US20080193955A1

US20080193955A1 - Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies

Info

Publication number: US20080193955A1
Application number: US11/883,499
Authority: US
Inventors: Peng Cui
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-02-02
Filing date: 2005-05-10
Publication date: 2008-08-14
Also published as: CN101160413B; CN101287989A; US7658930B2; WO2006081701A1; CN101287989B; WO2006081718A1; US20090029348A1; EP1845378A4; EP1845378A1; CN101160413A; JP2008529015A

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

The present invention pertains generally to virus diagnostics. In particular, this invention relates to HCV antibody diagnostic kit and its preparation method.

BACKGROUND

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.
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.
ELISA reagents for diagnosis of HCV have developed into the third generation. 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. Thereafter Elisa-2 kits came out to be substitute for Elisa-1 kits. Ortho Diagnosis Company and Abbott Diagnosis Company (U.S.A) used CORE, NS3 and NS4 antigens in the Elisa-2 kits and found its sensitivity and specificity improved in comparison with the Elisa-1 kits. Clinic use showed that positive rate was 92˜95% when Elisa-2 kits were applied in chronic hepatitis patients. In addition, the average window phase of HCV seropositive conversion was about 10 weeks which was much shorter than the Elisa-1 kits (16 weeks). Elisa-3 kits were developed by Ortho Diagnosis Company (U.K), Abbott Diagnosis Company (U.S.A) and Sanofi Diagnosis Company (France). Antigens used in these kits included CORE, NS3, NS4 and NS5. Detection of HCV in blood donors and subjects who was susceptible to be infected showed that the sensitivity increased to 97% and the average window phase of HCV seropositive conversion was about 7˜8 weeks.
Elisa can be automation, is easy for operation and analysis & saving data, moreover, it presents good reproducibility and the cost is relatively low. Therefore, commercial reagents for detection of HCV antibody are mainly Elisa-2 and Elisa-3 kits nowadays. Unfortunately even Elisa-3 has many disadvantages, false positive, false negative and uncertain results still exist. It may do to reagents used in this indirect method. They have many shortcomings as following:
1. Poor specificity. Non-specific binding labeled second antibody (SA) was employed in the indirect method, which lead to low specificity.
2. Low sensitivity. 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.
3. Long window phase. The common SA is anti-IgG antibody, which can only detect the IgG in HCV antibody. It's unable to diagnose other antibodies especially the IgM emerged in the early period. Then a lasting window phase is needed.
At present, 70% of individuals developing post-transfusion hepatitis were those infected with HCV after blood transfusion. Among HCV carriers 80˜90% are infected after blood transfusion. This serious phenomenon suggests that our available reagents for diagnosis have much deficiency and highly sensitive and specific kits are requested.
Double antigen sandwich assay use labeled antigen as a substitution for labeled SA in the indirect method, so disadvantages due to indirect method can be solved. It is commonly applied nowadays, but for screening of HCV antibody, there is no kits using double antigen sandwich assay. There are two main reasons:

1. The Activity Loss Following Enzymatic Labeling the HCV Protein Antigen

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. All those decrease the activity of labeled NS3 antigen substantially. The main active site in HCV CORE antigen epitope area contains lots of Lysine which are critical to the activity of CORE, and the free amino-group in the side chain is used as the target for HRP labeling by NaIO4 oxidization. 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. As if HCV antigen label indirectly, according orthodox methodic preliminary mix enzymatic mixture with secondary antigen and label, high molecular weight enzymatic label will cause very large effect to activity of the HCV secondary, antigen which is depending on its conformation.

2. The High Background

The CORE antigen has very powerful homologous and heterogenous bind capacity in vitro or in vivo (Matsumoto, M. etc., Virology, 218:P43-51, 1996; Kunkel, M. etc., Virology, 294: P239-245, 2002; Majeau, N. etc., Journal of General Virology, 85: P971-981, 2004)). The labeled antigen (the secondary antigen) in the sandwiched method of double antigens can bind homologously with the coating antigen (the primary antigen) which leads to the high background of diagnostic kit.

3. The Conformation Change of Recombinant HCV Secondary Antigen Causing by Possible Existing Mismatching of Disulfide

NS3 antigen, the main epitope segment of HCV, contains several cysteine residues (there is seven Cys in a.a.1196-1473), in case of recombinant expression, the protein may forms mismatching of disulfide, and then change its natural conformation to result in the activity loss of NS3 antigen, and result to the activity loss because of insufficient revealing of the CORE epitope.
America patent U.S. Pat. No. 6,096,319, U.S. Pat. No. 6,270,960, U.S. Pat. No. 6,306,579, all refer to the double antigen sandwiched assay detecting HCV antibody using recombinant NS3 antigen and introduce using sulfhydryl reagent to raise activity of NS3 antigen, wherein electrochemical detecting method is take secondary antigen after label ruthenium mixture directly as labelled antigen in embodiment. 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. And have not mention as enzyme as label in enzyme linked immunosorbent assay of HCV double antigen sandwich detecting method, therefore, there is not refer to the problem of the activity loss of enzyme when raise the activity of HCV antigen by/using sulfhydryl reagent (such as natural horseradish peroxidase has four disulfide bonds, respectively form, by Cysteine residues of 11-91, 44-49, 97-301, 177-209) and the treatment of the problem.
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. 2, don't refer to the method using in raise the activity of HCV antigen.
In patent, CN 1548958A, the described also introduce double antigen sandwich assay for HCV antigen. And the patent has not published specific segments in HCV antigen using, and never mentioned the way to raise the activity of HCV antigen by adding reducing agent in the reaction system.
The method introduce in Baochang Lee et.al., Journal of Experimental Hematology, 12(3):P359-362, 2004, research HCV antigen biotinylation, but they have not prefer the specific solving method about the mentioned problem of developing HCV antibody diagnostic reagent using in double antigen sandwich method, and just say in the antigen level research.
America-patent U.S. Pat. No. 6,613,530, have not refer to the method of raising the activity of HCV antigen in the HCV double antigen sandwich method.
In order to solve activity loss and high background problems following HCV protein label, the patent PCT/CN2005/00149, which is applied on 2 Feb., 2005, publishes the indirect label method of HCV recombinant antigen, and produce sandwich kit using the antigen, and this is better than the commercial indirect ELISA kit.
This invention solve the technological problem existing in present double antigen sandwich kit, especially take secondary antigen and enzymatic label as two independence component of the kit to respectively package. And perform step-by-step detection by two steps adding in reaction system, and then incubate respectively. These not only solve the problem of the activity loss of secondary antigen which is cause by enzymatic label and secondary antigen conjugate to affect secondary; antigen conformation in secondary antigen diluent and enzymatic dilution adding in one container by one step or pre-mix of the two respective package solution in detecting, but also add reducing agent in secondary antigen diluent to raise the activity of secondary antigen and also avoid the destroy the enzymatic activity of reducing agent, and than, preferred raise sensitivity of the kit.

