CN101266249B - Reagent kit and method for measuring HCV antibody - Google Patents
Reagent kit and method for measuring HCV antibody Download PDFInfo
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- CN101266249B CN101266249B CN2008100847154A CN200810084715A CN101266249B CN 101266249 B CN101266249 B CN 101266249B CN 2008100847154 A CN2008100847154 A CN 2008100847154A CN 200810084715 A CN200810084715 A CN 200810084715A CN 101266249 B CN101266249 B CN 101266249B
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Abstract
A reagent kit for measuring an HCV antibody is described herein. This reagent comprises a first and second reagents. The first reagent comprises an HCV synthetic peptide antigen which has a solid phase binding site. The second reagent comprises a solid phase. On the solid phase is immobilized a binding substance and/or an HCV recombinant HCV antigen. The binding substance can bind to the solid phase binding site. The invention also provides a method for measuring an HCV antibody in a sample. This method comprises a step for contacting the sample, the first reagent and the second reagent, and a step for detecting a first complex comprising HCV synthetic peptide antigen and HCV antibody formed on solid phase and/or a second complex comprising HCV gene recombinant antigen and HCV antibody.
Description
Technical field:
The mensuration that the present invention relates to a kind of HCV (HCV) antibody is with kit and assay method.
Background technology:
Hepatitis C is a kind of communicable disease that is caused by HCV (HCV).
HCV is the virus of strand (+chain) rna gene group with total length 9.5kb, and its genome structure is made up of 5 ' end non-translational region, translation area and 3 ' end non-translational region.
Have corresponding to core protein and envelope protein matter (E1, E2) territory of these two structural proteins in the translation area that constitutes by about 3000 amino acid residues.Also has territory corresponding to NS2, NS3, NS4, these nonstructural proteins of NS5.
As the detection method of HCV is known the immunological method that detects in the sample with the corresponding antibody of HCV antigen (HCV antibody) arranged.This method is the antigen-antibody complex that generates of the HCV antigen in the detectable and the HCV antibodies in the sample normally.Use in the immunology detection of HCV antibody and contain HCV antigen, so that can detect the multiple HCV antibody that to discern different types of HCV protein corresponding to various HCV protein sequences.As everyone knows, HCV antigen has material of being separated by natural material and the material of producing through methods such as method of gene recombination and chemical synthesiss.As this HCV antigen such as disclosed things such as No. the 5308750th, United States Patent (USP) is arranged No. 5350671, No. the 5910404th, United States Patent (USP) and United States Patent (USP)s.
The traditional kit that is used for immunology detection HCV antibody all contains several HCV antigens that are fixed on the solid phase.Let HCV antibody response in these antigens and the sample, on this solid phase, generate antigen-antibody complex, detect compound, measure HCV antibody with this.
Summary of the invention:
Inventor of the present invention uses in the process of HCV TPPA with reagent of HCV antigenic synthetic peptide and HCV gene recombinant antigens in research, and the stability and the reactivity that just contain the reagent of these antigens are inquired into.The result finds that its stability of HCV antigenic synthetic peptide and HCV gene recombinant antigens is with reactive different.
The present invention provides a kind of HCV TPPA to use kit, comprising: contain first reagent of the HCV antigenic synthetic peptide that has added the solid phase binding site and second reagent of the solid phase that contains the bound substances that is fixed with the HCV gene recombinant antigens and can be incorporated into said solid phase binding site.
The present invention also provides a kind of HCV TPPA to use kit, comprising: contain first reagent of the HCV antigenic synthetic peptide that has added the solid phase binding site and contain first solid phase that is fixed with the HCV gene recombinant antigens and second reagent that is fixed with second solid phase of the bound substances that can be attached to said solid phase binding site.
Said HCV antigenic synthetic peptide is present in first reagent with free state.
Said HCV gene recombinant antigens contains HCV nonstructural proteins peptide.
Said HCV gene recombinant antigens is be selected from the group that constitutes with genetic recombination NS3 antigen, genetic recombination NS4 antigen, genetic recombination NS5 antigen and genetic recombination cAg a kind of at least; Said HCV antigenic synthetic peptide is be selected from the group that constitutes with synthetic peptide NS4 antigen, synthetic peptide NS5 antigen and synthetic peptide cAg a kind of at least.
Said first reagent contains and different types of the 2nd HCV antigenic synthetic peptide of said HCV antigenic synthetic peptide, and said the 2nd HCV antigenic synthetic peptide contains the solid phase binding site that can combine with said bound substances.Said HCV antigenic synthetic peptide contains HCV structural protein peptide, and said the 2nd HCV antigenic synthetic peptide contains HCV nonstructural proteins peptide.Said HCV antigenic synthetic peptide is a cAg, and said the 2nd HCV antigenic synthetic peptide is NS4 antigen or NS5 antigen, and said HCV gene recombinant antigens is a NS3 antigen.
Said first solid phase and second solid phase are the micropores of magnetic particle, latex particle or microtiter plate.
Said solid phase binding site contains biotin, and said bound substances is an antibiosis protein class.
Described kit also comprise contain be labeled substance markers and can with the 3rd reagent of the material of HCV antibodies.Said can be antibody with the material of HCV antibodies.Said label is an enzyme, also comprises the 4th reagent that contains the substrate relative with said enzyme.
The present invention provides a kind of HCV TPPA method; Comprise: contact procedure, first reagent that let sample, contains the HCV antigenic synthetic peptide that has added the solid phase binding site contacts with second reagent that contains the solid phase that is fixed with HCV gene recombinant antigens and the bound substances that can be incorporated into said solid phase binding site; And detect step, detect the HCV antigenic synthetic peptide and first compound of HCV antibody and/or second compound of HCV gene recombinant antigens and HCV antibody that on solid phase, form.
The present invention provides another kind of HCV TPPA method; Comprise: contact procedure, first reagent that let sample, contains the HCV antigenic synthetic peptide that has added the solid phase binding site contacts with second reagent that contains first solid phase that is fixed with the HCV gene recombinant antigens and second solid phase that is fixed with the bound substances that can be attached to said solid phase binding site; And detect step, first compound of contained HCV antibody and/or second compound of HCV gene recombinant antigens that on said first solid phase, forms and the contained HCV antibody of said sample in HCV antigenic synthetic peptide that detection forms and the said sample on said second solid phase.
Kit is used in HCV TPPA provided by the invention, and its various HCV antigen storage stabilities that are used to measure are superior, and reactive good with antibody, and making accurately to measure for a long time becomes possibility.Use HCV TPPA provided by the invention can prevent that with kit the mensuration sensitivity that HCV antigen storage stability lowly causes from descending, thereby can measure more accurately.
