JP2001224371A - Method of detecting or measuring hepatitis c virus (hcv) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of simply detecting or measuring HCV with high sensitivity. SOLUTION: The monoclonal antibody is provided so that the specimen including HCV may be measured and the hybridoma is provided for producing the monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting or measuring HCV and a reagent therefor.

[0002]

2. Description of the Related Art Hepatitis C has not been identified for a long time, but its gene has been cloned (S
cence 244: 359-362, 1989),
With the development of a diagnostic method based on antibody measurement using a recombinant antigen made based on the gene (Sci
ence 244: 362-364, 1989); Japanese Patent Application Laid-Open No. 2-500880); HCV (hepatitis C virus, Hepatitis C Virus) as a causative factor; and blood and blood-related preparations as main infection routes. It became clear. The development of a so-called second-generation antibody test to which a recombinant core antigen and a recombinant NS3 antigen have been added has made it possible to distinguish most HCV-infected persons by serum tests. This has made it possible to almost eliminate infections caused by domestic blood donations.

However, HIV (human immunodeficiency virus)
Like general virus infections, there is an indeterminable period called the so-called wind period, which occurs until the initial stage of antibody development, and even in areas where blood sales are recognized and some parts of the country. There is still the risk of secondary infections due to blood-derived components that could not be determined by antibody testing. In addition, the antibody test has a problem in that it is impossible to determine whether the subject is a resident who has been cured after the infection or an active infected person, based on the principle.

[0004] Interferon (IFN) is currently used for the treatment of hepatitis C.
Since HCV antibody titer decreases 6 months after V is exterminated, some researchers have determined that the therapeutic effect can be determined only by measuring the HCV antibody titer. However, since the movement of the antibody titer does not decrease until after the antigen stimulation is reduced, that is, several months after the antigen extermination, it is possible to timely and accurately determine whether or not HCV has been eliminated by IFN administration only by performing an antibody test. Can not. That is, in order to monitor the treatment, a method for detecting HCV itself, not an antibody against HCV, is required.

[0005] HCV is derived from other viruses such as HBV (B
Because the amount of virus in blood is lower than that of hepatitis virus and the virus cannot be propagated in vitro or in animals or the like as a host. It was difficult to establish a method for detection. Therefore, instead of detecting a virus antigen, a PCR (polymerase chain reaction) method (Science 230: 1350-
1354, 1985) and a method for detecting viral genomic RNA by a branched-chain DNA probe method. However, the method for detecting a viral genome has several problems as compared with the method for detecting a viral antigen.

[0006] First, it has been pointed out that since the substance to be detected is RNA, the storage stability is low, and thus the freeze-thaw operation of serum causes a decrease in the quantitative value. For this reason, it is necessary to pay more attention to the storage of the sample than in the conventional serum test method.
Care must also be taken when transporting specimens.

[0007] For example, a test method using the PCR method is the most sensitive detection method for detecting a gene fragment, but when extracting HCV genomic RNA from a sample, or when reversing genomic RNA into template DNA. In order to obtain a stable quantitative value, loss is likely to occur at the time of copying, skill is required, and amplification is an important principle, so false contamination often occurs when contamination occurs Therefore, a large amount of specimen cannot be processed at one time.

[0008] Even if a simple method is used, the pretreatment time is required to be 2 hours or more, which is complicated because it involves a large number of centrifugation operations. In addition, such complicated operations increase the chances of contamination and increase the possibility of generating false positive samples. On the other hand, the branched DNA probe method has low detection sensitivity, and it takes about 20
Takes time (medicine and pharmacy 31: 961-970,19
94), problems remain in terms of sensitivity and operation time.

In order to solve the above problems of the method for detecting a viral genome, a method for directly detecting a virus antigen has been developed. As disclosed in JP-A-8-29427, a method for detecting a core antigen in serum using a monoclonal antibody having specificity for the core antigen of HCV has been developed. This report is published by Tanaka et al.
Hepatology 23: 742-745,19
95) and Kawai et al. (Medical and Pharmacy 36: 1065-1)
070, 1996) by detecting core antigens present in serum has been shown to have clinical utility similar to the above-described method for detecting a viral genome. However, as in the case of the virus genome detection method, major problems remain in several points.

One of the points is that it cannot be used for the final test in serum screening because of its lower sensitivity than the PCR method. Tanaka et al. (Journal of He
Patology 23: 742-745,199
5), indicating that the detection limit is between 10 ^{4 and} 10 ⁵ copies / ml as the amount of HCV RNA, and Kawai et al. (Medical and Pharmacy 36: 1065-1070, 199).
6), RNA is detected by CRT (Cope Tete Reverse Transcription) -PCR which is the most sensitive detection method.
It reports a positive rate of 67% in the pretreatment serum of 102 patients with chronic hepatitis C classified as positive. That is, the sensitivity is greatly inferior to that of the highly sensitive CRT-PCR method.

Further, the complicated and time-consuming process of sample processing for measurement poses a problem when used for applications such as screening. That is, for the treatment of a sample (serum), polyethylene glycol (PEG) treatment (4 ° C. for 1 hour) for concentration of virus particles and removal of serum components, centrifugation operation (15 minutes), removal of supernatant, urea treatment , An alkali treatment (37 ° C. for 30 minutes), and a multi-step treatment step such as addition of a neutralizing agent. Further, the dispersion step of urea treatment of the precipitate formed firmly and having increased viscosity by PEG is a very skillful operation. Therefore, skill is required to obtain reproducibility, and a processing time of at least about 2 hours is required. Further, since there are steps such as centrifugation operation and supernatant removal, automation and simultaneous large-scale processing are difficult, and the operation is not suitable for applications requiring large-scale processing such as screening.

On the other hand, the virus antigen detection system is superior to the high-sensitivity PCR method in the following points. That is, since an excessive amplification processing operation is not added in the detection process, it is extremely tolerant to contamination. Also RNA
Rather than detecting an unstable substance such as that described above, the antigen protein, which is a relatively stable substance, is detected. There is no need to use such special equipment, and the transport of the specimen is facilitated.

