JP2001215228A - Method of detecting or measuring virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily detect or measure a virus at high sensitivity. SOLUTION: In this measuring method, a virus antibody is measured by binding it with its probe under the presence of a surfactant having an alkyl group having 10 or more of carbon numbers, and having secondary, tertiary or quaternary amine, or a nonionic surfactant, or under the presence of the both.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting or measuring a virus and a reagent therefor.

[0002]

2. Description of the Related Art At present, various virus detection methods are used for screening for the presence of infectious viruses in blood or blood products, and for determining the presence or absence of viruses in disease patients. However, these methods are slightly different depending on the type of virus, but are not limited to those having high sensitivity and high specificity.
Many are expensive or require a long time, such as virus isolation culture. Hereinafter, hepatitis C will be mainly described and will be regarded as background art of the present invention.

[0003] The cause of hepatitis C has not been elucidated for a long time, but its gene has been cloned (Sc
gene 244: 359-362, 1989), and the development of a diagnostic method by antibody measurement using a recombinant antigen made based on the gene (Scie)
inc. 244: 362-364, 1989); JP-T-2-500880); HCV (hepatitis C virus, Hepatitis C Virus) as a causative factor; and infectious diseases mainly involving blood and blood-related preparations. It became clear that there was. 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 common viral infections, there is an indeterminable period called the so-called wind period, which occurs during the early stage of the infection, 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.

[0005] Interferon (IFN) is currently used for the treatment of hepatitis C.
Six months after V extermination, HCV antibody titers decrease, and some researchers believe that it is possible to determine the therapeutic effect only by measuring HCV antibody titers. 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.

[0006] HCV is derived from other viruses such as HBV (B
Virus particles (virus antigens) directly, because the amount of virus in the blood is lower than that of hepatitis virus, etc., and the virus cannot be propagated in vitro or in animals. 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.

[0007] First, it has been pointed out that since the substance to be detected is RNA, its storage stability is low, so that 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.

[0008] For example, a test method using the PCR method is the most sensitive detection method for detecting a gene fragment, but it is necessary to extract HCV genomic RNA from a sample, or to reverse genomic RNA to 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 when contamination occurs, false positives occur frequently. Therefore, a large amount of specimen cannot be processed at one time. Also, the pre-processing time is 2
It takes more than an hour, and is complicated because it involves many 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-chain DNA probe method has low detection sensitivity and requires about 20 hours to obtain a result (Medical Science and Pharmaceutical Sciences 31: 961-970, 199).
4) 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 Fujino 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 method for detecting the viral genome described above. However, as in the case of the viral genome detection method, there remain some major problems.

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 Hep)
atomology 23: 742-745, 1995).
Is 10 ⁴ to 10 ⁵ copies /
ml indicates the detection limit, and Fujino et al. (Medical and Pharmaceutical Sciences 36: 1065-1070, 1996) suggested that the most sensitive detection method is CRT (conipetive reverse transcription) -PCR. It is reported that the pretreatment serum of 102 patients with chronic hepatitis C classified as RNA-positive by the method has a positive rate of 67%. 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, in order to obtain reproducibility, skill is required, and a processing time of at least about 2 hours is required. In addition, since there are steps such as centrifugation operation and supernatant removal, automation is difficult,
Moreover, simultaneous mass processing is 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 has the following advantages over the high-sensitivity PCR method. That is, since an excessive amplification operation is not added in the detection process, it is very 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 requirements suitable for use in measuring a large number of samples, for example, in the blood business and health examination. However, as already pointed out, the disclosed core antigen detection method can be 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 example, they cannot be used for so-called screening applications that handle a large number of samples, and cannot take advantage of the advantages of the PCR method. In addition, a measurement method that is highly clinically useful is always subject to sensitivity, specificity, reproducibility, operability, and low cost, and it is necessary to develop vigorously to satisfy all of them. Regarding the detection of viral antigens other than HCV, especially in screening applications where a large number of samples are measured, the sensitivity is lower than that of the PCR method, and the useful pretreatment method and the antigen are not exposed. Many have not been put to practical use.

[0014]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a virus antigen detection method suitable for treating a large number of specimens for so-called screening applications such as blood business and health examination. That is, it has the same sensitivity and specificity as compared to the PCR method, and simplifies the pretreatment.
Another object of the present invention is to provide a virus antigen detection system that can be easily applied to a large-scale processing system such as automation without performing a pretreatment operation.

[0015]

According to a first aspect of the present invention, a virus particle is destroyed to sufficiently expose a virus antigen, and if an antibody against the virus antigen is present, the antibody is destroyed. Thus, means for detecting or measuring a virus by detecting or measuring a virus antigen is provided.
Therefore, the present invention provides ( 1 ) a sample containing a virus,
A virus-containing specimen characterized by being treated with a treatment solution containing (1) an anionic surfactant and (2) any of a zwitterionic surfactant, a nonionic surfactant and a protein denaturant. Provide a processing method.

[0016] The present invention also provides ( 2 ) a sample containing a virus, wherein (1) an anionic surfactant, (2) a zwitterionic surfactant, and (3) a nonionic surfactant or protein denatured. A method for treating a virus-containing specimen, wherein the method comprises treating with a treatment solution containing any of the agents. The present invention also provides ( 3 ) a sample containing a virus, wherein (1) an anionic surfactant, (2) a zwitterionic surfactant, (3) a nonionic surfactant, and (4) protein denaturation. The present invention provides a method for treating a virus-containing specimen, wherein the method comprises treating with a treatment solution containing an agent.

The present invention further provides ( 4 ) the above ( 1 ) to ( 3 ).
A method for measuring a virus, characterized by detecting or quantifying the presence of a virus antigen by reacting a probe that specifically recognizes a virus antigen using the sample processing method according to any one of the above. The present invention further provides a kit for determining the presence or absence of a virus in a sample, a kit for quantification, or a diagnostic agent, which comprises an anionic surfactant and is used for the immunoassay according to ( 4 ). The present invention further provides a kit for determining the presence or absence of a virus in a sample, a kit for quantification, or a diagnostic agent, which comprises the monoclonal antibody described below, which is used in the immunoassay method of the above ( 4 ).

According to a second aspect of the present invention, there is provided a method for detecting or measuring a virus antigen in a window period in which an antibody against the virus has not yet been produced. In this method, it is sufficient to destroy the virus particles to expose the virus antigen, and it is not necessary to destroy antibodies in the blood against the virus antigen. Therefore, the present invention further provides a method for measuring a virus in the presence of a surfactant or a nonionic surfactant having an alkyl group having 10 or more carbon atoms and a secondary to quaternary amine, or both. A method comprising measuring a viral antigen by binding to the probe is provided.

The present invention further provides HC11-14 (FE) which produces a monoclonal antibody suitable as a probe for detecting the HCV core antigen among the above-mentioned virus antigens.
RMBP-6006), HC11-10 (FERM B)
P-6004), HC11-3 (FERM BP-60
02), and HC11-7 (FERM BP-600)
3) providing a hybridoma cell line selected from the group consisting of: The present invention also relates to HC11-14 (FERM).
BP-6006), HC11-10 (FERM BP-
6004), HC11-3 (FERM BP-600)
2) A monoclonal antibody produced by a hybridoma selected from the group consisting of HC11-7 (FERM BP-6003).