Description of drawings:
Fig. 1 is the mode chart of HCV TPPA of the present invention with a kind of embodiment of kit;
Fig. 2 is reference example 1 result's a displayed map;
Fig. 3 is the result's that mensuration positive serum 1 is obtained in the reference example 2 a displayed map;
Fig. 4 is the result's that mensuration positive serum 2 is obtained in the reference example 2 a displayed map;
Fig. 5 is the result's that mensuration positive serum 7 is obtained among the embodiment 2 a displayed map;
Fig. 6 is the result's that mensuration positive serum 8 is obtained among the embodiment 2 a displayed map;
Fig. 7 is the result's that mensuration positive serum 9 is obtained among the embodiment 2 a displayed map;
Fig. 8 is the result's that mensuration positive serum 10 is obtained among the embodiment 2 a displayed map;
Fig. 9 is the result's that mensuration positive serum 11 is obtained among the embodiment 3 a displayed map;
Figure 10 is the result's that mensuration positive serum 12 is obtained among the embodiment 3 a displayed map;
Figure 11 is the result's that mensuration positive serum 13 is obtained among the embodiment 3 a displayed map;
Figure 12 is the result's that mensuration positive serum 14 is obtained among the embodiment 3 a displayed map.
Embodiment:
HCV (HCV) has the genome of the protein that coding is made up of about 3000 amino acid residues.The protein that is made up of about 3000 amino acid residues is by structural protein territory (core protein and envelope protein matter (E1; E2)) and nonstructural proteins territory (NS2, NS3, NS4, NS5) constitute, known these territories are the antigen that has an antigenic determinant at least.The sequence illustration in contained each territory is such as on the books on the United States Patent (USP) the 5350671st in the protein of the genome encoding of HCV and this protein.
HCV is the virus that highly makes a variation, and the protein sequence of its genome encoding cannot treat different things as the same.So-called in this manual " antigen ", the two includes with there being antigenic its to make a variation for have antigenic protein or the peptide of HCV genome encoding.
The HCV TPPA of this embodiment has first reagent and second reagent with kit.First reagent contains the HCV antigenic synthetic peptide that has added the solid phase binding site.Second reagent contains the two kinds of solid phases (first solid phase and second solid phase) that are fixed with the material that has nothing in common with each other.First solid phase is fixed with the HCV gene recombinant antigens, and second solid phase is fixed with the bound substances that can be incorporated into the above-mentioned solid phase binding site.
The HCV TPPA of another embodiment of the present invention has above-mentioned first reagent to fix the two second reagent of solid phase of HCV gene recombinant antigens and bound substances with containing with kit.
Explanation utilization HCV TPPA is with the principle of kit measurement HCV below.
Contained HCV antibody can combine with HCV antigenic synthetic peptide in first reagent and/or the HCV gene recombinant antigens in second reagent in the sample.The solid phase binding site that is attached on the first reagent HCV antigenic synthetic peptide can combine with the bound substances in second reagent.At this, sample, first reagent and second reagent one contact, and the HCV gene recombinant antigens promptly combines the HCV antibody in the sample, and the HCV antigenic synthetic peptide combines the HCV antibody in the sample.Be fixed on the HCV antigenic synthetic peptide that can combine to have added the solid phase binding site on the bound substances on the solid phase.
The HCV antibody that so captures on the solid phase can be used in the well-known method detection of this technical field.
Above-mentioned HCV antigenic synthetic peptide is from the synthetic peptide of native protein separation, cut-out or chemical synthesis, contains an antigenic determinant at least.The HCV antigenic synthetic peptide is advisable with the amino acid residue that contains 8 HCV protein, the amino acid residue of 60 following HCV protein at least.Preferably contain the amino acid residue of at least 10 HCV protein, the amino acid residue of 50 following HCV protein.HCV antigenic synthetic peptide molecular weight is between 900～7500, to be advisable, and is better between 1000～6000.
The HCV antigenic synthetic peptide can be used such as well-known traditional chemical synthesis such as solid-phase synthesis, fragment condensation, solution synthetic method and obtain.This antigenic synthetic peptide is preferably used Merrifield, J.Am.Chem.Soc., and 85, p.2149, the solid-phase synthesis of recording and narrating on 1963 is synthetic.
The preferably at least a group that constitutes with synthetic peptide NS4 antigen, synthetic peptide NS5 antigen and synthetic peptide cAg that is selected from of above-mentioned HCV antigenic synthetic peptide.These antigenic synthetic peptides can use more than two kinds.The sequence of these antigens such as can as No. the 5350671st, United States Patent (USP) the above, as long as but have antigenicity to be not limited to these specific sequences, also can contain variation.In this manual, such as when saying " synthetic peptide cAg ", refer to contain the antigenic synthetic peptide of the amino acid sequence of the amino acid sequence that derives from HCV protein core territory.
Above-mentioned HCV antigenic synthetic peptide is by the peptide of method of gene recombination preparation, contains an antigenic determinant at least.This HCV antigenic synthetic peptide is advisable to have 80 amino acid residues at least, has at least 90 amino acid residues better.The HCV gene recombinant antigens is to be advisable more than 9000 with molecular weight, and is better more than 10000.
This HCV gene recombinant antigens can be used traditional known recombination method preparation.Such as the amino acid sequences encoded DNA that can make up to want antigen, on suitable expression, make appropriate host cell on this carrier, carry out the conversion of shape matter this dna clone, prepare the HCV gene recombinant antigens with this.This HCV gene recombinant antigens prepares such as using United States Patent (USP) to go up the method for gene recombination of recording and narrating for No. 5350671.
The preferably at least a group that constitutes with genetic recombination NS3 antigen, genetic recombination NS4 antigen, genetic recombination NS5 antigen and genetic recombination cAg that is selected from of above-mentioned HCV gene recombinant antigens.The sequence of these antigens can be that United States Patent (USP) is gone up the sequence of recording and narrating No. 5350671, as long as but antigenicity is arranged, to these specific sequence indefinite, also variation can be arranged.In this manual, such as when saying " genetic recombination NS3 antigen ", refer to contain the gene recombinant antigens of amino acid sequence of the amino acid sequence in the NS3 territory that derives from HCV protein.
Above-mentioned solid phase binding site and bound substances do not have special qualification, so long as the combinations of substances that can specificity when under using the condition of kit, making its contact combines gets final product.These combinations are such as biotin and antibiosis protein class, haptens and antihapten antibody, nickel and histidine-tagged, glutathione and glutathione-S-transferase etc. are arranged.As haptens and antihapten antibody if any DNP and anti-DNP antibody.Suitable with being combined as of biotin and antibiosis protein class, preferably the solid phase binding site contains biotin, and bound substances is an antibiosis protein class.In this manual, " antibiosis protein class " refers to comprise antibiosis protein and streptococcus antibiosis protein.
