio of the number of samples determined to be negative using the test method according the embodiments of the present application to the negative samples actually involving in the evaluation, and the positive coincidence rate refers to the ratio of the number of samples determined to be positive using the test method according to the embodiments of the present application to the positive samples actually involving in the evaluation; the true negative and positive results of the samples came from hospital diagnoses.

In order to compare the HCV detection effect of a fusion protein of an enzyme and an HCV antigenic protein in the embodiments of the present application relative to that of a conjugate of the enzyme and the HCV antigenic protein produced by chemical cross-linking, the following three reagent combinations were prepared.

Combination <NUM>: utilizing the E. coli expression system described above, a Core+NS3+NS4+NS5 chimeric protein (or fusion antigen) was used as an HCV antigenic protein to fuse with alkaline phosphatase to prepare the second reagent Rb, where in the fusion protein of the second reagent Rb, alkaline phosphatase (Genebank: AF052227. <NUM> (https://www. gov/)) was fused at N-terminus of the Core+NS3+NS4+NS5 chimeric protein, and the Combination <NUM> was prepared according to the above "Reagent preparation method".

Combination <NUM>: the second reagent Rb was prepared using a conjugate of alkaline phosphatase and Core+NS3+NS4+NS5 chimeric protein linked chemically, and the rest was the same as combination <NUM>.

Next, <NUM> confirmed HCV negative samples and <NUM> confirmed positive samples from general hospitals were tested using combinations <NUM> and <NUM> according to the "Double-antigen sandwich assay method" described above, respectively.

It can be seen from Table <NUM> that the negative coincidence rate of combination <NUM> is <NUM>% and the positive coincidence rate is <NUM>%, which is higher than the conventional double antigen sandwich assay using chemical linking (combination <NUM>: negative coincidence rate is <NUM>%, positive coincidence rate is <NUM>%), and this result shows that: using the fusion protein of alkaline phosphatase and Core+NS3+NS4+NS5 can avoid the destruction of antigen activity or alkaline phosphatase activity caused by the preparation process using chemical linking method in the conventional double antigen sandwich assay. In the embodiments of the present application, both the alkaline phosphatase and the antigen in the fusion protein of alkaline phosphatase and Core+NS3+NS4+NS5 maintain high activity.

In addition, in order to prove the activity of the Core+NS3+NS4+NS5 chimeric protein used in the embodiments of the present application, reagent combination <NUM> was prepared: the second reagent Rb was prepared using a conjugate of alkaline phosphatase and anti-human IgG and anti-human IgM linked chemically, and the rest was the same as combination <NUM>.

According to the indirect assay method, the <NUM> confirmed HCV negative samples and the <NUM> confirmed positive samples above were tested using the combination <NUM> above. The specific steps of the "indirect assay method" were as follows:.

The results are shown in Table <NUM>. The indirect assay (combination <NUM>) can better distinguish the negative samples from the positive samples, indicating that the Core+NS3+NS4+NS5 chimeric protein used in the embodiments of the present application has a good activity.

In order to compare the HCV detection effect of the fusion protein of the enzyme and the antigenic protein in the embodiments of the present application relative to that of the conjugate of the enzyme and the HCV antigenic protein produced by chemical cross-linking, the following three reagent combinations were prepared:.

Next, the <NUM> HCV negative samples and the <NUM> positive samples in Example <NUM> were tested using combinations <NUM> and <NUM> according to the "Double antigen sandwich assay method" described above, respectively.

It can be seen from Table <NUM> that the negative coincidence rate of combination <NUM> is <NUM>% and the positive coincidence rate is <NUM>%, which is higher than the conventional double antigen sandwich assay using chemical linking (combination <NUM>: negative coincidence rate is <NUM>%, positive coincidence rate is <NUM>%), and this result shows that: using the fusion protein of horseradish peroxidase and Core+NS3+NS4+NS5 can avoid the destruction of antigen activity or horseradish peroxidase activity caused by the preparation process using chemical linking method in the conventional double antigen sandwich assay. In the embodiments of the present application, both the horseradish peroxidase and the antigen in the fusion protein of horseradish peroxidase and Core+NS3+NS4+NS5 maintain high activity.