These features are indispensable requirements for use in measuring a large number of samples, for example, in the blood business and in health examinations. However, as already pointed out, the disclosed core antigen detection method becomes a golden standard in applications where pretreatment is complicated and unsuitable for automation, and low in sensitivity, for example, in the blood business where sensitivity is required. For the reason that they cannot be obtained, they cannot be used for so-called screening applications that handle a large number of specimens, and their advantages over the PCR method cannot be utilized. Also,
Measuring methods that are clinically useful are always sensitive, specific,
There is a need for reproducibility, operability, and low cost.

[0014]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an HCV antigen detection method suitable for treating a large number of specimens such as those used in so-called screening applications such as blood business and medical examination. . That is, an object of the present invention is to provide a system for detecting and quantifying an HCV antigen which has the same sensitivity and specificity as the PCR method and simplifies the pretreatment, and can be easily applied to a mass processing system such as automation. Another object of the present invention is to provide a system for detecting and quantifying a virus antigen having a structure similar to that of HCV.

[0015]

Means for Solving the Problems HCV has a blood concentration of 10%.
From ² to 10 ⁶ copies / ml, HBV (10 ⁹ copies /
ml), it requires extremely high sensitivity to detect viral antigens. Generally, in a detection method represented by an immunological technique using an antibody as a probe, methods for increasing the detection sensitivity include: I) increasing the number of antigen molecules to be detected, and II) a probe binding to an antigen. For example, to increase the number of antibody molecules, III) a probe that defines the lower limit of detection sensitivity, for example, to reduce non-specific reactions caused by binding of the antibody to a substance other than the antigen,
IV) A method of increasing the detection sensitivity of a label used for detection is conceivable, and it is possible to increase the sensitivity by combining these methods.

As a method for increasing the number of antigen molecules, I
-1) It is most easily considered to increase the amount of the sample. However, in a generally used reaction system (for example, a 96-well immunoplate), the maximum volume that can be added is about 300 μl, and the upper limit is naturally determined. As specified
I-2) A method of increasing the number of molecules added to the reaction system by concentration is used.

In order to increase the number of detection probes that bind to an antigen, for example, the number of molecules of an antibody, II-1) increase the number of recognition epitopes by using a plurality of probes, for example, an antibody. This is a technique that can easily be considered to increase the number of antibodies bound per unit time by increasing the affinity (affinity and avidity) between a probe, for example, an antibody and an antigen. Here, as a method of improving the affinity between an antigen and a probe, for example, an antibody, a method of changing the composition of a buffer solution of a reaction system, a method of modifying a probe, and a combination thereof are considered. II-3) It is also conceivable to capture a large number of antigens by binding a large amount of the antibody to a carrier having a large surface area such as beads or magnetic particles to increase the reaction area with a limited amount of antigen. .

In the case of infectious diseases, it is expected that human antibodies exhibiting high affinity for binding to antigens are present in the specimen, and the epitope of these antibodies may overlap with the probe used for detection, for example, the epitope of the antibody. Since it is expected that a competitive reaction will occur and lead to a decrease in the number of antibodies used for detection, it is necessary to increase the number of antibody molecules for detection that bind to the antigen by removing the antibodies that inhibit the reaction in this sample. Connect (II-4).

Although it is difficult to generalize the method for reducing non-specific reactions, III-1) By changing the buffer composition, the affinity (affinity and avidity) of a probe, for example, an antibody with an antigen can be increased. Measures such as reducing the non-specific reaction by increasing the concentration, and III-2) removing the substance causing the non-specific reaction can be considered. As a method for increasing the detection sensitivity of a labeled substance, IV-1) a method using a labeled substance having a high detection sensitivity (such as a radioisotope) may be used.
-2) Amplify signals by using enzymes and catalysts as labels, IV-3) Modify enzyme substrates to more sensitive substrates, IV-4) Chemically convert substrate signals for enzyme reactions and chemical reactions Or electrical or mechanical amplification, IV-
5) Increasing the number of labels per antibody, IV-6) Increasing the sensitivity of equipment used for signal detection, and the like can be considered.

When analyzing the steps of the pretreatment method of the disclosed HCV core antigen detection method, the antigen is concentrated by adding polyethylene glycol to the sample and then collecting the HCV as a precipitate by centrifugation (I-2). At the same time, after performing the step of removing a part of the serum component (II-2), the human antibody present in the specimen is inactivated by resuspension in a solution containing urea and an alkaline agent (II- 3) HC
V. The step of releasing the core antigen from V, and the step of adding a solution containing a nonionic surfactant (Triton-X100) and a neutralizing agent to form a solution that reacts with the monoclonal antibody.

As already pointed out above, the centrifugation operation and the resuspension operation of the precipitate are complicated steps in operation, and are steps requiring skill. Accordingly, an object of the present invention is to provide a core antigen detection system which solves these operational problems. H
Although the appearance of CV itself has not yet been elucidated, its genomic structure, the structure of related virus particles, and information on common viruses indicate that HCV particles are
E1, E2 / NS in which NA is packed by core antigen and anchored to lipid membrane to surround it
It is presumed that it exists in a state surrounded by a coat protein consisting of one antigen.

Therefore, in order to detect the core antigen, it is necessary to remove the envelope so that a probe used for detecting the core antigen, for example, an antibody can bind thereto. It has been reported that virus particles have a complex structure surrounded by LDL (low-density lipoprotein) in blood and that antibodies against coat proteins are also present. It is expected that it also exists as an immune complex with the protein antibody. That is, in order to increase the number of antigen molecules to be detected, it is most important to efficiently remove envelopes and contaminants surrounding the virus particles from the virus particles and efficiently release the core antigen molecules.