In this manual, what is called refers to make the HCV antigenic synthetic peptide have the solid phase binding site through reaction at HCV antigenic synthetic peptide " additional solid phase binding site ".This reaction is as long as suitably select according to the kind of above-mentioned solid phase binding site, such as can being to have chemical bond such as combination.
The method that the solid phase binding site is appended to the HCV antigenic synthetic peptide is well-known.Such as, when the solid phase binding site contains biotin, can through with the HCV antigenic synthetic peptide in the base of responding property such as amino and mercapto make the antibiosis protein be attached to this antigen.As the base that responds with amino if any the NHS base, as the base that responds with mercapto if any the maleic amide base etc.
The above-mentioned method that bound substances is fixed on the solid phase also is well-known.Should fixing can take the methods such as combination of physisorphtion, total combined techniques, ions bind method and these methods to carry out.
When bound substances is an antibiosis protein time-like, such as taking physisorphtion directly the antibiosis protein to be fixed on the solid phase.Also can be combined with on the solid phase of material such as biotin that can combine, antibiosis protein class is fixed on the solid phase through antibiosis protein class is attached to antibiosis protein class.Can also disclose the 2006-226689 number last method of recording and narrating with Jap.P. is fixed on antibiosis protein class on the solid phase.
Make the solid phase that antibiosis protein class combines also can be from such as JSR Corp. and the purchases such as (Dynal Biotech) of Dai Nuo biotech company.
The method that the HCV gene recombinant antigens is fixed on the solid phase carrier is well-known.Should fixing can adopt the combination of physisorphtion, total combined techniques, ions bind method and these methods etc.
Also can the HCV gene recombinant antigens be fixed on the solid phase through the combination of biotin and antibiosis protein class.As to the fixing combination of media of HCV gene recombinant antigens of solid phase, except that above-mentioned biotin and antibiosis protein class, haptens and antihapten antibody, nickel and histidine-tagged, glutathione and glutathione-S-transferase etc. can also be arranged.As haptens and antihapten antibody if any DNP and anti-DNP antibody.
The means that the HCV gene recombinant antigens is fixed on the solid phase carrier also can be identical with the means that bound substances is fixed on the solid phase carrier, also can be different.
The material of above-mentioned first solid phase and second solid phase can be identical, also can be different, and should be from the simplicity that kit is produced with identical materials.This material is that the common solid phase material that can be used for immunoassays gets final product, and does not have especially to limit.Such as being latex; Rubber; Tygon; Polypropylene; Styrene resin; SB; PVC; Polyvinyl acetate (PVA); Polyacrylamide; Polymethacrylate; Styrene-methacrylate copolymer; Poly (glycidylmethacrylate--co-ethylene dimethacrylate); Acryl aldehyde-ethylene glycol dimethacrylate multipolymer; Kynoar; Polymeric material (silicon etc.); Agarose; Gelatin; Red blood cell; Silica gel; Glass; The non-activity alumina; Inorganic material (magnet etc.) etc.Also can be with a kind of or use that combines more than two kinds wherein.
, not having especially and limit so long as be used for the common solid shape of immunoassays as the shape of solid phase, can be microtiter plate, test tube, microballoon, particle, nano particle etc.As particle if any magnetic particle, resemble hydrophobic particles, particle surface the styrene resin latex copolymer latex particle, red blood cell, gelatine particle of hydrophilic groups such as amino, carboxyl etc. arranged.
Preferably solid phase is the micropore of magnetic particle, latex particle or microtiter plate.
Magnetic particle is to be the particle of base material with magnetic material.This magnetic particle is known in this technical field, as the known use of base material Fe
2O
3And/or Fe
3O
4, cobalt, nickel, ferrite, MAG etc.In order to make protein etc. be attached to the magnetic particle surface, preferably use the base material that encapsulates the surface with polymkeric substance etc.
Microtiter plate can suitably use the thing that generally in enzyme linked immunosorbent assay (ELISA) (ELISA), uses, the microwell plate that preferably is made up of materials such as styrene resin, polypropylene.
As stated, above-mentioned first reagent contains the HCV antigenic synthetic peptide with free state.First reagent preferably the HCV antigenic synthetic peptide be dissolved in do not influence HCV antigenic synthetic peptide stability and with reactive damping fluid of HCV antibody in liquid condition.Or also can through the solid state that freeze-dried HCV antigenic synthetic peptide obtains.In this state, this reagent will add appropriate solvents such as water when measuring, use with solution.
When first reagent is solution state, and when containing several HCV antigenic synthetic peptides, this first reagent can contain several HCV antigenic synthetic peptides in same damping fluid, can comprise that also several contain the damping fluid with a kind of HCV antigenic synthetic peptide.First reagent preferably contains several HCV antigenic synthetic peptides in a kind of damping fluid.
When first reagent was solution, the concentration of HCV antigenic synthetic peptide can suitably be selected according to the kind of HCV antigenic synthetic peptide and quantity thereof etc.
Above-mentioned pH of buffer value is advisable with 6.5～8.0, such as using phosphate buffer normal saline (PBS), triethanolamine hydrochloride damping fluid (TEA), trishydroxymethylaminomethane hydrochloride buffer (Tris-HCI) etc.This damping fluid also can contain well-known adjuvants such as surfactant, preservative agent, serum proteins.
Above-mentioned second reagent can contain the solid phase that has been fixed with several HCV gene recombinant antigens when containing several HCV gene recombinant antigens, also can contain several and fix the solid phase with a kind of HCV gene recombinant antigens, also can be this combination of two kinds.
Above-mentioned second reagent preferably provides solid phase with outstanding state turbid or that contact in suitable damping fluid.When second reagent comprised first solid phase and second solid phase, first solid phase can be hanged turbid with second solid phase or contacted different damping fluids, but used the simplicity of operating from kit, and is preferably outstanding turbid at same damping fluid.The outstanding turbid state at damping fluid of so-called solid phase hangs turbid state at damping fluid such as having as particles such as the magnetic particle of solid phase and latex particles.The state of so-called solid phase contact damping fluid is such as being the state that is added with buffering agent in the hole as the microtiter plate of solid phase.
When second reagent comprised first solid phase and second solid phase, the ratio of first solid phase and the second solid phase quantity can suitably be selected according to the kind and the quantity of HCV antigenic synthetic peptide in first reagent, the kind and the quantity that are fixed on the HCV gene recombinant antigens on first solid phase, the kind that is fixed on the bound substances on second solid phase and quantity thereof etc.Such as, when first solid phase and second solid phase were magnetic particle, contained magnetic particle quantity can be in first solid phase in second reagent: second solid phase=1～10: between 10～1.
Above-mentioned damping fluid can use and the same liquid of liquid that can be used for first reagent.