OBJECTIVES OF THE INVENTION

The present invention's objective, is to raise the activity of HCV antigen, especially the activity of secondary antigen, and combined with biotin-avidin magnifying system, to provide a new double-antigen sandwiched test kit for HCV antibodies, whose sensitivity and specificity are much better, than present commercial kits.

SUMMARY OF THE INVENTION

The present invention provides a HCV antibodies diagnosis kit. The kit completes the detection in the form of ‘support-primary antigen-HCV antibodies to be detected-secondary antigen-label recognizable signal’, wherein the second antigen carry on the combination reaction with the enzyme label in one step or several steps. The kit contains sample diluent, secondary antigen diluent and label diluent, said secondary antigen diluent and label diluent are packed separately.
In an embodiment of the present invention, said secondary antigen diluent contains reducing agent.
In an embodiment of the present invention, said reducing agents include, but are not limited to β-mercaptoethanol, 1,4-Dithiothreitol, dithio-erythritol, reduced glutathione, Cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride.
In an embodiment of the present invention, when sample to be detected and secondary antigen are added in the reaction system and incubated at the same time in detecting process, sample diluent and secondary antigen diluent can be the same solution, i.e. secondary dilution packaging in the same container, the β-mercaptoethanol concentration in secondary antigen diluent is 0.01-50‰, and prefers 0.05-20‰, and furthermore prefers 1-10‰, and most prefers 5‰.
In an embodiment of the present invention, when sample to be detected and secondary antigen add in the reaction system and incubate respectively by two steps, the β-mercaptoethanol concentration in secondary antigen diluent is 0.01-50‰, and prefers 0.05-30‰, and furthermore prefers 0.2-20‰, and furthermore prefers 0.5-1‰, and furthermore prefers 0.5-5‰, and most prefers 1‰.
In an embodiment of the present invention, said sample diluent includes reducing agent which can reduce disulfide bond, including but not limited to β-mercaptoethanol, 1,4-dithiothreitol, dithio-erythritol, reduced glutathione, cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride.
In an embodiment of the present invention, when sample to be detected and secondary antigen are added in the reaction system and incubated respectively by two steps, the β-mercaptoethanol concentration in sample diluent is 0.01-50‰, and prefers 0.1-20‰, and furthermore prefers 0.5-10‰, and furthermore prefers 5‰.
In an embodiment of the present invention, said kit includes pre-coating ELISA plate, secondary antigen, washing solution, developer solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box.
In an embodiment of the present invention, said secondary antigen is conjugate of HCV protein and tag, the tags include peptide tag or non-peptide tag.
In an embodiment of the present invention, said peptide tag is peptide or protein containing His Tag, T7 Tag, S Tag, Flag Tag, HA Tag or HCV peptide fragment.
In an embodiment of the present invention, said non-peptide tag is chemical compound, hapten, vitamin, steroid, dye, hormone, antibiotic, nucleic acid or conjugate of all above component with peptide or protein.
In an embodiment of the present invention, said chemical compound is dinitrophenol, bromodeoxyuridine; vitamin is biotin or derivatives thereof; steroid is Digoxin, dye is Acridinium Ester, Rhodamine, Dansyl Chloride, Dihydroxyfluorane, Oregon Green, Lucifer yellow, Alexa Fluor, or Cascade Blue.
In an embodiment of the present invention, said tag is biotin.
The present invention provides a method of detecting the HCV antibodies using the diagnosis kit of the present invention. The method completes the detection in the form of ‘support-primary antigen-HCV antibodies to be detected-secondary antigen-label-recognizable signal’, comprising following detecting steps:
A. Primary antigen captures antibody: add specimen to be detected and incubate to allow primary antigens to capture the specimen antibodies on pre-coating ELISA plate;
B. Antibody captures secondary antigen: add secondary antigens, and incubate to allow HCV antibodies to capture secondary antigens.
C. Detect: introduce enzyme label by tag in one or more incubating steps and then detect; wherein the step B is carried out in the reduction condition, step A and B can merge, i.e. specimen to be detected and the secondary antigen can be added into the reaction system simultaneously and incubated together, wherein said secondary antigen and enzyme label are added into reaction system in two steps successively and incubated respectively.

DESCRIPTION OF THE FIGURES

FIG. 1 is a sketch map of indirect detection of HCV antibody.

FIG. 2 is a sketch map of one step sandwiched detection of HCV antibody.

FIG. 3 is a sketch map of two steps sandwiched detection of HCV antibody.

FIG. 4 is a sketch map of three steps sandwiched detection of HCV antibody.

FIG. 5 is a plasmid profile of expression plasmid P2.

FIG. 6 is a plasmid profile of plasmid P2-HCVAg1 expressing primary antigen.

FIG. 7 is a plasmid profile of plasmid P2-X-Y expressing secondary antigen.