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION
A virus that forms a virus particle having a structure composed of a structural protein surrounding genomic RNA or DNA and a membrane protein or lipid membrane surrounding the structural protein. Representative examples of the above virus having RNA as a genome include hepatitis C virus (HCV) and HCV related viruses.

As HCV-related viruses, hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, hepatitis G virus, hand-foot-and-mouth disease virus, flavivirus (yellow fever virus, West Nile virus, Japanese encephalitis) Virus, dengue virus), togavirus (alphavirus, rubivirus, arterivirus, rubella virus), pestivirus (porcine cholera virus, bovine diarrhea virus), paramyxovirus (parainfluenza virus 1,2,3,4, dog distem virus) Virus,
Newcastle disease virus, respiratory syncytial virus, Linda pest virus, simian parainfluenza virus, measles virus, mumps virus), orthoxovirus (human influenza virus,

Avian influenza virus, equine influenza virus, swine influenza virus), rhabdovirus (rabies virus, vesicular stomatitis virus), picornavirus (poliovirus, coxsackievirus, echovirus, bovine enterovirus,
Porcine enterovirus, simian enterovirus, mouse encephalomyelitis virus, human rhinovirus, bovine rhinovirus, equine rhinovirus, foot-and-mouth disease virus, hepatitis A virus), coronavirus (human coronavirus,
Chicken infectious bronchitis virus, mouse hepatitis virus, swine infectious gastroenteritis virus), arenavirus (lymphocytic choriomeningitis virus, lhasavirus, Korean hemorrhagic fever virus), retrovirus (HTLV: human adult leukemia virus) , HIV: AIDS virus, feline leukemia sarcoma virus, bovine leukemia virus, rous sarcoma virus), reovirus (rotavirus), calicivirus (norwalk virus), Bunya virus (nephrotic symptomatic hemorrhagic fever virus), filovirus (Ebola) Virus,
Marburg virus).

Representative examples of the above viruses having DNA as a genome include hepatitis B virus (HBV),
And HBV-related viruses. As HBV-related viruses, poxviruses (vaccinia virus, alastorium virus, cowpox virus, smallpox virus), parvoviruses (human parvovirus, swine parvovirus, bovine parvovirus, canine parvovirus,
Feline leukopenia virus, mink aleutian virus, papova virus (papilloma virus, polyoma virus), adenovirus, herpes virus (herpes simplex virus, cytomegalovirus, varicella-zoster virus, EB virus, equine herpes virus, cat herpes) Virus, Marek's disease virus), African swine fever virus, and the like.

[0024] In addition to these, various pathogenic viruses are known, and unidentified viruses still exist. However, as described above, their viral structures are based on genomic RNA or DNA. As long as the virus forms a virus particle having a structure composed of a wrapping structural protein and a membrane protein and a lipid membrane surrounding the wrapping protein, the present invention can also release the virus into a state suitable for immunoassay by the treatment method. Is obvious.

Hereinafter, embodiments will be described focusing on the HCV core antigen. HCV has a blood concentration of 10 ² copies / ml to 10
And ⁶ copies / ml, since low compared to HBV (10 ⁹ copies / ml), which requires a very 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, increasing the number of antibody molecules, III) a probe defining the lower limit of detection sensitivity, for example, reducing non-specific reactions caused by binding of an antibody to a substance other than an antigen, and IV) labeling used for detection. Methods for increasing the detection sensitivity are conceivable, and it is possible to increase the sensitivity by combining these methods.

As a method for increasing the number of antigen molecules,
-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 molecules of a probe for detection, for example, an antibody, which binds to an antigen, II-1) the number of recognition epitopes is increased by using a plurality of probes, for example, antibodies. 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 an infectious disease, it is expected that human antibodies exhibiting high affinity for binding to an antigen 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. Connected (II-3). Although it is difficult to generalize the method for reducing non-specific reactions, III-
1) The nonspecific reaction is reduced by increasing the affinity (affinity and avidity) of the probe, for example, the antibody with the antigen by changing the composition of the buffer solution. III-2) Removing the causative substance of the nonspecific reaction There are other ways to do this.

As a method for increasing the detection sensitivity of a labeled substance, there are four methods: IV-1) using a labeled substance having high detection sensitivity (such as a radioisotope); and IV-2) using an enzyme or a catalyst for the labeled substance to obtain a signal. Amplify, IV-3) Modify enzyme substrate to a more sensitive substrate, IV-4) Amplify chemically, electrically or mechanically the signal of the substrate of the enzymatic reaction or chemical reaction, IV-5) Methods such as increasing the number of labels per antibody, and increasing the sensitivity of an instrument used for IV-6) signal detection are conceivable.

Analysis of the steps of the pretreatment method of the disclosed HCV core antigen detection method reveals that 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 sediment 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. In addition, it has been reported that virus particles have a complex structure surrounded by LDL (low-density lipoprotein) in blood and that there are also antibodies against coat proteins. 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 important to efficiently remove envelopes and contaminants surrounding the virus particles from the virus particles and efficiently release the core antigen molecules.
The same is true for viruses other than HCV, and it is necessary to efficiently release virus antigens.

Accordingly, the present invention relates to a method for treating a virus (a serum) 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, since a probe to be used for detection, for example, a human antibody that competes with the antibody for binding exists in the specimen at a high titer, an operation for removing this is important for increasing the sensitivity. Therefore, one embodiment of the present invention relates to a treatment method for simultaneously inactivating human antibodies present in a specimen by using a treatment method for easily releasing a virus antigen in a specimen.

By using the treatment method presented by the present invention, the viral antigens present in the sample are released from the virus particles or immune complexes in a state suitable for forming an immune complex with a probe, eg, an antibody. However, by simultaneously inactivating the human antibody present in the sample that inhibits the detection reaction, it becomes possible to detect the antibody easily and with high sensitivity by an immunoassay using a probe such as an antibody.

The probe used for detection, for example, an antibody specifically binds to a virus antigen, exhibits a certain high affinity, and does not induce a nonspecific reaction when added to a reaction system. Any probe may be used, but as shown in Example 4, it is preferable that one of the probes used for the primary reaction includes one that can recognize and bind to the C-terminal side of the HCV core antigen. Here, the C-terminal side of the HCV core antigen means
It refers to the 81st to 160th sequence of the sequence shown in SEQ ID NO: 2, or a part thereof. Furthermore, a probe for the N-terminal side of the HCV core antigen may be included here.
Here, the N-terminal side of the HCV core antigen refers to the 10th to 70th sequence 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 a hybridoma obtained by the method described above, or a spleen cell, a monoclonal antibody produced by a cell obtained by immortalizing blood leukocytes with an EB virus, H
An antibody produced by a human or chimpanzee or the like infected with CV; a variable region gene fragment obtained from a mouse or human immunoglobulin cDNA or chromosomal DNA; A variable region gene fragment constituted by combining the sequence with
Recombinant antibody comprising a variable region gene fragment constructed using an artificial gene sequence or a variable region gene fragment produced by using the same as a material by a gene recombination technique, and an immunoglobulin constant region gene fragment A recombinant antibody produced by a cell transformed by the gene;

A phage antibody produced by fusing the above-mentioned variable region gene fragment with, for example, a structural protein of bacteriophage, and combining the above-mentioned variable auditory region gene fragment with another applicable gene fragment, such as a part of the myc gene. Recombinant antibodies produced by cells transformed by the constructed recombinant antibody gene, molecules specifically binding to proteins such as probes and receptors produced by artificially introducing a variable region into a trypsin molecule. Any molecule that exhibits high specificity and affinity for the core antigen, such as a probe obtained by artificial modification or another probe produced by combinatorial chemistry technology, can be used.