When be fixed with on the contained solid phase of second reagent HCV gene recombinant antigens and bound substances the two time, as long as the bound substances that exists the contained HCV antigenic synthetic peptide of HCV gene recombinant antigens and enough first reagent of contained HCV antibodies in enough samples to combine on the solid phase.These quantity are than can suitably selecting according to kind and the kind of quantity and HCV gene recombinant antigens, the kind of bound substances etc. of HCV antigenic synthetic peptide in first reagent.Such as, the molecular weight that is fixed on HCV gene recombinant antigens and bound substances on the solid phase in the HCV gene recombinant antigens: bound substances=between 1: 10 to 10: 1.When the HCV gene recombinant antigens is that genetic recombination NS3 antigen, bound substances are antibiosis protein time-like, such as genetic recombination NS3 antigen: between antibiosis protein class=1: 2～8.
Above-mentioned HCV TPPA preferably also contains the 3rd reagent with kit, the 3rd reagent contain can with the material of the HCV antibodies that is labeled the material mark.
Can comprise with the material of HCV antibodies and the corresponding antibody of HCV antibody, HCV antigen etc.Also comprise antibody fragment and derivant thereof at this illustrative antibody.Specifically such as Fab fragment, F (ab ') fragment, F (ab) are arranged
2Fragment and sFv fragment etc.The classification of antibody can comprise IgG, IgM etc., but is not limited thereto.
The mark substance that above-mentioned mark substance uses in general immunoassay gets final product, and does not have especially to limit, if any enzyme, fluorescent material and radioactive isotope etc.As enzyme if any alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, acid phosphatase etc.As fluorescent material if any fluorescein isothiocynate (FITC), egfp (GFP), luciferin etc.As radioactive isotope if any
125I,
14C,
32P etc.
When above-mentioned mark substance was enzyme, above-mentioned HCV TPPA preferably also contained the 4th reagent with kit, contained in the 4th reagent and the corresponding substrate of this enzyme.
Above-mentioned substrate can use well-known substrate in about this technical field of above-mentioned enzyme.When using alkaline phosphatase to make above-mentioned enzyme; Can use the luminous substrate all known in this technical field and chromogenic substrate etc.; Have such as luminous substrate: CDP-star (registered trademark) (4-chloro-3-(the methoxyl spiral shell 1,2-dioxetane-3,2 '-(5 '-chlorine) three ring [3.3.1.1
3,7] decane-the 4-yl) disodium phenylphosphate), CSPD (registered trademark) (3-(4-methoxyl spiral shell 1,2-dioxetane-3,2-(5 '-chlorine) three ring [3.3.1.1
3,7] decane-the 4-yl) disodium phenylphosphate) etc.; Chromogenic substrate has: P-nitrobenzophenone phosphate, 5-bromo-4-chloro-3-indolylphosphate (BCIP), nitroblue tetrazolium (NBT) (NBT), iodonitrotetrazolium (INT) etc.
Above-mentioned the 4th reagent preferably is dissolved in the solution state in the suitable damping fluid with substrate.The concentration of substrate can suitably be selected according to the kind of substrate.The damping fluid that is used for the 4th reagent can suitably be selected according to the kind of substrate, can use the thing of generally acknowledging in this technical field.
The HCV TPPA with kit in, first reagent and second reagent are packed respectively.Fig. 1 is that first reagent is that solution, second reagent are kit one examples during with the outstanding turbid suspension in damping fluid of solid phase.Among Fig. 1, first reagent is placed in first reagent container 1, and second reagent is placed in second reagent container 2.The HCV TPPA also can have other reagent containers of other reagent of dress (such as above-mentioned the 3rd reagent and the 4th reagent) as required with kit.The HCV TPPA also can have a kind of or several damping fluids of a kind of reagent of dilution or several agents etc. as required with kit.
The method of the mensuration HCV antibody of this embodiment comprises: contact procedure makes sample, first reagent, the contact of second reagent; Detect step, detect the HCV antigenic synthetic peptide and first compound of HCV antibody and/or second compound of HCV gene recombinant antigens and HCV antibody that on solid phase, form.
Above-mentioned sample is the biological sample that picks up from animal, such as blood, serum and blood plasma etc.
Above-mentioned contact procedure both can let earlier sample with contact second reagent again after first reagent contacts, also can let earlier sample with contact first reagent again after second reagent contacts.Contact sample again after perhaps also can making first reagent earlier and second reagent contacts.HCV antibody from be free in sample be free in HCV antigen in the reagent and combine this point to consider easily, let sample with to contact second reagent again after first reagent contacts better earlier.Comprise compound sample, first reagent and second reagent in this what is called " contact ".
Above-mentioned contact procedure can be advisable under the temperature of sound response with the HCV antigen in the HCV antibody in sample and first reagent and second reagent, and 30～45 ℃ temperature is better.
Can suitably select duration of contact according to the kind of used HCV antigenic synthetic peptide, HCV gene recombinant antigens, solid phase binding site and bound substances.
Above-mentioned detection step can be undertaken by the immunological detection method that this technical field is generally acknowledged, preferably uses and can carry out with the material with the HCV antibodies of above-mentioned mark substance mark.
Above-mentioned detection step preferably let can with contacted with above-mentioned first compound and/or second compound by the material of the HCV antibodies of above-mentioned mark substance mark, detect above-mentioned mark substance.
The detection of mark substance can be measured because catalysis the light that substrate reactions sent corresponding with above-mentioned enzyme, look etc. with appropriate device during for enzyme when this mark substance.As this device such as spectrophotometer, chemiluminescence ELIASA etc. are arranged.
[embodiment]
The preparation method of each the HCV antigen that uses in an embodiment is described below.
(1) preparation of genetic recombination NS3 antigen
As the HCV gene recombinant antigens, preparation has the genetic recombination NS3 antigen of the interval amino acid sequence of jp patent publication H05-508219 number amino acid sequence shown in Figure 1 1192～1457 as the embodiment 1 of jp patent publication H05-508219 number records and narrates.
(2) preparation of synthetic peptide NS4 antigen
As antigenic synthetic peptide, has the synthetic peptide NS4 antigen (No. 2995216 peptide VIII of patent) of 1688～1707 amino acid sequences in No. 2995216 amino acid sequence shown in Figure 1 of Jap.P. according to the said method of peptide synthesis preparation of the embodiment of No. the 2995216th, Jap.P..With (Sulfo-0Su) the synthetic peptide NS4 antigen of ((strain) colleague chemistry institute) biotinylation of biotinylation kit (Biotinylation Kit).
(3) preparation of synthetic peptide cAg
As antigenic synthetic peptide, have the synthetic peptide cAg (No. 2995216 peptide II of patent) of the 7th～26 section amino acid sequence in No. 2995216 amino acid sequence shown in Figure 1 of Jap.P., the synthetic peptide cAg (No. 2995216 peptide V of patent) that has the synthetic peptide cAg (No. 2995216 peptide III of patent) of the 13rd～32 section amino acid sequence and have the 49th～68 section amino acid sequence according to the said method of peptide synthesis preparation of the embodiment of No. the 2995216th, Jap.P..Use biotinylation kit (Sulfo-0Su) ((strain) colleague chemistry institute) biotinylation to synthesize the peptide cAg again.