DEFINITIONS

The terms in the present invention have the following meanings unless stated otherwise.
The term used herein ‘reducing agent’ is the reducing agent for disulfide bond in protein, i.e. the one can reduce the disulfide bond formed by two cysteines' sulfhydryl groups inside protein or between the proteins to dissociated sulfhydryls. The reducing agent includes but not limited to β-mercaptoethanol, 1,4-dithiothreitol, dithio-erythritol, reduced glutathione, cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride.
The term used herein ‘primary antigen’ refers to coating antigen in the present invention.
The term used herein ‘secondary antigen’ is the conjugate of HCV protein and tag. The secondary antigen used herein can be one or more, and respective tags thereof satisfied the limitative condition in the present invention.
The terms used herein ‘one step method’ is a method in which after the combination of supporter and primary antigen, we add the specimen to be detected and the secondary antigen at the same time, then incubate them and wash the plate, add enzyme label. So, the HCV antibody double antigens sandwiched diagnostic kit developed in this manner is called one-step-method diagnostic kit.
The terms used herein ‘two steps method’ is a method in which after the combination of supporter, the primary antigen, and the HCV antibody to be detected and the plate washing, we add the secondary antigen and enzyme label or its complex, then incubate. So, the HCV antibody double antigens sandwiched diagnostic kit developed in this manner is called two-step-method diagnostic kit.
The terms used herein ‘three steps method’ is a method in which after the combination of supporter, the primary antigen, and the HCV antibody to be detected and plate washing, we add the secondary antigen solution and incubate it, then wash the plate, add enzyme label. So, the HCV antibody double antigens sandwiched diagnostic kit developed in this manner is called three-step-method diagnostic kit.
The term used herein ‘biotin or its derivatives’ refers to biotin or its functional derivatives, including, but are not limited to D-biotin, activated biotin, biocytin, ethylendiamine biotin, cadaverine biotin, desthiobiotin and so on, with common characters of containing imidazolone ring or functional similar structure which can bind with avidin.
The terms used herein ‘peptide’ and ‘protein’ have the common meaning as the ordinary one in the art understand, and can be used interchangeably.
The terms used herein ‘CORE’ and ‘NS3’ respectively refer to HCV core protein and non-structural protein 3. The former means the a.a.1-191 segment of protein encoded by whole gene (a.a. is amino acid simplified character, in the present invention a.a.A-B means segment from number A to number B amino acid in the HCV protein encoded by whole gene, wherein B>A), the latter means the a.a.1027-1657 segment of protein encoded by whole gene.
The term used herein ‘Biotin Receptor Protein’ refers to biotin carboxyl-carrier protein (BCCP) family or functional domain thereof, SEQ ID NO.3, polypeptide, or functional analogs thereof (detailed view Peter, J. S. et.al. Biotechnology, 11:1138-1143, 1993), in the present invention the biotin carboxyl-carrier protein selected can be one, several or combinations of above-mentioned peptides or proteins.
The term used herein ‘tag’ is an agent which can link with HCV protein and specifically bind to binding partner, and furthermore educe following detected reaction process, including peptide tag and non-peptide tag.
Said peptide tag is peptide or protein, including peptide of protein containing His Tag, T7 Tag, S Tag, Flag Tag, HA Tag (many peptide can be selected, detailed see Abeam company epitope tags sections product introduction http://www.abcam.com) or HCV peptide segment et.al., said HCV peptide segments are not present in primary antigen and do not have the binding capability of causing high background, whose corresponding binding partner peptide or protein existed or can be prepared. They are characterized in that the core substance which can specifically bind to binding partner, and furthermore educe following detected reaction process' is peptide or protein. Said His Tag means six successive His, i.e. 6×His.
Said peptide tag is Heterologous Protein, with the molecular weight smaller than 30 KD, preferable smaller than 20 KD, and furthermore preferable smaller than 15 KD, and furthermore preferable smaller than 10 KD, and furthermore preferable smaller than 5 KD.
Said peptide Heterologous Protein tag has a molecular, weight smaller than 5 KD when it is at the N-terminal of HCV protein; when said peptide tag is at C-terminal, its molecular weight is smaller than 20 KD.
Said non-peptide tag is chemical compound (such as dinitrophenol, bromodeoxyuridine), hapten, vitamin (such as biotin or derivatives thereof), steroids (such as Digoxin), dye (such as Acridinium Ester, Rhodamine, Dansyl Chloride, Dihydroxyfluorane, Oregon Green, Lucifer yellow, Alexa Fluor, Cascade Blue), hormones, antibiotics, nucleic acids and so on (many non-peptide can be selected, detailed see Invitrogen company molecular probe product introduction http://www.probes.com), whose corresponding binding partner non-peptide exist or can be prepared. They are characterized in that the core substance which can specifically bind to binding partner, and furthermore educe following detected reaction process is non-peptide or non-protein.
Tag can be one, several or combination of several tags, can exist at HCV antigen N-terminal, C-terminal, both terminals or in inside
Tag should be as possible as small, in order to reducing HCV antigen activity reduction.
The term used herein ‘binding partner’ refers to a ligand or receptor which can bind to tag in the present invention. When choosing non-peptide tag, its binding partner are tag specific receptor or antibody; if choosing biotin or derivatives thereof or their conjugate with peptide or protein as tag, their binding partner are avidin, streptavidin, neutron-streptavidin, their analogue, antibiotin or derivatives thereof antibody; when choosing peptide as tag, their binding partner are monoclonal antibody or polyclonal antibody against the peptide or protein.
The term used herein ‘flexibility’ and ‘rigidity’ main refer to an agent structure's distortion, when macro molecule protein such as HRP etc. is cross-linked with HCV antigen, said protein can kink with HCV antigen, resulting in the latter's structure being changed, and than affect activity thereof; this kind of agent is ‘flexibility’ agent; inversely, when polystyrol, gold colloid etc. is cross-linked with HCV antigen, both sides will not kink together, so HCV antigen conformational changed little, this kind of agent is ‘rigidity’agent.
the term used herein ‘expression host’ comprise prokaryotic, eukaryotic expression system host, including, but are not limited to bacterium, such as Escherichia coli, bacillus subtilis and so on; yeast such as Pichia methanolica, Saccharomyces cerevisiae and so on; eukaryotic cell such as entomo-cell, mammalian cell and so on; virus, tissue, organ, bio-reactor in vivo and so on.
The term used herein ‘support’ include, but are not limited to ELISA plate, bead, microparticle, slide, array, plastics, membrane and so on, prepared by materials such as polystyrene, polyethylene, cellulose, nitrocellulose, acetylcellulose, silicide and so on.
The term used herein ‘sample/specimen to be detected’ comprise all kinds of body fluid from species which can be infected by HCV, including, but are not limited to whole blood, serum, plasma, secretion, urine, saliva, cerebrospinal fluid, lymph fluid, tissue fluid, diachorema leachate, cell culture fluid or cell, tissue, organ homogenate and so on.
The Main Advantages of this Invention:
1, Reductant is added into the secondary antigen diluting solution, and during the testing procedure, the second antigens and labels are added into the reacting system in two separate steps then incubated, the advantages of this are as following:

- A, If the secondary antigens and labels are added at the same time or previously well-mixed then added for the incubation, then the labels with larger molecular weight specifically bind to secondary antigens, the binding make insufficient exposure of the epitopes of secondary antigens, and partially destroy the conformation of HCV antigens, leading the great reduction on secondary antigen activity. Therefore, adding the secondary antigens and labels into the reacting system in two steps and then incubating together can avoid this phenomenon.
- 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.
  2, The advantages of adding reductant into sample diluting solutions:
- A, it performs the reaction of the antigens in the sample to be tested with HCV antigen under a reducing environment, ensuring the high activity of HCV antigens.
- 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 dissociative IgM antigens and not to attend the detection reaction, which is disadvantageous for the IgM antibody detection, and further disadvantageous for reducing the window phase period. Adding reductant into sample diluting solutions can solve above problem.
  The Concentrations of Reductants Added into Buffers in the Reaction System

Adding reductant can open the mis-paired disulfide bonds within the HCV antigen molecules, which increases the activity of HCV antigens. Therefore, except for enzyme diluting solutions, each buffer used in this invention's reaction system all contain certain concentrations of reductants, said reductants can be β-mercaptoethanol, 1,4-Dithiothreitol, dithio-erythritol, reduced glutathione, Cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride and so on, or any reductant solutions which can reduce disulfide bonds.
The concentration ranges of β-mercaptoethanol in various buffer solutions are as follows:

- 1, the concentration range of β-mercaptoethanol in coating solutions, is 0.5 to 5.0‰, preferring 1‰,
- 2, the concentration range of β-mercaptoethanol in blocking solutions is 0.5 to 5.0‰, preferring 5‰,
- 3, in case that the sample diluting solutions and the secondary antigen diluting solutions used in one-step method are the same, the concentrations of β-mercaptoethanol is 0.01 to 50‰, preferring 0.05 to 20‰, more preferring 1 to 10‰, most preferring 5‰,
- 4, the concentration range of β-mercaptoethanol in sample diluting solutions in three-step method is 0.01 to 50‰, preferring 0.1 to 20‰, more preferring 0.5 to 10‰, the most preferring 5‰,
- 5, the concentration range of β-mercaptoethanol in three-step method secondary antigen diluting solutions is 0.01 to 50‰, preferring 0.1 to 50‰, more preferring 0.5 to 30‰, more preferring 0.2 to 20‰, more preferring 0.5 to 10‰, the most preferring 1‰.