Furthermore, the present invention provides a method for releasing a virus antigen or a virus complex from a specimen containing a virus antigen, in order to make the virus core antigen and a probe such as an antibody suitable for forming an immune complex. Treating the sample with a treating agent that simultaneously inactivates the human antibody present in the sample, which simultaneously inhibits the detection reaction, and detects and quantifies the released core antigen by an immunoassay using a probe such as an antibody. The present invention provides an assay method and a test kit.

Sample processing agents and treatments indicated by the present invention
Methods Samples in 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.

As is already known by SDS (sodium dodecyl sulfate) polyacrylamide electrophoresis (SDS-PAGE), most proteins are SDS
It is denatured by heat treatment in the presence and becomes a monomer except for a molecule bonded by a covalent bond. That is, when a treatment agent containing an anionic surfactant such as SDS is added to the measurement sample, the virus can be destroyed, and at the same time, the anti-core antibody in the sample can be denatured and the core antigen in the sample can be released. is there. This is because the SDS
When the HCV core antigen in the sample treated with the treating agent containing is subjected to molecular weight analysis using gel filtration, it was also confirmed that the HCV core antigen was detected at the position of a monomer expected from the theory.

Also, Kashiwakuma et al. (J. Immunological)
al Methods 190 190-89,199
As reported in 6), a sample consisting of the extract of the recombinant HCV-expressing cells was separated by SDS-PAGE, and the HCV core antigen was detected by Western blotting. Its immune activity is detected. It can be easily conceived that by adding a denaturant containing SDS to a sample, antigens can be efficiently released and the number of antigen molecules can be increased, as long as they belong to this field.

However, as is well known, SD
Since anionic surfactants such as S have a large protein denaturing effect, if they are directly added to a probe, for example, an immune complex formation reaction with an antibody in the presence thereof, the antibody also denatures the antibody, losing its function and reducing sensitivity. Lead. It is also known that treatment with an anionic surfactant such as SDS causes the loss of the epitope structure. As a result, the binding of the antibody is weakened, and the sensitivity is reduced. In order to eliminate these effects leading to a decrease in sensitivity, it is necessary to reduce the denaturing effect by some method after the SDS treatment.

Surfactants including anionic surfactants include:
For example, it is known that it can be removed by dialysis, ultrafiltration, gel filtration, electrophoresis, ion exchange, precipitation, membrane transfer, etc., as described above by Western blotting, gel filtration. The fact that the antigen is detectable
This shows that the antigen-antibody reaction can be performed by performing some operation after the SDS treatment. However, performing these processes is not a method suitable for the purpose of the present invention because it requires time and complicated operations.

Further, by diluting with an excess amount of the reaction solution, the reaction can be prevented from being affected by reducing the concentration to a concentration at which no denaturing action is exhibited. It is apparent that the method is not suitable for the purpose of the present invention because the method cannot be applied to an immunoassay in which the amount of a sample to be added is limited, such as a measurement method using the method. Therefore, the present inventor added a treating agent containing an anionic surfactant, and further added some additives, so that the denaturing effect of the anionic surfactant does not affect probes such as antibodies. It was examined whether it was possible to enhance the ability to release the HCV core antigen by an anionic surfactant.

Here, the present inventor weakened the denaturing effect of SDS on the immobilized antibody by adding a treating agent containing a surfactant other than an anionic surfactant such as SDS. It has been found that the sensitivity can be increased as compared with only the treating agent containing the same. Also, S
A similar effect can be obtained when a treating agent containing an anionic surfactant such as DS and a treating agent containing another surfactant or an agent for weakening a hydrogen ion bond such as urea are added at the same time. It has been found that the release of HCV core antigen is further enhanced by enhancing the release of HCV core antigen from virus particles and the inactivation of anti-core antigen antibody in the specimen. Furthermore, it has been found that the HCV core antigen can be detected with higher sensitivity by performing a heat treatment step after adding a treating agent containing SDS and other surfactants, thereby completing the present invention.

The anionic surfactant used for the treatment of the specimen is not limited to SDS, but may be sodium cetyl sulfate, other alkylated sulfates, alkylated sulfonates such as sodium dodecyl sulfonate, or alkylallyl sulfonate. Salts and the like are also possible, and as a surfactant to be added in addition to the anionic surfactant, CHAPS (3-
[3-Colamidopropyl) dimethylammonio] -1-
Propanesulfonic acid), CHAPSO (3- [cholamidopropyl) dimethylammonio] -2-hydroxy-1
-Propanesulfonic acid), amphoteric surfactants such as dodecyl-N-betaine, and polyoxyethylene isooctylphenyl ethers such as Triton X100;
Nonionic surfactants such as polyoxyethylene nonylphenyl ethers such as NP40, polyoxyethylene sorbitol esters such as Tween 80, polyoxyethylene dodecyl ethers such as Brij 58, and octyl glucoside are suitable.
Zwitterionic surfactants such as HAPS and Triton-
Includes a non-ionic surfactant such as X100. It is also effective to add a drug (protein denaturant) such as urea or thiourea, which acts to destroy the higher-order structure of the protein.

The concentration at the time of the treatment is 0.5% or more for SDS, 0.1% or more for CHAPS, 1M or more for urea,
More preferably, ritonX100 is used at 0.1% or more and 0.75% or less. The processing temperature of the above sample is 4 ° C. which is a range usually used in a general laboratory.
The temperature may be in the range of 100 ° C. or lower, but in the case of adding a nonionic surfactant, attention should be paid to the cloud point. Preferably, the temperature is 37 ° C. or higher, and a treatment at 50 to 60 ° C., which is generally used for inactivating serum, is effective.

By using the processing method of the present invention, H
From a sample containing virus particles having the same structure as CV,
It is clear that a virus antigen can be released in a state suitable for a so-called immunoassay using an antibody or the like as a probe. Here, the virus having a structure similar to HCV is a virus that forms a virus particle having a structure composed of a protein that packs genomic RNA and DNA and a membrane protein and a lipid membrane surrounding the protein. Related viruses include flaviviruses, retroviruses such as human immunodeficiency virus (HIV), and the like. DNA as the genome
Even those having the same structure include those having the same structure.

Further, HCV which is an RNA virus,
Both DNA viruses, HBV, have these genomic R
It is a virus that forms a virus particle having a structure consisting of a structural protein surrounding NA and DNA and a membrane protein and lipid membrane surrounding it. In any of the embodiments, by using the treatment method of the present invention, not only HCV and HBV but also virus particles having the same structure as those described above are destroyed, and the antigen of the virus is sufficiently exposed, and the antigen is exposed. It also provides for detecting or measuring the virus by detecting or measuring.