(4) the fixedly preparation of the magnetic particle of genetic recombination NS3 antigen
The magnetic particle (mean grain size 2 μ m) that market is sold adds in the 20mM sodium phosphate buffer (pH7.5), makes concentration reach about 5mg/mL, preparation magnetic particle suspension.The genetic recombination NS3 antigen of above-mentioned (1) preparation is dissolved in the 20mM sodium phosphate buffer (pH7.5), makes concentration reach about 1mg/mL, preparation NS3 antigenic solution.Mictomagnetism particle suspension 10mL and NS3 antigenic solution 0.1mL cultivated 30 minutes, genetic recombination NS3 antigen are fixed on the magnetic particle for 37 ℃.Then, Mixed Stationary has the magnetic particle and the confining liquid (1%BSA, 20mM sodium phosphate buffer, pH7.5) of NS3 antigen, cultivates 30 minutes, and encapsulates processing for 37 ℃.Abbreviate the magnetic particle that so obtains as NS3 antigen magnetic particle later on.
(5) preparation of the magnetic particle of fixing synthetic peptide NS4 antigen
In 20mM sodium phosphate buffer (pH7.5), add the Streptavidin of selling in market and encapsulate magnetic particle (to call the ST magnetic particle in the following text), make concentration reach about 5mg/mL, preparation ST magnetic particle suspension.The synthetic peptide NS4 antigen of above-mentioned (2) preparation is dissolved in the 20mM sodium phosphate buffer (pH7.5), makes concentration reach about 1mg/mL, preparation NS4 antigenic solution.Mix ST magnetic particle suspension 10mL and NS4 antigenic solution 0.01mL, cultivated 30 minutes for 37 ℃, synthetic peptide NS4 antigen is fixed on the magnetic particle.Then, Mixed Stationary has the magnetic particle and the above-mentioned confining liquid (1%BSA, 20mM sodium phosphate buffer, pH7.5) of NS4 antigen, cultivates 30 minutes down, encapsulates processing for 37 ℃.The magnetic particle that so obtains abbreviates NS4 antigen magnetic particle later on as.
(6) preparation of the magnetic particle of fixing synthetic peptide cAg
In 20mM sodium phosphate buffer (pH7.5), add the ST magnetic particle that sell in market, make concentration reach about 1mg/mL, preparation ST magnetic particle suspension.Mix each synthetic peptide cAg of above-mentioned (3) preparation, it is dissolved in the 20mM sodium phosphate buffer (pH7.5), preparation cAg solution.CAg formulations prepared from solutions concentration is that the concentration of contained various synthetic peptide cAgs is about 1mg/mL.Mix ST magnetic particle suspension 10mL and cAg solution 0.01mL, cultivated 30 minutes down for 37 ℃, synthetic peptide cAg is fixed on the ST magnetic particle.Then, Mixed Stationary has the magnetic particle and the confining liquid (1%BSA, 20mM sodium phosphate buffer, pH7.5) of cAg, cultivates 30 minutes down, encapsulates processing for 37 ℃.The magnetic particle that so obtains abbreviates the cAg magnetic particle later on as.
[reference example 1]
Reactive difference when at first, it is free in the solution with not fixing when being fixed in magnetic particle with regard to genetic recombination NS3 antigen investigation.
NS3 antigen in this use above-mentioned (1) preparation is handled the biotinylation genetic recombination NS3 antigen that obtains through biotinylation.The biotinylation of NS3 antigen is handled use biotinylation kit (Sulfo-0Su) ((strain) colleague chemistry institute).
Biotinylation NS3 antigen is fixed in the method for ST magnetic particle, except that with biotinylation NS3 antigen as HCV antigen, with the ST magnetic particle as the magnetic particle, other are all identical with above-mentioned (4).
The reagent that uses is in this example formed as follows:
(7) first reagent (A)
20mM sodium phosphate buffer (pH7.5)
(8) first reagent (B-1)
20mM sodium phosphate buffer (pH7.5)
2.5 μ g/mL biotinylation NS3 antigen
(9) first reagent (B-2)
With 10 times of above-mentioned first reagent (B-1) dilutions, process first reagent (B-2) with 20mM sodium phosphate buffer (pH7.5).
(10) first reagent (B-3)
With 50 times of above-mentioned first reagent (B-1) dilutions, process first reagent (B-3) with 20mM sodium phosphate buffer (pH7.5).
(11) second reagent (A)
20mM sodium phosphate buffer (pH7.5)
3mg/mL biotinylation NS3 antigen combines the ST magnetic particle
2mg/mL ST magnetic particle
(12) second reagent (B)
20mM sodium phosphate buffer (pH7.5)
5mg/mL ST magnetic particle
(13) the 3rd reagent
20mM sodium phosphate buffer (pH7.5)
0.1U/mL alkali phosphatase enzyme mark mouse-anti human IgG antibody (ALKALINEPHOSPHATASE-MOUSE, Zymed Laboratories)
(14) the 4th reagent
The CDP-star (registered trademark) (applying biological company product) that Sapphire II (trade mark) arranged
Assay method is following:
The reactivity of genetic recombination NS3 antigen when (15) being fixed on magnetic particle
At first, mix HCV positive serum 10 μ L and above-mentioned first reagent (A) 120 μ L, cultivated about 2 minutes for 42 ℃.In this mixed liquor, add above-mentioned second reagent (A) 30 μ L then, cultivated about 1 minute for 45 ℃, make HCV antibody and the NS3 antigen-reactive on the magnetic particle in second reagent (A) in the serum.Behind cleaning fluid (0.1%Tween20,20mM sodium phosphate buffer (pH7.5)) flushing magnetic particle, add above-mentioned the 3rd reagent 100 μ L, incubated at room temperature 5 minutes.Behind above-mentioned cleaning fluid flushing magnetic particle, add above-mentioned the 4th reagent 100 μ L, with LUMI-COUNTER700 (Japanese Microtec Nition company) mensuration luminous intensity.
As the sample use is two kinds of HCV positive serums (positive serums 1 and positive serums 2).Also use 20mM sodium phosphate buffer (pH7.5) to replace the HCV positive serum to measure as negative quality-control product.Same sample is measured respectively three times, asked its average canbdle power.The result is as shown in Figure 2.
The reactivity of genetic recombination BS3 antigen when (16) being free in the solution
With above-mentioned first reagent (B-1), (B-2) or (B-3) as first reagent, as second reagent, in addition, use with above-mentioned (15) identical method and measure with above-mentioned second reagent (B).First reagent (B-1) contains free NS3 antigen, and is identical with fix N S3 antigen concentration in second reagent A.The result sees Fig. 2.