Therefore, one of the requirements provided by the present invention is that
There is a method for treating HCV core antigen in a sample (serum) in a state suitable for detection using a probe without being concentrated by a complicated operation such as centrifugation. Further, as described above, the probe used for detection in the sample, for example, a human antibody that competes with the antibody for binding exists at a high titer,
The operation of removing this is important for increasing the sensitivity.
Therefore, one of the important requirements of the present invention is a treatment method for simultaneously inactivating human antibodies present in a specimen by a treatment method for easily releasing the HCV core antigen in the specimen.

By using the treatment method presented by the present invention, the HCV core antigen present in the sample can be converted from the virus particles or immune complex in a state suitable for forming an immune complex with a probe, eg, an antibody. By simultaneously inactivating and releasing human antibodies present in a sample that simultaneously inhibits the detection reaction, it is possible to detect easily and with high sensitivity by an immunoassay using a probe such as an antibody. .

The probe used for the detection, for example, the antibody is HC
V can be used for the primary reaction, as long as it specifically binds to the core antigen of V, exhibits a certain high affinity, and does not induce nonspecific reactions when added to the reaction system. One of the probes preferably contains a probe that can recognize and bind to the N-terminal side of the HCV core antigen. Here, the N-terminal side of the core antigen refers to one of the sequences shown in SEQ ID NO: 2.
Refers to the 0th to 70th sequence or a part thereof.
Further, a probe for the C-terminal side of the core antigen may be included here. Here, the C-terminal side of the core antigen refers to the sequence from sequence 81 to sequence 160 shown in SEQ ID NO: 2, or a part thereof.

The term "probe" used herein refers to a polyclonal antibody obtained by immunizing experimental animals such as mice, rabbits, chickens, goats, sheep, and cows. Separating spleen cells from immunized individuals and fusing them with myeloma cells. A monoclonal antibody produced by the hybridoma obtained by the method described above, or a spleen cell, a monoclonal antibody produced by a cell obtained by immortalizing blood leukocytes with the EB virus, H
CV-infected humans, monoclonal antibodies produced by chimpanzees, etc., mouse, human immunoglobulin cDNA, variable region gene fragments obtained from chromosomal DNA, or immunoglobulin cDNA, part of chromosomal DNA A variable region gene fragment composed by combining an artificially produced sequence, a variable region gene fragment composed by using an artificial gene sequence, or a variable region produced by genetic recombination using these as a material A gene fragment is fused with a recombinant antibody produced by a cell transformed with a recombinant antibody gene constituted by combining immunoglobulin constant region gene fragments, the above-described variable region gene fragment and a structural protein of bacteriophage, for example. Phage antibodies produced by The variable region is artificially produced in a recombinant antibody produced by a cell transformed by a recombinant antibody gene constituted by combining an auditory region gene fragment with another applicable gene fragment such as a part of a myc gene, and a trypsin molecule. High specificity for core antigens, such as probes produced by introduction into DNA, probes obtained by artificially modifying molecules that specifically bind to proteins such as receptors, and other probes produced by combinatorial chemistry technology If it is a molecule showing affinity, it can be used.

Further, the present invention provides a method for releasing a HCV core antigen from a sample containing a HCV core antigen from a virus particle or an immune complex so as to be in a state suitable for forming an immune complex between the HCV core antigen and a probe such as an antibody. A step of treating the specimen with a treating agent that simultaneously inactivates the human antibody present in the specimen that simultaneously inhibits the detection reaction, detecting the released core antigen by an immunoassay using a probe such as an antibody, and An assay method for quantification and a test kit are provided.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Sample processing shown by the present invention
Agents and Processing Methods Samples of the present invention include biological fluids such as whole blood, plasma, serum, urine, saliva, cerebrospinal fluid, and liver tissue. In the present invention, without complicated operation of the sample,
The most important requirement is a method of treating a core antigen in a sample in a state suitable for a binding reaction with a probe such as a monoclonal antibody. That is, in order to increase the number of antigen molecules, it is important to efficiently release core antigens contained in virus particles and the like.

[0029] Membrane proteins such as HCV coat proteins do not dissolve in water unless subjected to some treatment. In order to dissolve a protein having such a hydrophobic part as a part thereof in water, a method of using a surfactant and converting the hydrophobic part to hydrophilic is well known. Certain salts are known to have the property of making such poorly soluble proteins readily soluble in water. Ions generated from salts (chaotropic agents) exhibiting such properties are called chaotropic ions, and known cations include guanidine ion, thiocyanate ion, iodine ion, periodate ion, and chlorate ion. Salts that generate these ions have been used for solubilizing poorly soluble proteins. Chaotropic ions were predicted to have a function of efficiently releasing antigen from virus particles.

However, when such chaotropic ions are added, the secondary structure of the protein is partially destroyed, and as a result, the epitope structure is lost. Therefore, if it is directly added to an immunocomplex formation reaction with a probe, for example, an antibody in the presence of chaotropic ions, the binding of the antibody is weakened and the sensitivity is reduced, which is considered to be a major problem.

On the other hand, the denaturing action of chaotropic ions is often reversible, and the temporarily denatured structure may return to its original state by weakening the ionic strength by dialysis or dilution. This indicates another problem when a treatment agent using chaotropic ions such as guanidine is used. In other words, the target treatment method of the present invention not only efficiently releases the antigen contained in the virus particles and the like present in the sample, but also uses a high-titer antibody that binds to the antigen present in the sample at the same time. It needs to be deactivated. That is, in the case of solubilization using chaotropic ions, it is conceivable that the inactivation of the high-titer antibody present in the sample is insufficient, and the sensitivity is lowered by the influence of this antibody.

Therefore, the treatment method using chaotropic ions has two contradictory problems. In other words, the antigen-antibody reaction is inhibited under conditions that disrupt the structure due to chaotropic ions.On the other hand, the effect of chaotropic ions alone does not sufficiently inactivate the antibody that inhibits the reaction in the sample. In a situation where the reaction is not inhibited, the reaction is inhibited by the contaminating antibody. That is, in order to solve this conflicting problem, it is necessary to find conditions under which the epitope structure of the antigen is reversibly destroyed and the functional disruption of the contaminating antibody in the sample occurs irreversibly.