The technicians working in this field can follow the ordinary technique and the spirit of the present invention to choose other reductants and their concentrations.
The following examples can illustrate this invention further.

EXAMPLE 1

The Preparation of Primary Antigens

The present invention get the reconstruction vector pTO-T7 referring to method of Wenxin Luo et al, Chinese Journal of Biotechnology, 16(5): P578-581, 2000. We synthesize primers to amplification sequence between NdeI and BamHI sites, wherein forward primer has NdeI site and BamHI and EcoRI sites, and the reserve primer has BamHI site and EcoRI sites. After recovery (All molecular biology extraction and purification kits are bought from Watson Biotechnologies, Inc in the present invention), the fragment is cleaved by restriction endonuclease NdeI/EcoRI (all kinds of molecular biology enzyme are bought from TAKARA Biotechnology (Dalian) Co., Ltd. in the present invention) and ligated to the pTO-T7 vector cleaved by the same endonucleases. Positive clone is cleaved by restriction endonuclease BamHI, then recovered and ligated to each other (that is to remove T7 Tag gene which is between NdeI and BamHI sites in pTO-T7 vector), and we get the vector named P1.
Because upstream the T7 promoter of P1 vector there is one BgIII site, which is disadvantageous to clone of the present invention. Synthesis primers to amplify the segments between BgIII and XbaI sites of P1 vector, forward primer has BamHI site and reverse primer has XbaI site. The segment is cleaved by BamHI and XbaI and then ligated in P1 vector cleaved by the same endonuclease. So, the positive done is expression plasmid P2 in which BgIII site is blocked, and also is clone; vector used in the present invention. In applying the present invention, P1 and P2 are not the necessary expression vectors, and upstream enhancer thereof also has not substantial affection in the present invention, and construction of these vectors are just for convenience in cloning, many other expression vectors such as pET-24a(+) (America Novagen Co., Article No. 69749-3) can also be used in the present invention practice.
We amplify a.a.1201-1465 gene (the 1^stto 795^thnucleotide in SEQ ID NO.2) by PCR. The forward primer thereof has BamHI site; reserve primer has BgIII and EcoRI sites between which there is a termination condon TAA. Then, we amplify a.a.2-59 gene (the 4th to 177th nucleotide in SEQ ID NO.1) by PCR, and the forward primer thereof has BamHI site, reverse primer has BgIII and EcoRI sites between which there is a termination codon TAA. Ligating the segment of a.a.1201-1465 cleaved by BamHI/EcoRI endonucleases into expression vector P2 cleaved by the same endonucleases, we can get the plasmid P2-G. Insert a.a.2-59 gene cleaved by BgIII/EcoRI into the expression vector cleaved by the same endonucleases, we can obtain the positive clone P2-G-CORE (a.a.2-59) which is also the expression plasmid of the primary antigen in this invention, named P2-HCVAg1. The ER2566 strain (Escherichia coli ER2566/P2-HCVAg1) containing the plasmid has been stored in China Center for Type Culture Collection (Wuhan University, Luojia hill, Wuchang district, Wuhan, Hubei province, China) in Jan. 21, 2005, with store number of CCTCC M 205009, and the antigen expressed by this plasmid is named HCVAg1.
Transform P2-HCVAg1 into Escherichia coli ER2566 (New England Biolabs, Inc.) and spread the E. Coli on LB solid culture medium which contain 100 ug/ml Kanamycin (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., short for Sangon, Article No. KE170), cultivate it at 37° C. overnight, then pick single colony to a bottle of 500 ml LB liquid culture medium which contains the same concentration of Kanamycin to shake and cultivate to OD600 1.0 approximately in which; Then add IPTG (Sangon, Article No. IB0168) to induce, whose final concentration is 0.5 mM, at 37° C., 200 rpm for 4 hours; centrifugate induced cells at 5000 rpm for 20 minutes and resuspend the sediment in 10 ml split buffer (50 mM Tris-HCl; 1 mM EDTA, 100 mM NaCl, pH 8.0) in per liter culture of engineering bacteria/Break up the Cell by Ultrasonic; then collect inclusion body, after centrifugated at 12000 rpm for 20 minutes at 4° C., resuspend the sediment in Solution I (20 mM Tirs-HCl, 5 mM EDTA, 100 mM NaCl; pH8.5) containing 2% Triton X-100 (Sangon, Article No. T0694). Collect inclusion body by centrifugation at 12000 rpm for 20 minutes at 4° C., dissolve the inclusion body by 4M carbamide in Solution I and then dialyze it in 100 times voluminal PB buffer (20 mM, pH7.0)/exchange PB buffer 3 times, centrifugate it to remove sediment to get raw antigen. Equilibrate Sephacryl S-200 (America Amersham Biosciences Inc.) with the same PB buffer as above, then add above raw antigen; collect and pool the outflow which contains target protein, after that, we purify the outflow with CM-Sepharose (America Amersham Biosciences Inc.). The HCV antigen will be absorbed by CM-Sepharose, and the impurity protein will be eluted by the PBS buffer (20 mM PB, 50 mM NaCl, pH7.0). Then we elute target protein with PBS buffer (20 mM PB, 500 mM NaCl, pH7.0) and measure protein concentration and store it at 4° C. or −20° C. for further use.
Apart from HCVAg1, the primary antigens used in the patent application (application number is PCT/CN2005/00149) on 2 Feb., 2005, can be used as the primary antigen in this invention.