Among Fig. 2, the result when 1 expression genetic recombination BS3 antigen is fixed in magnetic particle, the result when 2～4 expression genetic recombination BS3 antigens dissociate in solution.The longitudinal axis representes to measure the gained luminous intensity.The result of the negative quality-control product of N.
Can know from Fig. 2, the HCV positive serum of appearance whatsoever, it is all high than the luminous intensity that lets genetic recombination NS3 antigen be free on to obtain in the solution that genetic recombination BS3 antigen is fixed in magnetic particle.By on can know, about the HCV gene recombinant antigens, be fixed on the magnetic particle specific ionization in solution, higher with the reactivity of HCV antibody.
[reference example 2]
Storage stability was different when it was free in the solution with not fixing when being fixed in magnetic particle with regard to the investigation of genetic recombination NS3 antigen below.
This example uses reference example 1 used second reagent (A) (containing fix N S3 antigen) and first reagent (B-2) (containing free NS3 antigen) to carry out accelerated test, measures the storage stability of reagent.In accelerated test, in the dark preserved second reagent (A) and first reagent (B-2) 0,1 or 5 day down for 37 ℃.Use each reagent of handling through accelerated test to measure HCV antibody.
So-called accelerated test refers to preserve reagent under than the high temperature of the temperature that should preserve, measure the test of storage stability etc.During the investigation storage stability, therefore the high more reagent storage stability that loses more easily of storage temperature, at high temperature preserves reagent through accelerated test, promotes the rotten of reagent.Like this, storage stability is i.e. forfeiture in the short time, can obtain the test findings of storage stability effect at short notice.
Assay method is following:
The stability of genetic recombination NS3 antigen when (17) being fixed in magnetic particle
Except that using second reagent (A) that passes through accelerated test as second reagent, other are used with the same method of reference example 1 said (15) and measure.Sample uses reference example 1 used two kinds of HCV positive serums (positive serum 1 and positive serum 2).Result such as Fig. 3 and shown in Figure 4.
The stability of genetic recombination NS3 antigen when (18) being free in the solution
Except that using first reagent (B-2) that passes through accelerated test as first reagent, other are used with the same method of reference example 1 said (16) and measure.Sample uses reference example 1 used two kinds of HCV positive serums (positive serum 1 and positive serum 2).Result such as Fig. 3 and shown in Figure 4.
Fig. 3 is the result when being sample with positive serum 1, and Fig. 4 is the result when being sample with positive serum 2.In Fig. 3 and Fig. 4, the result when 1 expression is fixed in magnetic particle with genetic recombination NS3 antigen, the result when 2 expressions let genetic recombination NS3 antigen be free in the solution.The longitudinal axis is for getting the number percent that average canbdle power is 100%, respectively preserves the average canbdle power of fate to survey the reagent place of (the 0th day) before the accelerated test, and transverse axis is represented the preservation fate of accelerated test.
Can find out from the result of Fig. 3 and Fig. 4, preserve with the state that lets genetic recombination NS3 antigen be free in the solution, poor stability, one day afterreaction property of accelerated test just sharply descends.And being fixed in magnetic particle, preserves genetic recombination NS3 antigen, good stability, and accelerated test still keeps very high stability after five days.By on learn, about the HCV gene recombinant antigens, be fixed on magnetic particle and preserve than let it and be free on storage stability height in the solution.
[embodiment 1]
The difference of the storage stability when being free in the solution with not fixing when being fixed in magnetic particle with regard to synthetic peptide NS4 antigen and the investigation of synthetic peptide cAg below.
At this, use synthetic peptide NS4 antigen of above-mentioned (2) preparation and the free HCV antigen of synthetic peptide cAg conduct of above-mentioned (3) preparation.The cAg magnetic particle of NS4 antigen magnetic particle and above-mentioned (6) preparation of NS3 antigen magnetic particle, above-mentioned (5) preparation that uses above-mentioned (4) preparation is as fixing HCV antigen.
The reagent that this example is used is formed as follows.What in addition, the 3rd reagent and the 4th reagent used is the reagent that reference example 1 uses.
(19) first reagent (C)
20mM sodium phosphate buffer (pH7.5)
1 μ g/mL synthesizes peptide NS4 antigen
Each synthetic peptide cAg of 10 μ g/mL
(20) first reagent (D)
20mM sodium phosphate buffer (pH7.5)
(21) second reagent (C)
20mM sodium phosphate buffer (pH7.5)
3mg/mL NS3 antigen magnetic particle
2mg/mL ST magnetic particle
(22) second reagent (D)
20mM sodium phosphate buffer (pH7.5)
3mg/mL NS3 antigen magnetic particle
1mg/mL NS4 antigen magnetic particle
1mg/mL cAg magnetic particle
In this example, carry out accelerated test, investigate the storage stability of reagent with first reagent (C) (contain free NS4 antigen and free cAg) and second reagent (D) (contain fix N S4 antigen and fixedly cAg).In accelerated test, in the dark preserved first reagent (C) and second reagent (D) 0 or 1 day down for 45 ℃.Use through each reagent of accelerated test and measure HCV antibody.
Assay method is following:
The stability of synthetic peptide NS3 antigen and synthetic peptide cAg when (23) in solution, dissociating
Divided by being first reagent through first reagent (C) of accelerated test, being outside second reagent that all the other use (15) same method of recording and narrating with reference example 1 to measure with above-mentioned second reagent (C).Sample is a kind of HCV negative serum (negative serum 1) and four kinds of HCV positive serums (positive serum 3～6).The result sees table 1.
The stability of synthetic peptide NS3 antigen and synthetic peptide cAg when (24) being fixed on magnetic particle
Divided by above-mentioned first reagent (D) be first reagent, to be outside second reagent through above-mentioned second reagent (D) of accelerated test, all the other use (15) same method of recording and narrating with reference example 1 to measure.Sample is a kind of HCV negative serum (negative serum 1) and four kinds of HCV positive serums (positive serum 3～6).The result sees table 1.
[table 1]
Shown the average canbdle power that records with (the 0th day) before the accelerated test and the 1st day reagent of accelerated test in the table 1.And the average canbdle power that records with the reagent that used the 0th day is 100%, shown the number percent of the average canbdle power that the reagent that uses the 1st day records.
Learn from table 1, will synthesize peptide NS4 antigen and synthetic peptide cAg and be fixed on the magnetic particle and preserve, poor stability, accelerated test after one day the reactive of total positives serum (positive serum 3～6) sharply descend.To synthesize peptide NS4 antigen and synthetic peptide cAg and be free in the solution and preserve, have good stability, accelerated test after five days total positives serum still keep high response.Learn by above,, let it be free in the solution than being fixed on storage stability height on the solid phase for the HCV antigenic synthetic peptide.