Conditions for inactivating the activity of the antibody include an alkali treatment and an acid treatment. When serum is treated with acid, some serum proteins are irreversibly denatured and precipitate, which often hinders pipetting, for example, after sample treatment. The entrained precipitate may be adsorbed and detected as turbidity, which may cause false positives. In addition, the target antigen is non-specifically involved in these precipitates, which causes a problem such as a decrease in sensitivity due to a decrease in the amount of reaction with the probe.

The present inventor has found that by combining acid treatment and guanidine treatment, the problems of acid treatment such as formation of a precipitate and the contradictory problems of guanidine treatment can be solved, and the present invention has been completed. Reached. It has also been found that it is more preferable to add a surfactant to a treating agent comprising chaotropic ions such as guanidine and an acidifying agent. Suitable acidifying agents include hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, and the like.

As the surfactant, CHAPS is used.
(3- [3-cholamidopropyl) dimethylammonio]
Zwitterionic surfactants such as -1-propanesulfonic acid), CHAPSO (3- [cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonic acid), dodecyl-N-betaine, and Triton X10
Polyoxyethylene isooctyl phenyl ethers such as 0, polyoxyethylene nonyl phenyl ethers such as NP40, polyoxyethylene sorbitol esters such as Tween 20, polyoxyethylene dodecyl ethers such as Brij 58, and nonionic such as octyl glucoside Surfactants are suitable. Further, a drug such as urea which acts to weaken the hydrogen ion bond to partially destroy the higher-order structure of the protein may be added.

In particular, when the guanidine hydrochloride concentration is 2M or more,
Triton X100 concentration is 2% or more, Tween2
The 0 concentration is 0.02% or more, and it is more preferable to use it in the range of 4 ° C to 45 ° C. By using the treatment method of the present invention, a virus antigen can be released from a sample containing virus particles having a structure similar to that of HCV to a state suitable for a so-called immunoassay method using an antibody or the like as a probe. it is obvious. Here, viruses having the same structure as HCV include genomic RNA and DNA.
Is a virus that forms a virus particle having a structure composed of a protein that packs a protein, a membrane protein surrounding the protein, and a lipid membrane. Examples of the virus include flaviviruses, which are related to HCV, and human immunodeficiency virus (HI).
V) and the like. Further, even those having DNA as a genome include those having a similar structure.

As a probe shown by the present invention,
Monoclonal antibody The HCV structural protein gene fragment referred to in the present invention is HC
V is a gene fragment containing the core region of the structural protein gene of V, and is a DNA fragment having a nucleotide sequence encoding a polypeptide containing at least the amino acid sequence of the N-terminus at positions 1 to 160 of HCV. Specifically, SEQ ID NO: 2
And a gene fragment containing a base sequence encoding the amino acid sequence of

As used herein, the term "polypeptide having HCV antigen activity" refers to a fusion polypeptide or polypeptide which immunologically reacts with an anti-HCV antibody, and is used for preparing the hybridoma of the present invention and the monoclonal antibody obtained therefrom. Used as an antigen for use. Specifically, it is a fusion polypeptide having HCV antigen activity including the amino acid sequence of SEQ ID NO: 1 or a polypeptide having HCV antigen activity including a part of the amino acid sequence of SEQ ID NO: 1; An extra amino acid sequence may be added.

[0039] Monoclonal antibodies against the fusion polypeptide of the present invention and the polypeptide containing the amino acid sequence shown in SEQ ID NOs: 3 to 5 can be easily prepared by those skilled in the art. The production of monoclonal antibodies by hybridomas is well known. For example, B
The above fusion polypeptide or polypeptide (hereinafter, the present antigen) is intraperitoneally or intradermally in ALB / c mice and the like.
Is used as an antigen alone or as an antigen conjugated with BSA, KIH, or the like, and is mixed with an adjuvant such as simple or Freund's complete adjuvant to periodically immunize. When the antibody titer in the blood rises, this antigen is administered into the tail vein as a booster, the spleen is aseptically removed, and the cell is fused with an appropriate mouse myeloma cell line to obtain a hybridoma. This method is based on the method of Kohler and Milstein (Natu
re 256: 495-497, 1975).

The hybridoma cell line obtained by the above method is cultured in an appropriate culture medium, and then a hybridoma cell line that produces an antibody showing a specific reaction to the present antigen is selected and cloned. For cloning of antibody-producing hybridomas, besides limiting dilution, soft agar (Eu)
r J Immunol. 6: 511-519, 197
6) can be used. Then, the produced monoclonal antibody is purified by a method such as column chromatography using protein A or the like. Molecules used as probes other than the above monoclonal antibodies can be produced. For example, recombinant antibodies are described in detail in a review by Hoogenboon (Trends in Biotechnolo).
gy, 15: 62-70, 1997).

Detection System Using Probe Monoclonal antibodies prepared according to the present invention are HC
For use as a test reagent in enzyme-linked immunosorbent assays (ELISAs), enzyme immunodot assays, radioimmunoassays, agglutination-based assays, or other well-known immunoassays for the detection and quantification of V-core antigens Can be. When a labeled antibody is used for detection, examples of the labeling compound include a fluorescent substance, a chemiluminescent substance, a radioactive substance, an enzyme,
Dyeing substances and the like are used.

For example, when a method based on a sandwich reaction system is used to detect HCV core antigen in a sample (serum), the diagnostic kit to be used is a solid support (for example, the inner wall of a microtiter well). Includes one or more monoclonal antibodies of the invention coated and one or more monoclonal antibodies or fragments thereof conjugated to a label. The combination of the monoclonal antibody immobilized on the solid support and the monoclonal antibody to be labeled is free, and a highly sensitive combination can be selected.

Examples of solid supports that can be used include microtiter plates made of polystyrene, polycarbonate, polypropylene and polyvinyl, test tubes, capillaries, beads (latex particles, red blood cells, metal compounds, etc.), membranes (liposomes, etc.), filters, etc. No.