EXAMPLE 2

Preparation of Secondary Antigen and Enzyme Label

1. The Preparation of Biotinylation Secondary Antigen

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.
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.
We extract Escherichia coli ER2566 whole genome as the template to amplifie Escherichia coli K12 BirA gene Y (SEQ ID NO.4) encoding biotin-[acetyl-CoA carboxylase] synthase. Amplification primers are as following:

primer 1:
5′-CCC GAA TTC ATG AAG GAT AAC ACC GTG CC-3′

primer 2:
5′-GGG AAG CTT TTA TTT TTC GGC ACT ACG CAG GGA TAT

TTC ACC-3′

The forward primer thereof has EcoRI site, the reverse one has HindIII site and termination condon TAA. The amplified segment Y is cleaved by EcoRI/HindIII, then cloned into P2-X expression plasmid cleaved by BgIII/EcoRI, and we obtain the positive clone named P2-X-Y, which is also preferred expression plasmid of secondary antigen in the present invention, ER2566 strain containing the plasmid has been stored in China Center for Type Culture Collection (Wuhan University, Iuojia hill, Wuchang district; Wuhan, Hubei province, China) in Jan. 21, 2005, with the store number of CCTCC M 205010, classification name: Escherichia coli ER2566/P2-X-Y. There is one termination codon between gene X and Y, so in P2-X-Y, gene X and Y make up the bi-cistron under control of the same expression regulatory element.
Transform Escherichia coli ER2566 with P2-X-Y, and spread it in LB plate which contain 100 ug/ml Kanamycin. Stay overnight to cultivate at 37° C., and then pick separate colony to shake cultivation to OD600 approximate 1.0 in 500 ml LB culture which contain the same concentration of Kanamycin and D-biotin of final concentration of 20 uM (America Sigma-Aldrich Co., Ltd., Article No. B-4501). Then add IPTG (final concentration of 0.5 mM) to induce; the induction condition is 37° C., 200 rpm and 4 hours. Then collect the cells; suspend the cells in 1 liter in 10 ml lysis buffer. Crack cells by ultrasonic, collect supernatant by centrifugation at 12000 rpm for 20 minutes at 4° C. Ammonium sulfate is added into the supernatants up to 10%-30% saturation to precipitate. After that, we re-dissolve the precipitate in PB buffer (100 mM, pH7.0), remove the sediment by centrifugation at 12000 rpm for 10 minutes at 4° C. We can use avidin column which contains SoftLink Soft Release Avidin resin to purify the target protein (Detailed purification process refers to America Promega Inc., Article No. V2012; instruction). Elute with PB buffer containing 5 mM D-biotin, and collect the outflow which is purified biotinylation antigen, then dialyze overnight against 100 times volume CB buffer (100 mM, pH9.6) to remove free biotin by exchange CG buffer 3 times. Centrifugate supernatant to measure protein concentration, the protein is stated at 4° C. or −20° C. for use in the future. The expressed and purified antigen is named HCVAg.
In this method of biotinylation, we try to insert SD sequence between the termination codon of gene X and start codon of gene Y (detail operation is the common technique of genetic engineering, can be referred to Tso, K. L. etc., Gene, 169; P59-6411.996), in order to increase the expression quantity, which may raise the efficiency of biotinylation and increase the sensitivity of the kit of present invention.
Besides HCVAg, the biotinylation secondary antigen mentioned in patent PCT/CN2005/00149 filed on Feb. 2, 2005 can be used for the secondary antigen in present invention
2. The Preparation of the Secondary Antigen with Peptide Tag
We amplify the HCV gene encoding antigen a.a.1-28 gene (the 1st to 84th nucleotide in SEQ ID NO.1). The forward primer has BamHI site, and the reserve one has EcoRI site and a termination codon TAA, upstream of which there a 6× his tag. Cleaved by BamHI/EcoRI, the gene is ligated to expression vector P2-G treated by BgIII/EcoRI. So, we can get the positive clone P2-G-CORE (a.a.1-28)-His, whose expression target protein is the secondary antigen with peptide tag. Alternatively, the secondary antigen with peptide tag mentioned in patent PCT/CN2005/00149 filed on Feb. 2, 2005 can be used for the secondary antigen.

3. The Preparation of Enzyme Label

The enzymatic labeling method of streptoavidin, the binding partner of biotin, is NaIO₄oxidation method. Weigh 10 mg HRP to dissolve into 1 ml ultrapure water, then slowly drop the 5 mg/ml NaIO₄(Sangon, No. ST1244) solution prepared by 1 ml ultrapure water, stir for 40 minutes gently avoiding light at room temperature, then add 0.05 ml 20% glycol (Sangon, NO. E0582) solution, stir for 40 minutes gently avoiding light at room temperature. Then add 1 ml 2.5 mg/ml streptoavidin (Sangon, No. SE497) solution which is dialyzed against 100 mM, pH 9.51 carbonate buffer for 2 hours, dialyze the mixture in 100 mM, pH 9.51 carbonate buffer over night at 4° C. free from light. The next day, drop the fresh 0.1 ml 4 mg/ml NaBH₄(Sangon, No. ST1268) solution to the mixture, blend, stand for 2 hours at 4° C. Place the mixture into dialysis bag and dialysis against PBS buffer (150 mM, pH7.4) over night at 4° C., then add enzyme protective agent and the glycerol of final concentration of 50%, then conserve it at −20° C. from light for future use.

EXAMPLE 3

SELECTION OF THE CONCENTRATION OF REDUCING AGENT

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 1

The effect of β-mercaptoethanol in coating
buffer to sensitivity of HCV indirect kit

final concentration of	20‰	10‰	5‰	2‰	1‰	0.5‰	0.2‰	0.1‰	0
β-mercaptoethanol in
coating buffer
average OD mean	5.10	5.62	5.89	6.31	6.54	6.19	5.78	5.12	5.20
value/Co of the
positive serums

Indication: Cut Off Value = 0.200

On the other way, dilute HRP to a certain ratio, add different final concentration of reducing agent to HRP solution, then directly add substrate to develop color. We can get the result that with increasing of reducing agent concentration, the HRP activity loses, even disappear in the end. The result indicates reducing agent can destroy HRP activity, and also indicates that the four internal disulfide bonds of HRP are critical to keep it activity.
In a word, one aspect is that the reducing agent can greatly raise HCV antigenic activity the other aspect is that it also can destroy HRP activity. When the secondary antigen was added to the reaction system together with the enzyme conjugate, in order to reach best detection effect of the kit performing detection via such a method, we need to search one suitable working concentration of reducing agent.
We add certain final concentration of β-mercaptoethanol into secondary antigen diluent, buffer and enzyme label dilution buffer, to detect sensitivity of the kit (data view tab 2). Finally, we conclude that working concentration of β-mercaptoethanol is 0.075‰, the sensitivity is the topmost at this condition.

