JP3510232B2 - Virus detection or measurement method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting or measuring a virus and a reagent therefor.

[0002]

2. Description of the Related Art Currently, various virus detection methods are used for screening for the presence of infectious virus in blood and blood products, and for determining the presence or absence of virus in diseased patients. However, those methods are slightly different depending on various viruses, but not only when they have high sensitivity and high specificity, and even if so,
Many are expensive and require a long time, such as virus isolation culture. Hereinafter, hepatitis C will be mainly described and used as the background art of the present invention.

Although the causative factor of hepatitis C has not been clarified for a long time, its gene has been cloned (Sc
244: 359-362, 1989), the development of a diagnostic method by antibody measurement using a recombinant antigen prepared based on the gene (Scie).
nce 244: 362-364, 1989); Tokuhyo 2-500880), HCV (hepatitis C virus, Hepatitis C Virus), which is a causative factor, and blood and blood-related products which are the main infection routes. It became clear that there is. With the development of the so-called second-generation antibody test method in which recombinant core antigen and recombinant NS3 antigen were added, it became possible to discriminate most HCV-infected persons by serum test. As a result, it has become possible to almost eliminate the infection caused by domestic blood donation.

However, HIV (human immunodeficiency virus)
Like general viral infections such as, there is an unidentifiable period called the so-called wind period until the antibody in the initial stage of infection occurs, and even in regions where domestic blood is allowed to be sold and even in parts of Japan. There is still a risk of secondary infection due to blood-derived components that could not be identified by antibody testing. Further, the antibody test has a problem that it is impossible to determine whether it is a resident who has been cured after the infection or an active infection by its principle.

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

HCV is another virus such as HBV (B
The virus amount in blood is lower than that of hepatitis C virus, etc., and the virus cannot be propagated in vitro or in an animal as a host. It was difficult to establish the method of detection. Therefore, instead of detecting the viral antigen, the PCR (Polymerase Chain Reaction) method (Science 230: 1350-
1354, 1985) and a branched-chain DNA probe method, a method for detecting viral genomic RNA was developed. However, the method of detecting a viral genome has some problems as compared with the method of detecting a viral antigen.

First, since the substance to be detected is RNA, its storage stability is low, and it has been pointed out that problems such as a decrease in quantitative value due to freeze-thawing operation of serum are pointed out. Therefore, it is necessary to pay attention to the preservation of the sample rather than the conventional serum test method.
It is also necessary to pay close attention to the transportation of specimens.

[0008] For example, a test method using the PCR method is the most sensitive detection method for detecting gene fragments, but when extracting HCV genomic RNA from a sample, and reversing from genomic RNA to template DNA. False positives occur frequently when contamination occurs, because skill is required to obtain stable quantitative values easily during copying, and amplification is an important principle. However, it is impossible to process a large amount of samples at one time. Moreover, even if the simple method is used, the pretreatment time is 2
It takes more time 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 it takes about 20 hours to obtain a result (Medical Science and Pharmacy 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 viral antigen has also 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 HCV core antigen has been developed. This report is Tanaka et al. (Journal of
Hepatology 23: 742-745, 19
95) and Fujino et al. (Medicine and Pharmacy 36: 1065-1)
070, 1996), the detection of core antigens present in serum has been shown to have clinical utility similar to the above-mentioned method for detecting viral genome. However, similar to the method for detecting a viral genome, there are still some major problems.

[0010] One point is that the sensitivity is lower than that of the PCR method, so that it cannot be used for the final test of serum screening. Tanaka et al. (Journal of Hep
atology 23: 742-745, 1995).
Is an HCV RNA amount of 10 ⁴ to 10 ⁵ copies /
It is shown that the detection limit is between ml, and Fujino et al. (Medical and Pharmaceutical Sciences 36: 1065-1070, 1996) showed that CRT (conipetive reverse transcription) -PCR, which is the most sensitive detection method. It has been 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, when compared with the highly sensitive CRT-PCR method, it is significantly inferior in terms of sensitivity.