[0050] When using the serum or the like as removing a sample for measurement of interference by hemoglobin, red blood cells contained in the sample is hemolyzed during the pretreatment hemoglobin is released, the modified hemoglobin interfere with the measurement There are cases. Therefore, in the first aspect of the present invention, it is preferable to eliminate such interference with the measurement. It has been found that it is preferable to add at least one of urea, an imidazole ring-containing compound and an indole ring-containing compound as an additive for this purpose.

Examples of the imidazole ring-containing compound include imidazole, histidine, imidazole acrylic acid, imidazole carboxaldehyde, imidazole carboxamide, imidazole dione, imidazole dithiocarboxylic acid, imidazole dicarboxylic acid, imidazole methanol, imidazolidinthion, imidazolidone, pyridine, and the like. Can be

Further, as the indole ring-containing compound,
Tryptophan, indole acrylic acid, indole,
Indoleacetic acid, indoleacetic acid hydrazide, indoleacetic acid methyl ester, indolebutyric acid, indoleacetonitrile, indolecarbinol, indolecarboxaldehyde, indolecarboxylic acid, indoleethanol, indolelactic acid, indolemethanol, indolepropionic acid, indolepyruvic acid,
Indolyl methyl ketone, indomycin, indoleacetone, indomethacin, indoprofen, indolamine and the like can be mentioned.

The appropriate amount of urea is 0.5M to 5M, and indoleacrylic acid is 5 mM to 50 mM.
Is appropriate, and other additives are 0.05M to
A concentration of 0.5M is appropriate.

[0054] According to a second aspect of the exposure invention of viral antigens, it relates to a method for the detection of viral antigens in a sample taken in the window period, because the antibody has not yet generated to viral antigens in this process, virus It is sufficient to break the particles to expose the viral antigens, and not the antibodies present in the sample. Therefore, the pretreatment of the sample described above is not required, and it is sufficient that a virus particle disrupting agent for exposing the virus antigen is present in the measurement reaction solution. The virus antigen described herein means an antigen present inside a virus particle, and a core antigen is a representative thereof.

It is considered that a virus particle has a structure in which a nucleic acid as a genome and a core antigen form a complex to form a particle, and the particle is covered with an outer membrane composed of a lipid membrane and an envelope protein. There are many things. Furthermore, it is thought that it exists in the form of a complex with low-density lipoprotein (LDL) and an antibody against a virus in blood. Therefore, the probe cannot recognize and bind to a virus antigen typified by a core antigen present inside the virus particle if the virus particle is present in the blood. Therefore, in order to detect the core antigen, it is necessary to perform a process such as removal of these structures surrounding the core antigen so that the probe recognizes the core antigen.

That is, in the present invention, a reaction condition comprising a core antigen in a virus particle contained in a specimen, which is exposed so that a probe for recognizing the core antigen can be recognized, a reaction method comprising a reaction system, and a reaction method comprising: Also provided is a reagent comprising a system for causing the reaction. A reaction system suitable for antigen detection in the system provided by the present invention is a virus particle which is a complex structure present in a sample, under mild conditions that do not lose the function of an antibody against a virus antigen epitope Therefore, the system comprises conditions that sufficiently expose the region recognized by the antibody that is the probe that recognizes the virus antigen.

Virus particles already separated by ultracentrifugation (Takahashi et al., 1992, J. Am.
Gen. Virol, 73: 667-672)), and treating the HCV particles coagulated and precipitated with polyethylene glycol with a nonionic surfactant such as Tween 80 or Triton X100 (Kas).
hiwakuma et al. , 1996, J. Mol. Im
Munological methods: 190: 79
-89), it has been shown that the core antigen can be detected. However, the former has insufficient detection sensitivity, and it is doubtful whether the antigen is sufficiently exposed. In the latter, the antibody is inactivated by adding another treating agent, and the effect of the surfactant itself is not mentioned.

In the present invention, first, the conditions are examined based on a surfactant, and the reaction solution is made to have a composition centered on the surfactant. The antigen in the virus particles can be efficiently detected by simply diluting the sample in the reaction solution without applying a pretreatment method including operations such as centrifugation and heating. It is necessary to effectively extract core antigens from virus particles, suppress interaction with various substances in serum, and provide conditions under which probes and antigens can react efficiently. As the effective surfactant in this case, a surfactant having an alkyl group having 10 or more carbon atoms and a secondary, tertiary or quaternary amine in the same molecule, or a nonionic surfactant is used. No.

In the surfactant having an alkyl group and a secondary, tertiary or quaternary amine, the alkyl group is preferably a straight-chain alkyl group, and the number of carbon atoms thereof is preferably 10 or more, more preferably 10 or more. ~ 20. As the amine, a tertiary amine or a quaternary amine (ammonium) is preferable. Specific surfactants include dodecyl-N-sarcosine acid, dodecyltrimethylammonium salt, cetyltrimethylammonium salt, 3- (dodecyldimethylammonio) -1-propanesulfonic acid,
3- (tetradecyldimethylammonio) -1-propanesulfonic acid, dodecylpyrimidium salt, cetylpyridium salt, decanoyl-N-methylglucamide (MEGA
-10), dodecyl-N-betaine and the like. Dodecyl-N-sarcosine acid and dodecyltrimethylammonium salt are preferred.

The above-mentioned nonionic surfactant is 12
Preferred are those having a hydrophilic / hydrophobic ratio of from about 14 to about 14, such as polyoxyethylene isooctyl phenyl ethers such as Triton X100 and Triton X114, or polyoxyethylene noniphanyl ethers such as Nonidet P40 and Triton N1.
01, Nikkol NP and the like. In the present invention, the above two types of surfactants may be used alone, but it is more preferable to use them in combination, and a synergistic effect can be obtained by using them in combination. Further, a factor that changes the water environment, such as urea, may be added.

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

The polypeptide having HCV antigen activity referred to in the present invention means a fusion polypeptide or polypeptide which immunologically reacts with an anti-HCV antibody, and is used for producing the hybridoma of the present invention and the monoclonal antibody obtained therefrom. It can be 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.

[0063] 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 6 can be easily prepared by those skilled in the art. The production of monoclonal antibodies by hybridomas is well known. For example,
The above fusion polypeptide or polypeptide (hereinafter, the present antigen) is used alone or in combination with BSA, KLH, or the like as an antigen in an intraperitoneal cavity or intradermally of a BALB / c mouse or the like, and mixed with an adjuvant such as simple or complete Freund's adjuvant. And immunize regularly. 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 Koehler and Milstein (N
Attitude 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 Hoogenboon's review and the like (Trends in Biotechnology).
y, 15: 62-70, 1997).

Detection System Using Probes Monoclonal antibodies prepared according to the present invention are based on enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, radioimmunoassay, and agglutination for the detection and quantification of viral structural proteins. Assay or other well-known immunoassays as test reagents. When a labeled antibody is used for detection, examples of the labeling compound include a fluorescent substance, a chemiluminescent substance, a radioactive substance,
Enzymes, dyes and the like are used.