[embodiment 2]
Contain first reagent of synthetic peptide NS4 antigen and synthetic peptide cAg and carry out accelerated test, the storage stability of investigation reagent in order to free state with second reagent that the state on the magnetic particle of being fixed on contains genetic recombination NS3 antigen.In accelerated test, in the dark preserved first reagent and second reagent 0,2 or 7 days down for 37 ℃.Each reagent with having implemented accelerated test is measured HCV antibody.In this example, be first reagent with used first reagent (C) among the embodiment 1, be second reagent with embodiment 1 used second reagent (C).
Assay method is following:
Divided by being first reagent through first reagent (C) of accelerated test, being outside second reagent that all the other use (15) same method of recording and narrating with reference example 1 to measure with above-mentioned second reagent (C).Sample uses four kinds of HCV positive serums (positive serum 7～10).Positive serum 7 be contain the HCV antibody corresponding with NS3 antigen, with the corresponding HCV antibody of NS4 antigen and with the serum of the corresponding HCV antibody of cAg; Positive serum 8 is the serum that contains the HCV antibody corresponding with NS3 antigen; Positive serum 9 is the serum that contains the HCV antibody corresponding with cAg, positive serum 10 be contain the HCV antibody corresponding with NS3 antigen and with the serum of the corresponding HCV antibody of NS4 antigen.Replacing the HCV positive serum with 20mM sodium phosphate buffer (pH7.5) measures as negative quality-control product.Result such as table 2.
[table 2]
Shown the average canbdle power that the reagent with (the 0th day) and accelerated test before the accelerated test the 2nd day and the 7th day records in the table 2.And use Fig. 5～8 to show that the average canbdle power that records with the reagent that used the 0th day is 100% with regard to positive serum 7～10, the number percent of the average canbdle power that records with the reagent of the 2nd day and the 7th day.Fig. 5 shows the result who measures positive serum 7, and Fig. 6 shows the result who measures positive serum 8, and Fig. 7 shows the result who measures positive serum 9, and Fig. 8 shows the result who measures positive serum 10.In Fig. 5～8, the longitudinal axis is represented to be 100%, respectively to preserve the average canbdle power number percent of fate with the average canbdle power that the reagent that used the 0th day records, and transverse axis is represented the preservation fate of accelerated test.
From table 2 and Fig. 5～8, learn, can detect the various samples that contain the antibody that reacts with each site of HCV protein well with the kit that contains first reagent and second reagent.Wherein, first reagent contains the HCV antigenic synthetic peptide under the free state, and second reagent contains the HCV gene recombinant antigens that is fixed on the magnetic particle.The storage stability of also knowing the antigen in this kit is very superior.
[embodiment 3]
Second reagent that contains first reagent of synthetic peptide NS4 antigen and synthetic peptide cAg and contain the magnetic particle that is fixed with genetic recombination NS3 antigen and Streptavidin with outstanding turbid state in order to free state carries out accelerated test, the storage stability of investigation reagent.In accelerated test, in the dark preserved first reagent and second reagent 0,4 or 7 days down for 37 ℃.Use each reagent of handling through accelerated test to measure HCV antibody.In this example, be first reagent with used first reagent (C) among the embodiment 1.
The magnetic particle that genetic recombination NS3 antigen and Streptavidin are fixed on the magnetic particle is prepared as follows.
Earlier the ST magnetic particle of market sale is put into 20mM sodium phosphate buffer (pH7.5), make its concentration reach about 5mg/mL, preparation ST magnetic particle suspension.The genetic recombination NS3 antigen of above-mentioned (1) preparation is dissolved in the 20mM sodium phosphate buffer (pH7.5), and concentration reaches 1mg/mL, preparation NS3 antigenic solution.Mix ST magnetic particle suspension 10mL and NS3 antigenic solution 0.06mL, cultivated 30 minutes, genetic recombination NS3 antigen is fixed on the ST magnetic particle at 37 ℃.Handle without biotinylation at the genetic recombination NS3 of this use antigen, therefore, it is through the method for physisorption to magnetic particle that genetic recombination NS3 antigen is fixed on the ST magnetic particle.Therefore, in the magnetic particle of this preparation, be fixed with genetic recombination NS3 antigen and Streptavidin respectively.
Then, Mixed Stationary has magnetic particle and the above-mentioned confining liquid of NS3 antigen and Streptavidin (1%BSA, 20mM sodium phosphate buffer pH7.5), was cultivated 30 minutes, and encapsulated processing for 37 ℃.The magnetic particle that so obtains abbreviates NS3 antigen/ST magnetic particle later on as.
Used following second reagent is called second reagent (E) in this example.
20mM sodium phosphate buffer (pH7.5)
5mg/mL NS3 antigen/ST magnetic particle
Assay method is following:
Divided by being first reagent through first reagent (C) of accelerated test, being outside second reagent that all the other use (15) same method of recording and narrating with reference example 1 to measure with second reagent (E) through accelerated test.Sample uses a kind of HCV negative serum (negative serum 2) and four kinds of HCV positive serums (positive serum 11～14).Positive serum 11 be contain the HCV antibody corresponding with NS3 antigen, with the corresponding HCV antibody of NS4 antigen and with the serum of the corresponding HCV antibody of cAg; Positive serum 12 is the serum that contains the HCV antibody corresponding with NS3 antigen; Positive serum 13 is the serum that contains the HCV antibody corresponding with cAg, positive serum 14 be contain the HCV antibody corresponding with NS3 antigen and with the serum of the corresponding HCV antibody of NS4 antigen.Measure result such as table 3.
[table 3]
Shown the average canbdle power that the reagent with (the 0th day) before the accelerated test, accelerated test the 4th day and the 7th day records in the table 3.And use Fig. 9～12 to show that the average canbdle power that records with the reagent that used the 0th day is 100% with regard to positive serum 11～14, use the number percent of the average canbdle power that the reagent of the 4th day and the 7th day records.Fig. 9 shows the result when measuring positive serum 11, and Figure 10 shows the result when measuring positive serum 12, and Figure 11 shows the result when measuring positive serum 13, and Figure 12 shows the result when measuring positive serum 14.In Fig. 9～12, it is 100% respectively to preserve the average canbdle power number percent of fate that the longitudinal axis is represented with the average canbdle power that the reagent with (the 0th day) before the accelerated test records, and transverse axis is represented the preservation fate of accelerated test.
From table 3 and Fig. 9～12, learn, can detect the various samples that contain the antibody that reacts with each site of HCV protein with the kit that contains first reagent and second reagent.Wherein, first reagent contains the HCV antigenic synthetic peptide under the free state, and second reagent contains the HCV gene recombinant antigens that is fixed on the magnetic particle.The storage stability of also knowing the antigen in the kit is very superior.