Likewise, in order to prove the activity of the Core+NS3+NS4+NS5 chimeric protein used in the embodiments of the present application, reagent combination <NUM> was prepared: the second reagent Rb was prepared using a conjugate of horseradish peroxidase and anti-human IgG and anti-human IgM linked chemically, and the rest was the same as combination <NUM>.

According to the indirect assay method above, the <NUM> confirmed HCV negative samples and the <NUM> confirmed positive samples above were tested using the combination <NUM> above. The results are shown in Table <NUM>. The indirect assay (combination <NUM>) can better distinguish the negative samples from the positive samples, indicating that the Core+NS3+NS4+NS5 chimeric protein used in the embodiments of the present application has a good activity.

In order to investigate the influence of the fusion position of the enzyme on the fusion protein in the embodiments of the present application, the following three reagent combinations were prepared:.

Next, the <NUM> confirmed HCV negative samples and the <NUM> confirmed positive samples from Example <NUM> were tested using combinations <NUM>, <NUM> and <NUM> according to the "Double antigen sandwich assay method" described above, respectively, and the results are shown in Table <NUM> below.

It can be seen from Table <NUM> that when alkaline phosphatase is fused at N-terminus (combination <NUM>), C-terminus (combination <NUM>) and middle (combination <NUM>) of the antigenic protein, both of the negative coincidence rate and the positive coincidence rate of the samples can reach <NUM>%. Furthermore, while not wishing to be bound by theory, the inventors believe that, in the case of fusing the enzyme in the middle of the HCV antigenic protein, the active center of the enzyme would be partially blocked by other proteins, so fusion of the enzyme in the middle of the HCV antigenic protein is slightly less effective than fusion of the enzyme at N-terminus and C-terminus of the HCV antigenic protein.

It can be seen from Table <NUM> that when horseradish peroxidase is fused at N-terminus (combination <NUM>), C-terminus (combination <NUM>) and middle (combination <NUM>) of the antigenic protein, both of the negative coincidence rate and the positive coincidence rate of the samples can reach <NUM>%. Furthermore, while not wishing to be bound by theory, the inventors believe that, in the case of fusing the enzyme in the middle of the HCV antigenic protein, the active center of the enzyme would be partially blocked by other proteins, so fusion of the enzyme in the middle of the HCV antigenic protein is slightly less effective than fusion of the enzyme at N-terminus and C-terminus of the HCV antigenic protein.

In order to investigate the influence of the order of the antigenic proteins on the fusion protein in the embodiments of the present application, the following three reagent combinations were prepared:.

As can be seen from Table <NUM>, when using the antigenic protein fused in the order of Core+NS3+NS4+NS5, NS3+NS4+core+NS5 or NS3+core+NS4+NS5, both of the negative coincidence rate and the positive coincidence rate of the samples can reach <NUM>%.

In order to investigate the HCV detection effect when using mutants of the enzyme, the following reagent combinations were prepared:.

Next, the <NUM> HCV negative samples and the <NUM> positive samples of Example <NUM> were tested using combinations <NUM> and <NUM> according to the "Double antigen sandwich assay method" described above, respectively, and the results are shown in Table <NUM>.

It can be seen from Table <NUM> that when using wild-type alkaline phosphatase or mutant alkaline phosphatase, both of the negative coincidence rate and positive coincidence rate of the samples can reach <NUM>%. In addition, detection discrimination is better when using mutant alkaline phosphatase compared to wild-type alkaline phosphatase.

In order to investigate the influence of different expression technologies on the HCV detection effect in the embodiments of the present application, a fusion protein of alkaline phosphatase and Core+NS3+NS4+NS5 was further prepared by yeast expression system, and combination <NUM> was prepared according to "Reagent preparation method".

The specific method of producing a fusion protein by yeast expression system was as follows:.

Claim 1:
A kit for detecting an HCV antibody comprising:
a fusion protein of an enzyme for labelling and an HCV antigenic protein,
wherein the antigenic protein contains one or more of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen, or a fusion antigen of two or more of the above-mentioned antigens; and
the enzyme for labelling is alkaline phosphatase or horseradish peroxidase.