For example, when a method based on a sandwich reaction system is used to detect a virus antigen in a sample (serum), a diagnostic kit to be used is coated on a solid support (for example, the inner wall of a microtiter well). Of the present invention and one or more monoclonal antibodies or fragments thereof conjugated to a labeling substance. 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, erythrocytes, metal compounds, etc.), membranes (liposomes, etc.), filters and the like. No.

[0068]

According to the method of the present invention, a virus antigen can be easily released from virus particles in a state suitable for a so-called immunoassay method in which an antibody or the like is detected as a probe. In addition, by processing a sample containing virus particles 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. Becomes In addition, it is possible to prepare a kit for determining the presence or absence of a virus in a sample, a kit for quantification, and a diagnostic agent using an immunoassay using the sample processing method according to the present invention.

[0069]

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 and 1 μg of each DNA of pUC · C10-E12 were 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 EcoRI
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 EcoRI-ClaI fragment and an approximately 920 bp ClaI-KpnI fragment.

These two DNA fragments and pUC119 were
A 10 × ligase buffer [660 mM Tris-HCl (pH 7.0) was added to the vector digested with coRI and KpnI.
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.
-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: 7), 5'-TTAGTCCTCCAGAACCC
PCR was performed using GGAC-3 ′ (SEQ ID NO: 8). PCR is performed using GeneAmpTM (DNA Amplification Reagent
Kit, manufactured by Perkin Elmer Cetus) under the conditions of DNA denaturation at 95 ° C for 1.5 minutes, annealing at 50 ° C for 2 minutes, and DNA synthesis at 70 ° C for 3 minutes, and the obtained DNA fragment was subjected to 0.8% agarose gel electrophoresis. It was separated by electrophoresis and purified by the glass powder method (Gene Clean).

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 (pH7.
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.
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 E
coRI and 15 units of BamHI enzyme] at 37 ° C.
The digestion reaction was performed for 1 hour, followed by 0.8% agarose gel electrophoresis, and about 490 bp EcoRI-Bam.
The HI 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
coRI 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 resulting aqueous layer was precipitated with ethanol, and the precipitate was dried. The obtained EcoRI-BamHI-treated vector DNA (1 μg) and the above-mentioned core 140 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 [Mann.
iatis et al., Molecular Cloning: A Laboratory Manual,
(1982)].

1 μg of the obtained plasmid DNA was added to a restriction enzyme reaction solution (20 μl [150 mM NaCl, 6 mM Tri).
s-HCl (pH 7.5), 6 mM MgCl ₂ , 15 units of EcoRI and 15 units of BamHI enzyme].
After digestion at 7 ° C for 1 hour, agarose gel electrophoresis was performed to select a Trp / TrpE core 160 expression plasmid in which an EooRI-BamHI fragment of about 490 bp was generated.

(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 fusion polypeptide was solubilized and extracted by adding 10 ml of buffer A containing 6 M urea to the fraction. 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 was performed every two weeks, and about two weeks later, TrpC11 dissolved in physiological saline was dissolved.
10 μg was administered into the tail vein.

On the third day after the final booster immunization, the spleen was aseptically removed from the immunized animal, cut into pieces with scissors, and the spleen was loosened into individual cells using a mesh.
Washed three times with 1640 medium. After washing the myeloma cell line SP2 / 0Ag14 in the logarithmic growth phase in the same manner as described above, 2.56 × 10 ⁷ cells and 1.64 × 10 ⁸ spleen cells were placed in a 50 ml centrifuge tube and mixed. Centrifugation was performed at 200 × g for 5 minutes, the supernatant was removed, and 1 ml of RPMI-1640 medium containing 50% polyethylene glycol (PEG) 4000 (manufactured by Merck) maintained at 37 ° C. was added.
Further, 10 ml of RPMI-1640 medium was added for 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 hybridomas were subjected to single cloning according to a conventional limiting dilution method, and the obtained hybridomas were named HC11-14, HC11-10, HC11-3, and HC11-7. The four types of hybridomas were sent to FERM BP on July 4, 1997 by the
-6006, FERM BP-6004, FERM B
Deposited as P-6002 and FERM BP-6003.

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.

The monoclonal antibodies C11-14 and C1 produced from each of the five hybridomas
The isotypes of 1-10, C11-7 and C11-3 are rabbit anti-mouse Ig isotype antibodies (Zyme
d11), C11-1 was obtained.
0, C11-7 are IgG2a, C11-14, C11-
3 was found to be IgG1. 4 obtained
As a result of epitope analysis of 20 kinds of monoclonal antibodies using a synthetic peptide consisting of 20 amino acids synthesized by a sequence derived from the HCV core region, Table 1 was obtained.
As shown in the figure, the monoclonal antibody was found to specifically recognize a part of the core region.

[0086]

[Table 1]

Embodiment 4 FIG . Examination of sample treatment conditions 1) Examination of SDS concentration 100 µl of healthy human serum and HCV-RNA positive serum
In addition, SDS dissolved at various concentrations and 0.6% CHAPS
100 μl of a treatment solution containing was added. Place in a warm box set at 56 ° C and treat for 30 minutes.
0 μl was used as a measurement sample. The results obtained by the measurement method described below are shown in FIG.

2) Examination of CHAPS concentration 100 μl of healthy human serum and HCV-RNA positive serum
Was added with 100 μl of a treatment solution containing CHAPS and 5% SDS dissolved at various concentrations. Place in a warm box set at 56 ° C and treat for 30 minutes.
1 was used as a measurement sample. The result of the measurement method described below,
The CHAPS concentration during the treatment reaction is shown on the horizontal axis, and is shown in FIG.

3) Examination of urea concentration 100 μl of healthy human serum and HCV-RNA positive serum
A treatment solution containing urea dissolved at various concentrations (5% S
DS, 0.6% CHAPS). 5
The sample was placed in a heat insulation box set at 6 ° C. and treated for 30 minutes, and 80 μl of the sample was used as a measurement sample. The results by the measurement method described below, the urea concentration at the time of the treatment reaction on the horizontal axis,
As shown in FIG.

4) Examination of Triton X100 concentration 100 μl of healthy human serum and HCV-RNA positive serum
Then, 100 μl of a treatment solution (5% SDS, 0.6% CHAPS, 6 M urea) containing Triton X100 dissolved at various concentrations was added to the mixture. The sample was placed in a heat insulation box set at 56 ° C. and treated for 30 minutes, and 80 μl thereof was used as a measurement sample. The results obtained by the measurement method described below are shown in FIG. 4 with the Triton X100 concentration at the time of the treatment reaction taken on the horizontal axis.

5) Examination of reaction temperature 100 μl of healthy human serum and HCV-RNA positive serum
, 100 μl of a treatment solution (5% SDS, 0.6% CHAPS, 6M urea, 0.75% Triton X100) was added. 4 ° C, room temperature (23 ° C), 37 ° C, 45 ° C, 56
The mixture was treated at 70 ° C. and 30 ° C. for 30 minutes, and 80 μl thereof was used as a measurement sample. FIG. 5 shows the results of examination using the measurement method described below.