[0044]

According to the method of the present invention, it is possible to easily release a virus antigen from virus particles such as HCV in a state suitable for a so-called immunoassay method for detecting an antigen using an antibody or the like as a probe. . In addition, by processing a sample containing virus particles such as HCV by the method shown by the present invention, a virus antigen can be easily and sensitively detected and quantified by a so-called immunoassay method for detecting an antigen using an antibody or the like as a probe. It becomes possible. In addition, it is possible to prepare a kit for determining the presence or absence of a virus such as HCV in a sample, a kit for quantifying the same, and a diagnostic agent using an immunoassay using the sample processing method according to the present invention.

[0045]

The following examples illustrate the invention but do not limit the scope of the invention. Embodiment 1 FIG. Expression and Purification of HCV-Derived Polypeptide (A) Construction of Expression Plasmid An expression plasmid corresponding to the core region of HCV was constructed by the following method. C11-C21 clone and C10-
The E12 clone (Japanese Unexamined Patent Application Publication No. 6-38765) was
No. 9 plasmid pUC.C11-C
1 μg of each DNA of pUC12 and pUC · C10-E12 was added to 20 μl of a restriction enzyme reaction solution [50 mM Tris-HCl
(PH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 100 mM NaCl, 15 units of EooRI
And 15 units of ClaI enzyme] and [10 mM
Tris-HCl (pH 7.5), 10 mM MgCl ₂ ,
1 mM dithiothreitol, 50 mM NaCl, 15 units of ClaI and 15 units of KpnI enzyme].
After digestion at 7 ° C. for 1 hour, 0.8% agarose gel electrophoresis was performed to purify an approximately 380 bp EoRI-ClaI fragment and an approximately 920 bp ClaI-KpnI fragment.

The two DNA fragments and pUC119 were
The vector digested with ooRI and KpnI was added to a 10 × ligase buffer [660 mM Tris-HCl (pH 7.
5), 66 mM MgCl2, 100 mM dithiothreitol, 1 mM ATP] 5 μl, T4 ligase 1 μl (3
(50 units / μl), water was added to make up to 50 μl, and the mixture was kept at 16 ° C. overnight to carry out a ligation reaction. Escherichia coli JM109 was transformed with this plasmid, and plasmid pUC was transformed.
-C21-E12 was obtained.

This plasmid pUC · C21-E12
1 ng of DNA was used for two primers (5'-GAATTCA).
TGGGCACGAATCCTAAA-3 '(SEQ ID NO: 6), 5'-TTAGTCCTCCAGAACCC
PCR was performed using GGAC-3 '(SEQ ID NO: 7). PCR is performed using GeneAmpTM (DNA Amplification Reagent K
it, Perkin Elmer Cetus), DNA denaturation at 95 ° C for 15 minutes, annealing at 50 ° C for 2 minutes, DNA synthesis 7
The reaction was carried out at 0 ° C. for 3 minutes, and the obtained DNA fragment was
% Agarose gel electrophoresis and purified by the glass powder method (GeneClean).

On the other hand, pUC19 was digested with the restriction enzyme SmaI, and the DNA fragment obtained by the PCR method was digested with a 10 × ligase buffer [660 mM Tris-HCl (pH 7.
5), 66 mM MgCl ₂ , 100 mM dithiothreitol, 1 mM ATP] 5 μl, T4 ligase 1 μl (3
(50 units / μl), water was added to make up to 50 μl, and the mixture was kept at 16 ° C. overnight to carry out a ligation reaction. Escherichia coli JM109 was transformed with this plasmid, and plasmid pUC was transformed.
19 C21-E12 SmaI was obtained. 1 μg of this plasmid DNA was added to 20 μl of a restriction enzyme reaction solution [150 mM
NaCl, 6 mM Tris-HCl (pH 7.5), 6 mM
MgCl ₂ , 15 units of EoRI and 15 units of B
amHI enzyme] for 1 hour at 37 ° C., followed by 0.8% agarose gel electrophoresis,
A 0 bp EooRI-BamHI fragment was separated and purified by the glass powder method.

Next, the expression vector Trp.TrpE
1 μg of the DNA (JP-A-5-84085) was added to 20 μl of a restriction enzyme reaction solution [150 mM NaCl, 6 mM Tris-
HCl (pH 7.5), 6 mM MgCl ₂ , 15 units of E
ooRI and 15 units of BamHI enzyme] at 37 ° C.
Digestion for 1 hour, and add 39 μl of water to the reaction solution.
After heat treatment at 5 ° C. for 5 minutes, 1 μl (250 units / μl) of bacterial alkaline phosphatase (BAP) was added, and the mixture was incubated at 37 ° C. for 1 hour.

Phenol was added to this reaction solution to perform phenol extraction, and the obtained aqueous layer was precipitated with ethanol, and the precipitate was dried. 1 μg of the resulting EooRI-BamHI-treated vector DNA and the above-mentioned core 160 fragment were digested with 10 ×
Ligase buffer [660 mM Tris-HCl (pH 7.
5), 66 mM MgCl ₂ , 100 mM dithiothreitol, 1 mM ATP] 5 μl, T4 ligase 1 μl (3
(50 units / μl), water was added to make up to 50 μl, and the mixture was kept at 16 ° C. overnight to carry out a ligation reaction.

Using 10 μl of this reaction solution, E. coli HB
101 strains were transformed. Sensitive E. coli strains used for transformation were prepared by the calcium chloride method [Mandel, M. and Higa, A., J. M.
ol. Biol., 53, 159-162 (1970)]. LB containing 25 μg / ml ampicillin
Plate (1% tryptone, 0.5% NaCl, 1.5%
% Agar) and kept at 37 ° C overnight. Take a platinum loop of a bacterial colony formed on the plate, and add 25 μg / ml
Was transferred to an LB medium containing ampicillin, and cultured overnight at 37 ° C.