TABLE 2

The effect of β-mercaptoethanol in secondary antigen diluent
buffer of two step method to the invention kit's sensitivity

β-mercaptoethanol final	5‰	3‰	2‰	1‰	0.5‰	0.2‰	0.1‰	0.05‰	0.02‰	0.01‰	0
concentration in
secondary antigen
diluent buffer
average OD mean	4.35	4.96	5.27	5.62	5.88	6.11	6.59	7.31	6.25	5.34	3.59
value/Co of the
positive serums

Indication: Cut Off Value = 0.150

In order to research the effect of different concentration of β-mercaptoethanol to the invention kit's sensitivity, this invention also added β-mercaptoethanol to secondary antigen diluent buffer of one step method and the kits of there step method. The result of table 3 and table 4 indicate that: the average OD mean value/Co of the positive serum of one step method increase from 7.24 to 16.54, the average OD mean value/Co of the positive serum of three step method increase from 7.35 to 17.26. The β-mercaptoethanol of various concentrations displays positive effects, which greatly increases the sensitivity of the testing kits. The result further indicate that β-mercaptoethanol can break up the mismatching disulfide of HCV antigen, and restore the natural conformation of the HCV antigen, and increase HCV antigenic activity to a certain extent. From the result of table 3, we conclude that the β-mercaptoethanol concentration in the secondary antigen diluent buffer range is 0.01-50‰, prefers 0.05-20‰, further prefers 1-10%*, most prefers 1‰. From the result of table 4, we conclude that the β-mercaptoethanol concentration in the enzymatic conjugate dilution buffer range is 0.01-50‰, prefers 0.05-30‰, further prefers 0.2-20‰, further prefers 0.5-10‰, further prefers 0.5-5‰, most prefers 1‰.

TABLE 3

The effect of β-mercaptoethanol in one step method secondary
antigen diluent buffer to the invention kit's sensitivity

β-mercaptoethanol

50‰

30‰

20‰

10‰

5‰

1‰

0.5‰

0.2‰

0.1‰

0.05‰

0.01‰

0

final concentration

in secondary

antigen diluent

buffer

average OD mean

12.81

14.93

15.67

16.05

16.54

16.19

15.60

14.87

13.70

12.54

11.30

7.24

value/Co of the

positive serums

Indication: Cut Off Value = 0.150

TABLE 4

the effect of β-mercaptoethanol in three step method secondary
antigen diluent buffer to the invention kit's sensitivity

β-

50‰

30‰

20‰

10‰

5‰

1‰

0.5‰

0.2‰

0.1‰

0.05‰

0.01‰

0

mercaptoetha-

nol final

concentration

in secondary

antigen diluent

buffer

average OD

12.33

14.85

15.48

16.12

16.84

17.26

16.63

17.87

14.22

12.67

11.45

7.35

mean value/

Co of the

positive

serums

Indication: Cut Off Value = 0.150

We tried to add β-mercaptoethanol in the sample diluent buffer of the three-step method kit as well as adding β-mercaptoethanol in the secondary antigen diluent buffer, the result is in the table 5. From table 5, we conclude that the β-mercaptoethanol concentration in the sample diluent buffer range is 0.01-50‰, prefers 0.1-20‰, further prefers 0.5-10‰, further prefers 0.5-5‰, most prefers 1‰.

TABLE 5

the effect of β-mercaptoethanol in three step method sample
diluent buffer to the invention kit's sensitivity

β-mercaptoetha-

50‰

30‰

20‰

10‰

5‰

1‰

0.5‰

0.2‰

0.1‰

•0.05‰

0.01‰

0

nol final

concentration in

secondary

antigen diluent

buffer

average OD

12.56

14.03

15.46

16.61

17.30

16:81

15.94

15.12

•14.79

13.92

12.53

7.30

mean value/

Co of the

positive serums

Indication: Cut Off Value = 0.150

For antibody against CORE antigen, we further search the different β-mercaptoethanol concentration in secondary antigen diluent buffer and sample diluent buffer of the three-step method, and the result is similar. The antibody against CORE was polyclonal antibody against CORE, prepared by purification from serum of different sufferer using the CORE antigen as the material of affinity chromatography. This result indicates that β-mercaptoethanol not only can break up the mismatching disulfide of NS3 antigen, and increase NS3 antigenic activity, but also can increase CORE antigenic activity. We conjecture that it is due to the aright match of the mismatched disulfide of NS3 antigen and the more sufficient exposure of epitope of CORE.
Otherwise, we tried DTT(1 4-Dithiothreitol) to replace β-mercaptoethanol as reducing agent. However, we found that it have worse stability than β-mercaptoethanol in stability test as reported, and its ability of reducing disulfide bond is equivalent to β-mercaptoethanol, not excels β-mercaptoethanol much more as in some reports.

EXAMPLE 4

PRODUCTION AND TEST OF THE PRESENT INVENTION KIT

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

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 is packed in aluminum film bag with drier.
The test can be performed according to one step method, two step method or three step method. Concretely as followed:

1.1 One Step Method

Add 50 ul 5% oβ-mercaptoethanol, 0.5‰ TritonX-100, 20% neonatal calf serum, 5‰casein, 150 mM NaCl and biotin labeled HCV secondary antigen (diluted before using) which was diluted by pH7.4, 20 mM PB with a certain ratio, then add 50 ul specimen to be detected, negative (health adult negative serum), positive (HCV antibody positive serum) control, incubate for 90 minute at 37° C., wash each well five times with PBST washing buffer, then desiccate the wells by patting.
Add 100 ul certain ratio diluted streptavidin labeled HRP (diluted before using) pH7.4, 20 mM PB contain 1‰casein, 20% neonatal calf serum, incubate for 30 minute at 37° C., wash each well five times with PBST washing buffer, then desiccate the wells by patting.
Respectively add 50 ul color A and color B into per well, wherein color A contains 0.5‰hydrogen peroxide carbamide (Sangon, Article No. UB1753), 4.76‰sodium acetate trihydrate, 0.9‰acetic acid and color B contains 0.32‰ TMB (Sangon, Article No. TB0514), 5 mM citric acid, 0.5 mM EDTA-2 Na, 5% methanol, 2‰N,N-Dimethyl formamide; and avoid light to color for 30 minute at 37° C., then we add 50 ul quench solution which contains 2M sulfuric acid into each well to stop reaction. After adjust the Zero with blank control in 450 nm wavelength (reference wavelength is 630 nm) of the enzyme micro-plate reader we use that to read OD value. Account Cutoff Value (COV): COV=mean of negative control OD value ×2.0 (If negative control OD value <0.075, account as 0.075, if negative control, OD value >0.075, account as actual measure value). When specimen to be detected OD. value ≧COV, it will be positive; when specimen to be detected OD value < COV, it will be negative.