Assay methods The samples obtained in the examination of the serum treatment method were each evaluated using the following assay methods. That is, anti-HCV core antigen monoclonal antibody (equivalent mixture of antibodies C11-3 and C11-7)
Was diluted with 0.1 M carbonate buffer (pH 9.6) to a final concentration of 6 μg / ml, and dispensed at 100 μl per well of a 96-well microplate (manufactured by Nunc). After standing at 4 ° C. overnight, 1 part containing 0.15 M NaCl
Wash twice with 0.35 ml of 0 mM sodium phosphate buffer (pH 7.3) and add 10% 0.5% casein-Na.
0.35 ml of a mM sodium phosphate buffer (pH 7.35) (hereinafter referred to as a blocking solution) was added, and the mixture was 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
160 μl of 100 mM sodium phosphate buffer (pH 7.3) containing 20% Tween 20 and a measurement sample obtained by each serum treatment method were added to each well, reacted at room temperature for 2 hours, and washed 5 times with 300 μl of a washing solution. Furthermore, a peroxidase (POD) labeled monoclonal antibody (C
100 μl of the mixture of 11-10 and C11-14) was added and reacted at room temperature for 30 minutes. After the reaction, the plate was washed 5 times with 300 μl of the above-mentioned washing solution, 100 μl of a substrate (orthophenylenediamine, hereinafter referred to as OPD) solution was added, and the mixture was reacted at room temperature for 30 minutes. The absorbance at 492 nm (OD492) was measured.

From FIGS. 1 to 4, the respective processing conditions were optimized, but it was difficult to detect the core antigen in the unprocessed sample. The detection of the core antigen became possible. In particular, the SDS concentration during the treatment reaction is 0.5% or more, and the CHAPS concentration is 0.1%.
1% or more, urea concentration is 1M or more, Triton X1
Use at a concentration of 0.1 to 0.75% at 4 ° C.
It was shown that the core antigen could be detected well in the temperature range of from 70 to 70 ° C.

Embodiment 5 FIG . Detection of structural region core antigen and
Assay method (1) Treatment solution (5% SDS, 0.6% CH
APS, 6M urea, 0.75% Triton X100)
Was added in an amount of 100 μl. The sample was placed in a heat insulation box set at 56 ° C. and treated for 30 minutes, and 120 μl of the sample was used as a measurement sample. Anti-HCV core antigen monoclonal antibody (C
11-3 and C11-7 in an equivalent amount) to a final concentration of 6 μg /
The mixture was diluted with 0.1 M carbonate buffer (pH 9.6) to a volume of 100 ml and dispensed at 100 μl per well of a 96-well microplate (manufactured by Nunc). After standing at 4 ° C overnight,
After washing twice with 0.35 ml of 10 nM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl, 0.35 ml of a blocking solution was added, and the mixture was allowed to stand at room temperature for 2 hours.

After removing the blocking solution, the reaction buffer 12
0 μl and the measurement sample obtained by the above-described treatment method were added to each well, and reacted at room temperature for 2 hours. Washing liquid 300μ
1 times, and further washed with peroxidase (POD) -labeled monoclonal antibodies (C11-10 and C11-1).
4: Equal volume) (100 μl) and reacted at room temperature for 30 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. 6 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 HCV core antigen could be detected or quantified.

Embodiment 6 FIG . Detection of HCV structural region core antigen
(2) Method Using Alkaline Phosphatase-Labeled Monoclonal Antibody A 96-well black microplate (Nunc) as a solid phase carrier, an alkaline phosphatase-labeled monoclonal antibody as a labeling antibody, CDPstar as a substrate (Emerald II as sensitizer) It was used. FIG. 7 shows a dilution line of panel serum 50 used as a standard serum.
The core antigen in the sample is measured in a concentration-dependent manner,
Detection of 5 mU / ml was possible. It has been clarified that the HCV core antigen can be detected or quantified by using the measurement method using the alkaline phosphatase-labeled monoclonal antibody.

Embodiment 7 FIG . Recognized by serum processing and assay
Analytical panel of molecular form 0.25 ml of serum 13 was treated by each 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 considered to recognize a molecule of 0 to 30 kDa, indicating that the core antigen in the virus was released by the pretreatment described above due to virus destruction and inactivation of anti-core antibodies present in serum. Was.

Embodiment 8 FIG . HCV structural region in serum sample
A. Antigen measurement method The amount of HCV-RNA was 10 ³ to 10 using the Amplicon HCV monitor kit (Roche), which is a PCR method.
^Using the serum measured at ⁷ copies / ml and the serum of a healthy individual, the HCV core antigen in the serum was quantified by the method described above. In addition, panel 50 serum (1 U / ml) was used as a standard serum.
) Was serially diluted with a 10 mM sodium phosphate buffer (pH 7.3) containing 1% BSA and treated in the same manner. Table 2 shows the measurement results. Among the samples measured this time, all healthy subjects were below the detection limit,
All of the PCR-positive cases could be detected. The correlation at this time is shown in FIG. 9, and the correlation coefficient with the PCR method was 0.8 or more, indicating a high correlation.

[0101]

[Table 2]

Embodiment 9 FIG . Reduces sensitivity loss due to hemolyzed serum
Examination of additives for serum The effect of serum components on sensitivity was examined, and it was confirmed that the sensitivity was significantly reduced when hemoglobin was added. Pretreatment with a pretreatment agent containing SDS, CHAPS or Triton X100 denatured hemoglobin, possibly resulting in the effect of released heme. Therefore, an additive capable of reducing the influence of denatured hemoglobin was examined.

HCV core antigen positive serum (panel serum No.
3), a high-concentration hemoglobin (manufactured by International Reagent: interference check) was added to prepare a model sample, urea was added to the above pretreatment agent, and core antigen measurement was performed according to Example 6, The effect of the addition was studied. Table 3 shows the core antigen activity of the 430 mg / dl hemoglobin-added group assuming that the core antigen activity of the control hemoglobin-free group was 100%. The core antigen activity of the hemoglobin-added group without urea was reduced to about 30%,
It was confirmed that the increase in the amount of added urea increased the amount of core antigen activity in the hemoglobin-added group, and reduced the interference effect of hemoglobin.

[0104]

[Table 3]

On the other hand, the interaction between various amino acids and heme, and the buffering effect due to amino groups and carboxyl groups were considered, and various amino acids were added and the effects were examined. The results are shown in Table 4.

[0106]

[Table 4]

[0107] Tryptophan and histidine were the ones that showed the greatest interference suppressing effect. Table 5 shows the results of examining the concentration dependence of these interference suppression effects.

[0108]

[Table 5]

Heme was coordinated by the histidine side chain in hemoglobin and retained in hemoglobin, suggesting that this effect was due to the side chain. Therefore, the effects of imidazole, a side chain of histidine, and indoleacrylic acid containing an indole ring, a side chain of tryptophan, were examined. The results are shown in Table 6.