1.5 ml of the bacterial culture was collected by centrifugation, and the mini-preparation of the plasmid DNA was performed by the alkaline method [Manniatis et al., Molecular Cloning: A Laboratory Ma
nual, (1982)]. 1 μg of the obtained plasmid DNA was added to 20 μl of a restriction enzyme reaction solution [150 mM N
aCl, 6 mM Tris-HCl (pH 7.5), 6 mM
gCl ₂ , 15 units of EoRI and 15 units of Ba
mHI enzyme] at 37 ° C. for 1 hour, and subjected to agarose gel electrophoresis to obtain about 490 bp of EooRI-Bam.
The Trp / TreE core 160 expression plasmid in which the HI fragment was generated was selected.

(B) Expression and Purification of Polypeptide Encoded by Clone Core 160 Escherichia coli HB101 strain having the expression plasmid Trp / TrpE core 160 contains 50 μg / ml ampicillin.
Inoculate ml of 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) and incubate at 37 ° C. for 9 hours. 1 ml of this culture was added to 100 ml of M9-CA medium (0.6% NA ₂ HPO containing 50 μg / ml ampicillin).
₄ , 0.5% KH ₂ PO ₄ , 0.5% NaCl, 0.1
% NH ₄ Cl, 0.1 mM CaCl ₂ , 2 mM MgSO
₄ , 0.5% casamino acid, 0.2% glucose) and cultured at 37 ° C. When OD600 = 0.3, indoleacrylic acid was added to a final concentration of 40 mg / l, and the cells were further cultured for 16 hours. The culture was centrifuged to collect the cells.

The cells were added to 20 ml of buffer A [50 mM Tri].
s-HCl (pH 8.0), 1 mM EDTA, 30 mM N
aCl] was added thereto, and the mixture was centrifuged again to obtain 2.6 g of expressed cells. The obtained cells were buffered with 10 ml of buffer A.
After suspending in E. coli and disrupting the E. coli membrane by sonication, centrifugation was performed to obtain an insoluble fraction containing a fusion polypeptide of the polypeptide encoded by HCV cDNA and TrpE. The fraction was solubilized and extracted by adding 10 ml of buffer A containing 6 M urea. The solubilized extract was subjected to ion exchange column chromatography using S-Sepharose to purify the fusion polypeptide.

Embodiment 2 FIG . Hybridoma preparation method The fusion polypeptide (TrpC1
1) After dissolving 6M urea, 1 containing 0.15M NaCl
A final concentration of 0.2 to 1 in 0 mM phosphate buffer (pH 7.3).
The mixture was diluted to 0 mg / ml and mixed with an equal amount of adjuvant (Titermax) to obtain a TrpC11 suspension. The suspension prepared to have a TrpC11 concentration of 0.1 to 0.5 mg / ml was intraperitoneally administered to BALB / c mice of 4 to 6 weeks of age. The same immunization is performed every two weeks, and about two weeks later, TrpC1 dissolved in physiological saline is used.
110 μg was administered into the tail vein.

On the third day after the final booster immunization, the spleen was aseptically excised from the immunized animal, cut into pieces with scissors, and the spleen was disentangled into individual cells using a mesh.
Washed three times with 1640 medium. After washing the mouse myeloma cell line sp2 / 0Ag14 in the logarithmic growth phase in the same manner as described above, 1.8 × 10 ^{7 of the} cells and 1.0 × 10 ^{8 of} spleen cells were added to 5 cells.
The mixture was placed in a 0 ml centrifuge tube and mixed. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and the mixture was kept at 37 ° C.
% Of polyethylene glycol (PEG) 4000 (manufactured by Merck), 1 ml of RPMI-1640 medium was added, and 10 ml of RPMI-1640 medium was further added to cause cell fusion.

The fused cells were subjected to centrifugation (200 × g, 5 minutes) to remove PEG, and then, using a 96-well plate, 10% fetal bovine serum hypoxanthine, aminopterin and thymidine (hereinafter abbreviated as HAT). R including
Only hybridomas were grown in PMI-1640 medium for about 10 days. Thereafter, clones producing the target antibody were searched for by ELISA, and hybridomas producing the monoclonal antibody of the present invention having the desired reaction specificity were obtained.

The obtained hybridoma was subjected to single cloning according to a conventional limiting dilution method, and the obtained hybridomas were named HC11-14, HC11-10 and HC11-11. Regarding the three types of hybridomas, the Institute of Biotechnology and Industrial Technology
On the date of March 4, FERM BP-6006, F
ERM BP-6004 and FERM BP-6005
Was deposited as

Embodiment 3 FIG . Preparation of Monoclonal Antibody The hybridoma obtained by the method described in Example 2 was transplanted into the peritoneal cavity of a mouse treated with pristane or the like, and a monoclonal antibody produced in ascites was obtained. For purification of the monoclonal antibody, an IgG fraction was separated by a Sepharose column to which protein A was bound. Monoclonal antibodies produced from each of the three hybridomas, C11-14, C11-10
And C11-11 were determined to be IgG2a, C1 by immunoassay using rabbit anti-mouse Ig isotype antibodies (Zymed).
It became clear that 1-14 and C11-11 were IgG1. Epitope analysis was performed on the obtained three types of monoclonal antibodies using a 20-amino acid synthetic peptide synthesized based on a sequence derived from the HCV core region. As a result, as shown in Table 1, specific recognition of a part of the core region was performed. Was found to be a monoclonal antibody.

[0060]

[Table 1]

Embodiment 4 FIG . Examination of sample treatment conditions Examination of treatment conditions 1) Examination of guanidine hydrochloride concentration Normal human serum and HCV-RNA positive panel serum 100
In ul, guanidine hydrochloride dissolved at various concentrations and 0.5
100 μl of a treatment solution containing NHCl was added. 3 at room temperature
The treatment was performed for 0 minutes, and 80 μl thereof was used as a measurement sample. The results obtained by the measurement method described below are shown in FIG. 1 by taking the concentration of guanidine hydrochloride during the treatment reaction on the horizontal axis.