1.2 Two Step Method

Add (add before use) certain dilution ratio of HCV biotinylation antigen to pH7.4, 20 mM PB enzyme dilution, which contain 0.5‰ TritonX-100, 20% neonatal calf serum, 5‰casein, 150 mM NaCl and certain dilution ratio of streptavidin enzyme tumor marked we get enzyme preparation solution.
Add 50 ul pH7.4, 20 mM PB sample diluent into each well to coating ELISA plate, which, contain 5‰ β-mercaptoethanol, 0.5‰ TritonX-100, 20% neonatal calf serum, 5‰casein and 150 mM NaCl, then add 50 ul specimen to be detected, negative (health adult negative serum), positive (HCV antibody positive serum) control, incubate for 60 minute at 37° C., wash each well five times with PBST washing buffer, then desiccate the wells by patting.
Add (before use) certain dilution ratio of β-mercaptoethanol whose final concentration is 0.075‰ in above enzyme preparation solution to prepare enzyme working solution, then add 100 ul enzyme working solution into ELISA plate per well, incubate for 30 minute at 37° C., wash each well five times with PBST washing buffer, then we desiccate the wells by patting.
The residuary steps are same to method of the one steps of 1.1

1.3 Three Step Method

Each well of the plate is added 50 ul sample diluent buffer (20 mM pH7.4 PB, contain 5‰ β-mercaptoethanol, 1‰ TritonX-100, 20% NBS) and 50 ul sample or negative or positive serum, incubated for 60 min at 37° C., then washed five times with PBST, dried by patting, then added 100 ul 20 mM pH7.4 PBS containing 1% β-mercaptoethanol, 0.5‰ TritonX-100, 20% NBS, 5‰ casein, 150 mM NaCl and biotin labeled HCV antigen (diluted before use) to each well. The residuary steps are same to method of the one steps of 1.1.
Add 100 ul certain ratio diluted streptavidin labeled HRP (diluted before using) pH7.4, 20 mM PB contain 1‰ casein, 20% neonatal calf serum, incubate for 30 minute at 37° C., wash each well five times with PBST washing buffer, then desiccate the wells by patting.
The residuary steps are same to method of the one steps of 1.1
Dilution ratio of biotinylated HCV antigen and enzyme labeled streptoavidin should be groped to the best effect based on the practice. The dilution ration and each step reaction time can also be adjusted in practice production to ensure detection effect and short detection time to raise working efficiency. For these techniques of grope and adjustment master by common technicians, and here we no longer detail them.

2. The Compare of the Kit's Different Detecting Process

In order to compare above mentioned three detecting ways, we take HCVAg1 as primary antigen, and HCVAg as secondary antigen; respectively use above three sandwich methods to detect 12 shares hepatitis C serum, (include two shares strong positive serum) and 10 shares negative serum. As results in tab 6, when secondary antigen and label added in together, the kit's sensitivity is lower, and approximate to the sensitivity of the indirect detecting, and can not play the advantages of sandwich method. These show that though we have confirmed the best concentration of 0.075‰ β-mercaptoethanol also effect to the activity of HRP at that concentration. We also do the experiments in which we do not add β-mercaptoethanol in the reaction process of three methods, and find that the sensitivity of the two-step method, is lower significant. These indicate the activity of secondary antigen lost very great following pre-mixing of enzyme label and secondary antigen. Therefore, the detecting process preferred three step and one step method. But one step method exists hook effect (hook effect, as the reaction to strong serum SP1 and SP2), so furthermore preferred three-step method in the present invention kit. If no specific introduce, the HCV antibody detecting kit of sandwich method is three-step method in the following mentioned of this invention.

TABLE 6

comparison of the kit's sensitivity in different detecting process

Test Method	One step method	Two steps method	Three steps method

SP1	0.318	*	*
SP1	0.056	*	*
P1	2.450	1.120	2.544
P2	2.232	1.043	2.318
P3	2.409	0.988	2.592
P4	2.388	1.093	2.501
P5	2.514	1.140	2.650
P6	2.291	1.026	2.411
P7	2.205	1.087	2.380
P8	2.470	1.160	2.562
P9	2.563	1.203	2.730
P10	2.311	1.103	2.423
N1	0.035	0.026	0.012
N2	0.032	0.022	0.010
N3	0.040	0.028	0.016
N4	0.046	0.031	0.020
N5	0.038	0.024	0.013
N6	0.029	0.019	0.009
N7	0.041	0.022	0.017
N8	0.047	0.030	0.021
N9	0.043	0.025	0.016
N10	0.037	0.020	0.011

note:
The number in the table is OD450 test value;
SP denotes positive serum with extremely high antibody titer (i.e. HOOK serum);
P denotes positive serum;
N denotes negative serum;
* denotes the OD value >3.0;

EXAMPLE 5

PERFORMANCE EVALUATION OF THE PRESENT INVENTION KIT

(1) Sensitivity

In order to compare the sensitivity between the sandwiched kit which is as biotinylation protein as secondary antigen and present commercial indirect ELISA kit, we use the present invention kit to detect 100 shares positive serum which is diluted with two shares health adult negative serum, and select HCV indirect diagnostic reagent of InTec products, Inc. (Xiamen) (short for InTec indirect kit), abbott's Murex Anti-HCV 4.0 as control, Result can be viewed in tab 7.
Result indicates that the sensitivity difference of the present invention kit and the two indirect ELISA kit to respectively dilution serum reaction has statistics significance (P<0.05). It is evident that the sensitivity of present invention reagent has great progress compared with existent indirect reagent.

TABLE 7

Sensitivity comparation of the present
invention kit and indirect ELISA kit

serum dilution (times)

Diagnostic kit	1	25	50	100	200

The present	100	100	99	97	84
invention kit
InTec indirect kit	100	72	59	37	26
Murex Anti-HCV 4.0	100	83	69	45	35

Note:
the number is positive detect number in 100 shares HCV positive serum

(2) Specificity

Because the present invention takes three steps specific recognition, it has great improvement than only one step specific recognition of indirect reagent. We respectively detect 3000 shares clinical negative serum using the present invention kit and HCV indirect kit of InTec products, Inc. (Xiamen). The false positive rate of this invention reagent is 0.1%, but that of InTec indirect kit is 0.67%. And we furthermore use Murex Anti-HCV 4.0 indirect ELISA kit to detect the tatter's 20 shares false positive serum, there also is 12 shares detected be positive, false positive minimal rate is 0.4% thereof.

(3) Suspicious Serum

Through screening, we get following 12 shares suspicious sera which are assessed as positive by own indirect reagent, as suspicious by HCV RIBA confirmation reagent (Singapore Genelabs Inc., Article No. 11130-018), as negative by PCR detection (Switzerland ROCHE Inc. COBAS Ampliscreen™ HCV 2.0), and thereof suppliers have not any hepatitis symptom (data are showed in tab 8). Beside C5, there is 11 shares serum are negative in sandwich method, and beside C4 and C6, there is 9 shares serum are NS4 single positive in RIBA result, and the two antigen of this sandwich method have not NS4 segment. In summary, we can conclude that 9 shares sera should be positive by our own indirect reagent. C4 and C6 all be negative detection by sandwiched reagent, Murex reagent and PCR, compare the clinic to conclude that the two are our own indirect reagent positive. Though C5 is positive in sandwich method detecting, the OD value of the sandwich reaction is conspicuous lower than indirect method, and conclude that these is common false positive of indirect and sandwich method. The same to deduction that besides C4 and C6 other 10 shares sera are Murex Anti-HCV 4.0 kit false positive. In the end, the accuracy of this invention kit assessment suspicious serum is higher than existent commercial reagent and our own indirect reagent.