[0110]

[Table 6]

When indole and indoleacrylic acid were added to the reaction solution, a concentration-dependent effect of inhibiting hemoglobin interference was observed as in the case where amino acids were added. From this, the reaction solution contains a substance containing an imidazole ring,
For example, it was found that by adding a substance containing a histidine or indole ring, for example, tryptophan, a core antigen can be detected with high sensitivity even in a sample containing hemoglobin. The effect of combining the various additives described above was studied. The results are shown in Table 7. The combination of histidine and tryptophan restored the recovery by 90% or more, and the combination of urea further increased the detection sensitivity.

[0112]

[Table 7]

Embodiment 10 FIG . Hepatitis B virus (HBV)
Detection of Core Antigen It was examined whether the treatment methods described in the above various examples can be applied to the detection of structural proteins in other viruses. HBV
Monoclonal antibody against the core antigen (Special Immune Laboratory) was adjusted to 3 μg / ml in 0.1 M carbonate buffer (pH
9.6) and add 1 to a 96-well microplate.
The solution was dispensed in an amount of 00 μl. After standing at 4 ° C. overnight, the plate was washed with a phosphate buffer, and 350 μl of a 1% BSA solution was dispensed. After standing at room temperature for 2 hours, the 1% BSA solution was removed by suction, and 200 μl of the reaction solution was added.

Using recombinant HBV core antigen as a standard, hepatitis B was diagnosed, HBe antigen positive and anti-H
Five serum samples from patients who were negative for Be antibody and 10 serum samples from healthy subjects were used as samples. To 100 μl of the sample, add a treatment reagent (7.5% SDS, 0.75% CHAPS, 0.15%
Triton X-100) was added and treated at 56 ° C. for 30 minutes. After the treatment, 50 μl of the mixture was added to a well filled with the reaction solution, and reacted at room temperature for 90 minutes.

As a comparison (without pretreatment), each sample 1
The solution was diluted to 00 μl with 50 μl of purified water, and 50 μl was used for the reaction. After washing 5 times with washing solution, biotin-labeled anti-HBV
Core monoclonal antibodies (HBc-2, HBc-5, H
Bc-14 equivalent) and reacted at room temperature for 30 minutes. After washing five times with the washing solution, avidin-labeled alkaline phosphatase was added, and the mixture was reacted at room temperature for 30 minutes. After washing five times with the washing solution, CDPstar (using Emerald II as a sensitizer) was added, reacted at room temperature for 15 minutes, and the relative luminescence intensity was measured.

A standard curve of the serially diluted recombinant HBV core antigen is shown in FIG. 10, and the amount of the core antigen in the sample measured is shown in Table 8. The detection limit is 21 ng / ml, and the cutoff value for discriminating between core antigen positive and negative is 60 ng / ml.
ml, healthy serum was pre-treated in all 10 cases,
The core antigen was negative in both cases without pretreatment, and was not detected in the serum of a hepatitis B patient without pretreatment. However, by performing the pretreatment, the core antigen was determined to be positive in all cases.

It is considered that the pretreatment of the serum of a patient with hepatitis B caused the destruction of the virus particles and the inactivation of the anti-HBc antibody, which became detectable. As mentioned above,
It has been confirmed that this sample pretreatment is useful not only in detecting structural proteins of viruses such as HBV having DNA as a genome but also of HCV.
Flaviviruses, viruses related to HCV, HI
It goes without saying that the same can be inferred for retroviruses such as V.

[0118]

[Table 8]

Embodiment 11 FIG . Efficient antigen preparation without pretreatment
A method for detecting HCV samples was diluted with a reaction solution to which a surfactant was added, and the detection efficiency of HCV core antigen was examined. The HCV core antigen was detected by a sandwich enzyme immunoassay (EIA) using a monoclonal antibody against the HCV core antigen. Among the monoclonal antibodies obtained in Example 3, C11-3 and C11-7 were used as antibodies to supplement the core antigen, and C11-10 and C11-14 were used.
Was used as an antibody to detect the supplemented core antigen.

The EIA was performed basically under the following conditions.
Solutions prepared by diluting monoclonal antibodies C11-3 and C11-7 to 4 μg / ml in an acetate buffer were added to a microtiter plate, and the mixture was incubated at 4 ° C. overnight. A blocking operation was performed by washing with a phosphate buffer and adding a phosphate buffer containing 1% BSA. There reaction solution 10
After adding 0 μl and 100 μl of the sample, stirring,
Allowed to react for hours. After removing unreacted substances by washing with a phosphate buffer containing a low concentration of a surfactant, the monoclonal antibody C11 labeled with alkaline phosphatase was used.
-10 and C11-14 were added and reacted at room temperature for 30 minutes.

After completion of the reaction, unreacted substances were removed by washing with a phosphate buffer containing a low-concentration surfactant, and a substrate solution (CDP-Star / emeraldll) was added. It was measured. Various surfactants were added to the reaction solution, and the effects were examined. Using an HCV antigen-positive serum in which the titer of the antibody against HCV is lower than the detection sensitivity and which is considered to contain almost no antibody against HCV, the core antigen activity depending on the amount of luminescence was reduced by 1.0 from the luminescence of healthy human serum. And expressed as a reaction ratio to that. The results are shown in Tables 9 and 10.

[0122]

[Table 9]

[0123]

[Table 10]

From these results, as typified by Triton X100, the addition of a nonionic surfactant having an HLB value of between 12 and 14, the HCV antigen-positive serum showed a higher luminescence than the healthy human serum. And the detection sensitivity was found to increase. Similarly, dodecyl-N-
By adding a surfactant having a linear alkyl group having 10 or more carbon atoms and a secondary, tertiary or quaternary amine at the same time, as represented by sodium sarcosinate and dodecyltrimethylammonium, HCV antigen It was also found that the detection sensitivity in positive serum was increased. Carbon number 8
The surfactant having the following alkyl group did not show such an effect of increasing the sensitivity. Further, by adding these two types of surfactants (in Table 8, 2% sodium dodecyl-N-sarcosine and 2% Triton X100 are added), the detection sensitivity in HCV antigen-positive serum is further increased. Also turned out.

Embodiment 12 FIG . Anti-HCV antibody after HCV infection
Detection of core antigen in specimen before appearance (wind period period) Commercial seroconversion panel PHV905 (B.
B. I. inc. ) In the primary reaction solution with 2% Trit
on X100 and 2% of sodium dodecyl N-sarcosinate were added, and the measurement was carried out according to Example 11. The PHV905 panel used here showed positive conversion by anti-HCV antibody test (ortho EIA.3.0) on day 21 (serum No. PHV905-7) after the start of observation, and the antibody titer was cut. Off index (S / CO)
And 1.0 or more is determined to be positive. The HCV core antigen activity (light emission amount) was expressed as a ratio (S / N) to the light emission amount of healthy human serum as 1.0.

As shown in Table 11, the core antigen activity was still observed before the anti-HCV antibody became positive, and the addition of this surfactant revealed the core antigenicity from the virus particles and immobilized on the solid phase. It was confirmed that the antibody was detected and reacted with the monoclonal antibody.