2) Examination of Triton X100 concentration Serum of healthy human and HCV-RNA positive panel serum 100
To μl, 100 μl of treatment solutions (6 M guanidine hydrochloride, 0.5 N HCl) containing Triton X100 dissolved at various concentrations were added. Treat at room temperature for 30 minutes,
80 μl thereof was used as a measurement sample. The results obtained by the measurement method described below are shown in FIG. 2, with the Triton X100 concentration at the time of the treatment reaction being plotted on the horizontal axis.

3) Examination of Tween 20 Concentration Serum of healthy human and HCV-RNA positive panel serum 100
μl of a treatment solution containing Triton X100 dissolved at various concentrations (6 M guanidine hydrochloride, 0.5 N HCl, 1
100% of 2.5% Triton X100) was added. The treatment was carried out at room temperature for 30 minutes, and 80 μl of the mixture was used as a measurement sample. The results obtained by the measurement method described below are shown in FIG. 3 with the Tween 20 concentration at the time of the treatment reaction taken on the horizontal axis.

4) Examination of reaction temperature Serum of healthy human and HCV-RNA positive panel serum 100
Add μl of treatment solution (6 M guanidine hydrochloride, 0.5 NHC
1,12.5% Triton X100, 0.75% Tw
een20) was added. 4 ° C., room temperature (23
° C), 37 ° C, and 45 ° C for 30 minutes, and 80 µl thereof was used as a measurement sample. FIG. 4 shows the results of examination using the measurement method described below.

Assay Methods The samples obtained by each of the serum treatment methods described above were evaluated using the following assay methods. That is, anti-HCV core antigen monoclonal antibodies (antibodies C11-14 and C11-1)
1) was diluted with 0.1 M carbonate buffer (pH 9.6) to a final concentration of 6 μg / ml, and 100 per well of a 96-well microplate (manufactured by Nunc).
Dispensed in μl. After standing at 4 ° C overnight, 0.15M N
10 mM sodium phosphate buffer containing aCl (pH 7.
3) Wash twice with 0.35 ml, and add 10% sodium phosphate buffer (pH 7.3) containing 0.5% casein-Na.
5), 0.35 ml (hereinafter referred to as a blocking solution) was added, and the mixture was further allowed to stand at room temperature for 2 hours.

After removing the blocking solution, 0.15 M Na
Cl, 1% BSA, 0.5% casein-Na, 0.05
A mixture of 140 μl of a 100 mM sodium phosphate buffer (pH 7.3) containing 20% of Tween 20 and 20 μl of 1 M Tris, a total of 160 μl (hereinafter referred to as a reaction buffer), and 80 μl of the measurement sample obtained by the above-described treatment method were used. Add to the wells and react for 2 hours at room temperature.
5 times, and 100 μl of peroxidase (POD) -labeled monoclonal antibody (C11-10)
Was added and reacted at room temperature for 30 minutes. After the reaction, the plate is washed 5 times with 300 μl of the above-mentioned washing solution, and 100 μl of a substrate (orthophenylenediamine, hereinafter referred to as OPD) solution is added thereto.
After reacting for 0 minutes, 100 μl of a 2N sulfuric acid solution was added, and the absorbance at a wavelength of 492 nm (OD492) was measured using the absorbance at a wavelength of 630 nm as a control.

From FIG. 1 to FIG. 4, the processing conditions were optimized, but it was difficult to detect the core antigen in the untreated sample in the panel serum. This dramatically enabled the detection of core antigens. In each case, no increase in signal was observed in healthy subjects. Particularly, the guanidine hydrochloride concentration is 2M.
As described above, it was shown that the core antigen can be detected favorably in the range of 4 ° C. to 45 ° C. by using Triton X100 at a concentration of 2% or more.

Embodiment 5 FIG . Detection and measurement method of core antigen 100 μl of serum was treated with a treatment solution (6M guanidine hydrochloride, 0.1 μl).
5N HCl, 12.5% Triton X100, 0.
100% of 75% Tween 20) was added. The treatment was performed at room temperature for 30 minutes, and 100 µl of the mixture was used as a measurement sample. Anti-HCV core antigen monoclonal antibody (C11-
14 and C11-11 equivalents) in a final concentration of 6 μg / ml
Was diluted with a 0.1 M carbonate buffer (pH 9.6), and dispensed in an amount of 100 μl per well of a 96-well microplate (manufactured by Nunc).

After standing at 4 ° C. overnight, 0.15 M NaCl
10 nM sodium phosphate buffer (pH 7.3)
After washing twice with 35 ml, the blocking solution was 0.35 ml.
Was added and the mixture was allowed to stand at room temperature for 2 hours. After removing the blocking solution, 150 μl of the reaction buffer and the measurement sample obtained by the above-described treatment method were added to each well, and the reaction was performed at room temperature for 2 hours.

The plate was washed five times with 300 μl of the washing solution, and 100 μl of a peroxidase (POD) -labeled monoclonal antibody (C11-10) was added, followed by a reaction at room temperature for 3 minutes. After washing five times with 300 μl of a washing solution, the substrate (OP
D) After adding 100 μl of the solution and reacting at room temperature for 45 minutes, 100 μl of a 2N sulfuric acid solution was added, and the absorbance at a wavelength of 492 nm was compared with the absorbance at a wavelength of 630 nm (OD).
492) was measured. In addition, the same treatment was carried out on a standard serum which was diluted stepwise with a 10 mM sodium phosphate buffer (pH 7.3) containing 1% BSA, using panel serum 50 as 1 U / ml, and measured.

FIG. 5 shows a dilution line of panel serum 50 used as a standard serum. The core antigen in the sample was measured in a concentration-dependent manner, and it was possible to detect about 0.5 mU / ml. That is, by using a combination of the extremely simple sample processing method of the present invention and a monoclonal antibody,
It became clear that the core antigen could be detected or quantified.