TABLE 4

Information of detecting suspicious sera (all be negative) by the present invention kit

		coating antigen	biotin labeled	Murex	Genelabs HCV
		indirect ELISA	antigen indirect	Anti-HCV	BLOT
sample	the present invention	detection	ELISA detection	4.0	Version 3.0	PCR

C1	−	+	+	+	NS4 +	−
C2	−	+	+	+	NS4 +	−
C3	−	+	+	+	NS4 +	−
C4	−	+	+	−	NS3-1 +	−
C5	weak−	+	+	+	NS4 +	−
C6	−	+	+	−	NS3-1 +	−
C7	−	+	+	+	NS4 +	−
C8	−	+	+	+	NS4 +	−
C9	−	+	+	+	NS4 +	−
C10	−	+	+	+	NS4 +	−
C11	−	+	+	+	NS4 +	−
C12	−	+	+	+	NS4 +	−

Note:
+ _positive;
− _negative

(4) Reproducibility

240 shares of negative and positive specimen to be detected respective, are detected in different time, in different patches, by different operators to test result reproducibility of the present invention kit. The result shows reproducibility of the present invention kit to negative and positive assessment result is 100%, which indicates that this method has good reproducibility of detection result and the assessment result is reliable.

(5) Stability

Store the whole set of reagent (include coating ELISA plate, specimen dilution, secondary antigen, secondary antigen diluent, streptavidin label, enzyme dilution, negative control positive control, color A and B, quench solution) for 6 days at 37° C., then take out these reagents to detect the same negative and positive quality control serum at the same condition with other reagents stored at 4° C. at the same time, thereof result showed in tab 9. The experiment indicate that the sample's OD value has not conspicuous different between the two addition, and indicates that the present invention has good stability.

TABLE 9

Experiment result of stability of the present invention kit

			mean of	mean of
			positive	negative
store condition	linearity	CV value	control	control

store at 4° C.	99.90%	9%	2.500	0.050
37° C., 72 h,	99.90%	8%	2.318	0.048

(6) Precision

10 or more wells parallel Repeated detection is carried out to the same known positive sample in the same reaction plate, and get OD value of each well, then calculate the CV value. The CV value is all lower than 15%, which indicates the present kit has good precision.
We also do the same test using the sandwiched kit prepared with direct enzyme-labeled HCV antigen, and get similar result, only the sensitivity is low slightly.

Claims

1.-17. (canceled)

18. A kit for detection of anti-HCV antibodies, said kit complete the detection in the form of ‘solid support-the primary antigen-HCV antibodies to be detected-the secondary antigen-enzyme label-recognizable signal’, wherein the secondary antigen carry on the combination reaction with the enzyme label in one step or several steps, said kit comprise sample diluent, the secondary antigen diluent and enzyme label diluent, wherein said secondary antigen diluent and enzyme diluent are packed separately.

19. The kit of claim 18, wherein diluent of the secondary antigen contains the reducing agent.

20. The kit of claim 19, wherein the reducing agent is β-mercaptoethanol, 1,4-dithiothreitol, dithio-erythritol, reduced glutathione, cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride.

21. The kit of claim 20, wherein the reducing agent is β-mercaptoethanol.

22. The kit of claim 21, wherein when specimen to be detected and the secondary antigen are added into reaction system simultaneously and incubated together, specimen diluent and the secondary antigen diluent can be the same solution, i.e. the secondary antigen diluent in one container, wherein the concentration of β-mercaptoethanol in the secondary antigen diluent is 0.01-50‰, prefers 0.05-20‰, further prefers 1-10‰, most prefers 5‰.

23. The kit of claim 21, wherein specimen to be detected and the secondary antigen are added into reaction system by two steps successively and incubated respectively, wherein the concentration of β-mercaptoethanol in said secondary antigen diluent is 0.01-50‰, prefers 0.05-30‰, prefers 0.2-20‰, further prefers 0.5-10‰, further prefers 0.5-5‰, most prefers 1‰.

24. The kit of claim 18, wherein specimen diluent contain reducing agent.

25. The kit of claim 23, wherein specimen diluent contain reducing agent.

26. The kit of claim 24, wherein the reducing agent is β-mercaptoethanol, 1,4-Dithiothreitol, dithio-erythritol, reduced glutathione, Cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride.

27. The kit of claim 26, wherein the reducing agent is β-mercaptoethanol.

28. The kit of claim 27, wherein the concentration of β-mercaptoethanol in specimen diluent is 0.01-50‰, prefers 0.1-20‰, further prefers 0.5-10‰, most prefers 5‰.

29. The kit of claim 18, said kit include pre-coating ELIS A plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box.

30. The kit of claim 23, said kit include pre-coating ELISA plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box.

31. The kit of claim 27, said kit include pre-coating ELISA plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box.

32. The kit of claim 18, said secondary antigen is conjugate of HCV protein and tag, said tag is peptide tag or non-peptide tag.

33. The kit of claim 32, wherein peptide tag is the peptide or protein containing His Tag, T7 Tag, S Tag, Flag Tag, HA Tag or HCV peptide fragment.

34. The kit of claim 32, wherein the non-peptide tag is compound, hapten, vitamin, steroid, dyestuff, hormone, antibiotic, nucleic acid or conjugate of all above component with peptide or protein.

35. The kit of claim 32, wherein the compound is dinitrophenol, bromodeoxyuridine; vitamin is biotin or derivatives thereof; steroid is digoxin; dyestuff is acridinium ester, rhodamine, dansyl chloride or dihydroxyfluorane.

36. The kit of claim 32, wherein the tag is biotin.

37. A method of detection of a kit of claim 1, wherein said method complete the detection in the form ‘solid support-the primary antigen-HCV antibodies to be detected-the secondary antigen-enzyme label-recognizable signal’, and comprise the following procedure:

A. the primary antigen catch antibody: add the specimen to be detected and incubate, then the primary antigen on pre-coating ELSIA plate catch the HCV antibody of specimen;

B. the antibody catch the secondary antigen: add the secondary antigen solution and incubate, HCV antibody catch the secondary antigen;

C. detection: introduce enzyme label by tag in one or more incubating steps and then detect;

wherein the step B is carried out in the reduction condition, step A and B can merge, i.e. specimen to be detected and the secondary antigen can be added into the reaction system simultaneously and incubated together, wherein said secondary antigen and enzyme label are added into reaction system in two steps successively and incubated respectively.