Learn by above; Storage stability and reactivity are more superior under the state of the solid phase of HCV gene recombinant antigens in being fixed on damping fluid; In contrast, storage stability is more superior down at the state that is not fixed on the solid phase in the damping fluid (free state) for the HCV antigenic synthetic peptide.
Claims (15)
1. kit is used in a HCV TPPA, comprising: contain first reagent of the HCV antigenic synthetic peptide that has added the solid phase binding site and second reagent of the solid phase that contains the bound substances that is fixed with the HCV gene recombinant antigens and can be incorporated into said solid phase binding site.
2. kit is used in a HCV TPPA, comprising: contain first reagent of the HCV antigenic synthetic peptide that has added the solid phase binding site and contain first solid phase that is fixed with the HCV gene recombinant antigens and second reagent that is fixed with second solid phase of the bound substances that can be attached to said solid phase binding site.
3. according to claim 1 or claim 2 kit, it is characterized in that: said HCV antigenic synthetic peptide is present in first reagent with free state.
4. according to claim 1 or claim 2 kit, it is characterized in that: said HCV gene recombinant antigens contains HCV nonstructural proteins peptide.
5. according to claim 1 or claim 2 kit is characterized in that: said HCV gene recombinant antigens is be selected from the group that constitutes with genetic recombination NS3 antigen, genetic recombination NS4 antigen, genetic recombination NS5 antigen and genetic recombination cAg a kind of at least;
Said HCV antigenic synthetic peptide is be selected from the group that constitutes with synthetic peptide NS4 antigen, synthetic peptide NS5 antigen and synthetic peptide cAg a kind of at least.
6. according to claim 1 or claim 2 kit; It is characterized in that: said first reagent contains and different types of the 2nd HCV antigenic synthetic peptide of said HCV antigenic synthetic peptide, and said the 2nd HCV antigenic synthetic peptide contains the solid phase binding site that can combine with said bound substances.
7. kit as claimed in claim 6 is characterized in that: said HCV antigenic synthetic peptide contains HCV structural protein peptide, and said the 2nd HCV antigenic synthetic peptide contains HCV nonstructural proteins peptide.
8. kit as claimed in claim 6 is characterized in that: said HCV antigenic synthetic peptide is a cAg, and said the 2nd HCV antigenic synthetic peptide is NS4 antigen or NS5 antigen, and said HCV gene recombinant antigens is a NS3 antigen.
9. kit as claimed in claim 2 is characterized in that: said first solid phase and second solid phase are the micropores of magnetic particle, latex particle or microtiter plate.
10. according to claim 1 or claim 2 kit, it is characterized in that: said solid phase binding site contains biotin, and said bound substances is an antibiosis protein class.
11. kit according to claim 1 or claim 2 is characterized in that also comprising: contain be labeled substance markers and can with the 3rd reagent of the material of HCV antibodies.
12. kit as claimed in claim 11 is characterized in that: said can be antibody with the material of HCV antibodies.
13. kit as claimed in claim 11 is characterized in that: said label is an enzyme, also comprises the 4th reagent that contains the substrate relative with said enzyme.
14. HCV TPPA method; Comprise: contact procedure, first reagent that let sample, contains the HCV antigenic synthetic peptide that has added the solid phase binding site contacts with second reagent that contains the solid phase that is fixed with HCV gene recombinant antigens and the bound substances that can be incorporated into said solid phase binding site; And detect step, detect the HCV antigenic synthetic peptide and first compound of HCV antibody and/or second compound of HCV gene recombinant antigens and HCV antibody that on solid phase, form.
15. HCV TPPA method; Comprise: contact procedure, first reagent that let sample, contains the HCV antigenic synthetic peptide that has added the solid phase binding site contacts with second reagent that contains first solid phase that is fixed with the HCV gene recombinant antigens and second solid phase that is fixed with the bound substances that can be attached to said solid phase binding site; And detect step, first compound of contained HCV antibody and/or second compound of HCV gene recombinant antigens that on said first solid phase, forms and the contained HCV antibody of said sample in HCV antigenic synthetic peptide that detection forms and the said sample on said second solid phase.
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| JP2007-324927 | 2007-12-17 | ||
| JP2007324919A JP4975600B2 (en) | 2007-03-16 | 2007-12-17 | Reagent kit for HCV antibody measurement and HCV antibody measurement method |
| JP2007-324919 | 2007-12-17 | ||
| JP2007324927A JP4975601B2 (en) | 2007-03-16 | 2007-12-17 | Reagent kit for HCV antibody measurement and HCV antibody measurement method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993000365A3 (en) * | 1991-06-24 | 1993-04-29 | Chiron Corp | Hepatitis c virus (hcv) polypeptides |
| CN1130910A (en) * | 1994-07-25 | 1996-09-11 | 伯伦格·曼海姆有限公司 | Hapten-tagged peptides |
| WO2002004484A2 (en) * | 2000-07-07 | 2002-01-17 | Medmira Inc. | Hcv mosaic antigen composition |
| CN1694971A (en) * | 2002-09-09 | 2005-11-09 | 希龙公司 | HCV detection |
| EP1365240B1 (en) * | 2002-05-22 | 2006-09-13 | Sysmex Corporation | Immunoassay methods, immunoassay apparatuses, and reagents for immunoassays |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH03190898A (en) * | 1989-12-21 | 1991-08-20 | Shima Kenkyusho:Kk | Synthetic polypeptide and reagent for hcv antibody analysis using same polypeptide |
| DK0489968T3 (en) * | 1990-12-14 | 1997-03-24 | Innogenetics Nv | Synthetic antigens for detection of antibodies against hepatitis C virus |
| WO1993006247A1 (en) * | 1991-09-16 | 1993-04-01 | Abbott Laboratories | Hepatitis c assay |
| JP3715027B2 (en) * | 1996-05-07 | 2005-11-09 | シスメックス株式会社 | Diagnostic agent for hepatitis C virus infection |
| JP4233382B2 (en) * | 2002-05-22 | 2009-03-04 | シスメックス株式会社 | Immunoassay method, immunoassay device, and immunoassay reagent |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993000365A3 (en) * | 1991-06-24 | 1993-04-29 | Chiron Corp | Hepatitis c virus (hcv) polypeptides |
| CN1130910A (en) * | 1994-07-25 | 1996-09-11 | 伯伦格·曼海姆有限公司 | Hapten-tagged peptides |
| WO2002004484A2 (en) * | 2000-07-07 | 2002-01-17 | Medmira Inc. | Hcv mosaic antigen composition |
| EP1365240B1 (en) * | 2002-05-22 | 2006-09-13 | Sysmex Corporation | Immunoassay methods, immunoassay apparatuses, and reagents for immunoassays |
| CN1694971A (en) * | 2002-09-09 | 2005-11-09 | 希龙公司 | HCV detection |
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