Embodiment 6 FIG . Recognized by serum processing and assay
Analytical panel of the molecular form 0.25 ml of the serum 13 was treated by the serum treatment method, and was applied to a gelloca column (Superdex200HR, 1).
x30), and the anti-core immunoreactivity in those fractions was measured. The results are shown in FIG. Molecular weight about 2
It is believed that it recognizes a 0-30 kDa molecule, and the core antigen in the virus is released from various interactions by the pretreatment described above, due to virus destruction and inactivation of anti-core antibodies present in serum. Was shown.

Embodiment 7 FIG . HCV-RNA amount using the measurement method PCR method in which Amplicor HCV Monitor kit (Roche) core antigen in the serum sample 10 ³ to 10 ⁷
Using the serum measured at copy / ml and the serum of a healthy individual, the HCV core antigen in the serum was quantified by the method described above.
As a standard serum, panel 50 serum (set to 1 U / ml) was serially diluted with 10 mM sodium phosphate buffer (pH 7.3) containing 1% BSA and treated in the same manner. 2 shows the measurement results. Among the samples measured this time, the samples of healthy subjects were all below the detection limit, and PC
All R method positive cases could be detected. The correlation at this time is shown in FIG. 7, and the correlation coefficient with the PCR method was 0.8 or more, indicating a high correlation.

[0074]

[Table 2]

[0075]

[Sequence List] (2) SEQ ID NO: 1: (i) Sequence characteristics: (A) Length: 177 (B) Type: amino acid (D) Topology: linear (ii) Sequence type: peptide Met Lys Ala Ile Phe Val Leu Lys Gly Ser Leu Asp Arg Asp Pro Glu 5 10 15 Phe Met Gly Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr 20 25 30 Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val 35 40 45 Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg 50 55 60 Ala Thr Arg Lys Thr Ser Lys Arg Ser Gln Pro Arg Gly Gly Arg Arg 65 70 75 80 Pro Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro 85 90 95 Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly 100 105 110 Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp 115 120 125 Pro Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr 130 135 140 Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Phe Arg Val Gly Ala Phe 145 150 155 160 Leu Gly Gly Ala Ala Arg Ala Leu Ala His G ly Val Arg Val Leu Glu 165 170 175 Asp

(2) SEQ ID NO: 2: (i) Sequence properties: (A) Length: 160 (B) Type: amino acid (D) Topology: linear (ii) Sequence type: peptide Met Gly Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Lys Arg Ser Gln Pro Arg Gly Gly Arg Arg Pro 50 55 60 Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Phe Arg Val Gly Ala Phe Leu 130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160

(2) SEQ ID NO: 3: (i) Sequence characteristics: (A) Length: 20 (B) Type: amino acid (D) Topology: linear (ii) Sequence type: peptide Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 5 10 15 Leu Pro Arg Arg 20

(2) SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 10 (B) Type: amino acid (D) Topology: linear (ii) Sequence type: peptide Gly Pro Arg Leu Gly Val Arg Ala Thr Arg 5 10

(2) SEQ ID NO: 5: (i) Sequence properties: (A) Length: 21 (B) Type: amino acid (D) Topology: linear (ii) Sequence type: peptide Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg His Arg 5 10 15 Ser Arg Asn Val Gly 20

[Brief description of the drawings]

FIG. 1 is a graph showing the results of examining the effect of guanidine hydrochloride addition concentration on sample treatment. Healthy human serum (n
normal) and HCV-RNA positive panel serum 1
3,50 were used.

FIG. 2 is a diagram showing the results of examining the effect of Triton X100 addition concentration in sample processing. Normal human serum (normal) and HCV-RNA positive panel serum 13,50 were used.

FIG. 3 is a diagram showing the results of examining the effect of Tween 20 addition concentration in sample processing. Healthy human serum (n
normal) and HCV-RNA positive panel serum 1
3,50 were used.

FIG. 4 is a diagram showing the results of examining the effect of temperature during sample processing. Normal human serum (normal) and HC
13,50 V-RNA positive panel sera were used.

FIG. 5 is a diagram showing a dilution calibration curve and detection sensitivity when a panel serum 50 of standard serum is serially diluted to 1 U / ml, and a sample treated with a sample is measured by a sandwich immunoassay reaction system.

FIG. 6 shows the results obtained by subjecting panel serum 13 to sample treatment, fractionating it using a gel-loca column, and measuring core antigen activity in the fractionation. The molecular weight is about 150 kD for IgG and about 68 kD for albumin.

FIG. 7 shows the correlation between the value obtained by measuring the released core antigen activity after the sample treatment of the present invention and the amount of HCV-RNA determined by the amplicor HCV monitor (PCR method) for a sample that showed a positive result with the amplicor HCV monitor kit. FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/577 C12R 1:91) // (C12N 15/02 ZNA (C12P 21/08 C12R 1:91) C12R 1:91) (C12N 5/10 C12N 15/00 ZNAC C12R 1:91) 5/00 B (C12P 21/08 C12R 1:91) C12R 1:91) (72) Inventor Tatsuharu Kimura Iruma-gun, Saitama 1-3-1, Oimachi Nishitsurugaoka, Tonen Co., Ltd. (72) Inventor Kumiko Iida 1-31, Nishitsurugaoka, Oimachi, Iruma-gun, Saitama Prefecture Tonen Co., Ltd. (72) Inventor Shintaro Yagi 1-3-1 Nishitsurugaoka, Oi-machi, Iruma-gun, Saitama, Japan

Claims (6)

[Claims] 1. The amino acid number 10 of the HCV polypeptide.
An antibody that recognizes 0-120. 2. The antibody according to claim 1, which is a monoclonal antibody. 3. HC11-11 (FERM BP-60)
05). 4. HC11-11 (FERM BP-60)
05) is a monoclonal antibody produced by the hybridoma. 5. A method for determining the presence or absence of HCV in a sample or quantifying HCV, comprising using the antibody according to claim 1, 2, or 4. 6. A method for determining the presence or absence of HCV in a sample, comprising the antibody according to claim 1, 2, or 4,
Kit or diagnostic agent to quantify.