[0127]

[Table 11]

[0128]

[Sequence list] SEQUENCE LISTING <110> Tonen Corporation <120> Method for Detection or Measurement element of Hepatitis C Virus <160> 8 <210> 1 <211> 177 <212> Prt <1> Pit <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> PRT <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <2> Prt <1> Pit <2> PRT <2>) 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 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 Gly Val Arg Val Leu Glu 165 170 175 Asp

<210> 2 <211> 160 <212> TRP <213> Hepatitis Virus <400> 2 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

<210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> <400> 3 Asp Val Lys Phe Pro Gly Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 5 10 15 Leu Pro Arg Arg 20

<210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> <400> 4 Gly Pro Arg Leu Gly Val Arg Ala Thr Arg 5 10

<210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> <400> 5 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

<210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <230> <400> 6 Asp Pro Arg His Arg Ser Arg Asn Val Gly Lys Val Lle Asp Thr Leu 5 10 15 Thr Cys Gly Phe 20

<210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> Probe <230> Synthetic DNA <400> 7 gaattcatgg gcacgaatcc taaa 24

<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> Probe <230> Synthetic DNA <400> 8 ttagtcctcc agaacccggac 21

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of examining the effect of SDS addition concentration in sample processing. Healthy human serum (norma
l) and 13,50 HCV-RNA positive panel sera were used.

FIG. 2 is a diagram showing the results of examining the effect of CHAPS addition concentration in sample processing. Healthy human serum (nor
mal) and HCV-RNA positive panel sera 13,5
0 was used.

FIG. 3 is a diagram showing the results of examining the effect of urea addition concentration in sample processing. Healthy human serum (norma
1) and HCV-RNA positive panel sera 13,44,
50 were used.

FIG. 4 is a diagram showing the results of examining the effect of Triton X100 addition temperature on sample processing. Healthy human serum (normal) and HCV-RNA positive panel serum 13,44,50 were used.

FIG. 5 is a diagram showing the result of examining the effect of temperature during sample processing. Normal human serum (normal) and HC
V-RNA positive panel sera 13,44,50 were used.

FIG. 6 is a diagram showing a dilution calibration curve and a detection sensitivity of a sandwich reaction system using the monoclonal antibody of the present invention, in which a panel serum of a standard serum is diluted stepwise with 50 as 1 U / ml and a sample treated is used. .

FIG. 7 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. A luminescent substance is used as a substrate.

FIG. 8 shows the results obtained by subjecting panel serum 13 to sample treatment, fractionating the fraction using a gel-loca column, and measuring core antigen immunological activity in the fractionation. The molecular weight is IgG
Is about 150 kD and albumin is about 68 kD.

FIG. 9 shows the results obtained by measuring the released core antigen activity of a PCR-positive sample after the sample treatment of the present invention, and amplicore HCV.
It is a figure which shows correlation with HCV-RNA amount calculated | required by the monitor (PCR method).

FIG. 10 shows a standard curve when recombinant hepatitis B core antigen (HBV) was measured by the method of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12N 5/00 B C12P 21/02 15/00 C 21/08 ZNAA (72) Inventor Kumiko Iida 1-31, Nishitsurugaoka, Oi-machi, Iruma-gun, Saitama Prefecture Inside the Tonen Research Laboratory (72) Inventor Tatsuharu Kimura 1-3-1, Nishitsurugaoka, Oi-machi, Iruma-gun, Saitama Tonen Research Center In-house (72) Inventor Shintaro Yagi 1-3-1, Nishi Tsurugaoka, Oi-machi, Iruma-gun, Saitama

Claims (9)

[Claims] In a method for measuring a virus, the presence of a surfactant having an alkyl group having 10 or more carbon atoms and a secondary, tertiary or quaternary amine, or a nonionic surfactant, or both of them. Below, measuring the viral antigen by binding to its probe. 2. A surfactant having an alkyl group and a secondary, tertiary or quaternary amine, wherein the surfactant has 10 to 20 carbon atoms.
The method according to claim 1, wherein the surfactant is a surfactant having three alkyl groups and a tertiary or quaternary amine. 3. The tertiary or quaternary amine surfactant is dodecyl-N-sarcosine acid, cetyl or dodecyltrimethylammonium salt, 3- (dodecyldimethylammonio) -1-propanesulfonic acid, dodecylpyri. The method according to claim 1 or 2, which is a medium salt or decanoyl-N-methylglucamide (MEGA-10). 4. The method according to claim 1, wherein the nonionic surfactant comprises 12 to 1
A surfactant having a hydrophilic-hydrophobic ratio (HLB) of 4;
The method according to any one of claims 18 to 20. 5. The method according to claim 1, wherein the nonionic surfactant is polyoxyethylene isooctyl phenyl ether or polyoxyethylene nonyl phenyl ether.
4. The method according to any one of the above items 3. 6. The probe for a viral antigen,
The method according to any one of claims 1 to 5, which is an antibody against a viral antigen. 7. The method according to claim 7, wherein the virus is genomic RNA or DN.
The method according to any one of claims 1 to 6, which is a virus that forms a virus particle having a structure composed of a structural protein surrounding A and a membrane protein or a lipid membrane surrounding the structural protein. 8. The hepatitis C virus (H)
CV), hepatitis D virus, hepatitis E virus, hepatitis G virus, hand-foot-and-mouth disease virus, flavivirus (yellow fever virus, West Nile virus, Japanese encephalitis virus, dengue virus), togavirus (alpha virus, rubivirus, Arterivirus, rubella virus), pestivirus (porcine cholera virus, bovine diarrhea virus), paramyxovirus (parainfluenza virus 1,2,3,4, canine stempa virus, Newcastle disease virus, respiratory syncytial virus, lindapestovirus, monkey Parainfluenza virus, measles virus, mumps virus), orthoxovirus (human influenza virus, avian influenza virus,
Equine influenza virus, swine influenza virus), rhabdovirus (rabies virus, vesicular stomatitis virus), picornavirus (poliovirus, coxsackievirus, echovirus, bovine enterovirus, swine enterovirus, sarenterovirus, mouse encephalomyelitis virus, human rhino Virus, bovine rhinovirus, equine rhinovirus, foot-and-mouth disease virus, hepatitis A virus), coronavirus (human coronavirus, chicken infectious bronchitis virus, mouse hepatitis virus, swine infectious gastroenteritis virus), arenavirus (lymphocytes) Choroidal meningitis virus, Lhasa virus, Korean hemorrhagic fever virus), retrovirus (HTLV: human adult leukemia virus, HIV: AIDS virus, cat leukemia sarcoma virus) Bovine leukemia virus, Rous sarcoma virus), reovirus (rotavirus), calicivirus (norwalk virus), Bunya virus (nephrotic hemorrhagic fever virus), filovirus (Ebola virus, Marburg virus), hepatitis B virus ( HB
V), poxvirus (vaccinia virus, alastorium virus, cowpox virus, smallpox virus),
Parvovirus (human parvovirus, swine parvovirus, bovine parvovirus, canine parvovirus, feline leukopenia virus, mink aleutian disease virus),
Papova virus (papilloma virus, polyoma virus), adenovirus, herpes virus (herpes simplex virus, cytomegalovirus, varicella-zoster virus, EB virus, equine herpes virus, cat herpes virus, Marek's disease virus) or African swine fever virus 25. The method of claim 24. 9. The method according to claim 1, wherein the virus is HCV or HBV.