Further, the complicated sample processing step for measurement and the time-consuming are problems when it is intended to be used for screening or the like. That is, for treatment of a sample (serum), polyethylene glycol (PEG) treatment (4 ° C. 1 hour) for concentration of virus particles and removal of serum components, centrifugation operation (15 minutes), removal of supernatant, urea treatment , Multi-step treatment steps such as alkali treatment (37 ° C. for 30 minutes) and addition of neutralizing agent are required. In addition, the dispersion process of urea, which is strongly formed and whose viscosity is increased by PEG, by urea treatment is a task that requires a great deal of skill. Therefore, to obtain reproducibility, skill is required, and a processing time of at least about 2 hours is required. Furthermore, since there are steps such as centrifugation and removal of the supernatant, automation is difficult,
Moreover, simultaneous mass processing is difficult, and it is not suitable for applications requiring large-scale processing such as screening in terms of operation.

On the other hand, the virus antigen detection system is superior to the high-sensitivity PCR method in the following points. That is, it is extremely tolerant to contamination, because an excessive amplification treatment operation is not added in the detection process. RNA
Since it detects an antigen protein, which is a relatively stable substance, rather than detecting an unstable substance as described above, it does not require undue care in the preservation of the sample, It is not necessary to use such special equipment, and the sample can be easily transported.

These features are requirements suitable for use in measuring a large number of samples, such as 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 not suitable for automation, sensitivity is low, and sensitivity such as blood business is required. Due to the fact that it is not available, it cannot be used for so-called screening in which a large number of specimens are handled, and it is not possible to take advantage of its superiority to the PCR method. Further, a clinically highly useful measurement method always has the problems of sensitivity, specificity, reproducibility, operability, and low cost, and it is necessary to develop earnestly to satisfy all of these. Regarding the detection of viral antigens other than HCV, particularly in screening applications for measuring a large number of specimens, the sensitivity is lower than that of the PCR method, and the useful pretreatment method and the exposure of 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 method for detecting viral antigens suitable for treating a large number of specimens for so-called screening applications such as blood services and medical examinations. That is, it has the same sensitivity and specificity as the PCR method and simplifies the pretreatment.
Alternatively, it 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 the first aspect of the present invention, the virus particles are destroyed to sufficiently expose the viral antigen, and when an antibody against the viral antigen is present, the antibody is destroyed. And a means for detecting or measuring a virus by detecting or measuring a viral antigen.
Therefore, the present invention provides a sample containing ( 1 ) virus,
(1) A virus-containing specimen characterized by being treated with a treatment liquid containing any one of an anionic surfactant and (2) an amphoteric surfactant, a nonionic surfactant or a protein denaturing agent. Provide a processing method.

The present invention also provides ( 2 ) a sample containing a virus, (1) an anionic surfactant, (2) a zwitterionic surfactant, and (3) a nonionic surfactant or protein denaturation. There is provided a method for treating a virus-containing specimen, which comprises treating with a treatment liquid containing any of the agents. The present invention also provides ( 3 ) virus-containing specimens with (1) anionic surfactant, (2) zwitterionic surfactant, (3) nonionic surfactant, and (4) protein denaturation. Disclosed is a method for treating a virus-containing specimen, which comprises treating with a treatment liquid containing an agent.

The present invention further includes ( 4 ) the above ( 1 ) to ( 3 ).
There is provided a method for measuring a virus, which comprises detecting or quantifying the presence of a viral antigen by reacting a probe that specifically recognizes a viral antigen with the method for treating a sample according to any one of 1. The present invention further provides a kit for determining the presence or absence of virus in a sample, a kit for quantifying or a diagnostic agent, which comprises an anionic surfactant, for use in the immunoassay method of ( 4 ) above. The present invention further provides a kit for determining the presence / absence of a virus in a sample, a kit for quantifying, or a diagnostic agent, which comprises the monoclonal antibody described below, for use in the immunoassay method of ( 4 ) above.

According to the second aspect of the present invention, there is provided a method for detecting or measuring a viral 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 viral particles to expose the viral antigen, and it is not necessary to destroy the antibody in the blood against the viral 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 of them. A method is provided, characterized in that the viral antigen is measured by binding to its probe.

The present invention further provides HC11-14 (FE) which produces a monoclonal antibody suitable as a probe for the detection of HCV core antigen among the above viral antigens.
RMBP-6006), HC11-10 (FERM B
P-6004), HC11-3 (FERM BP-60)
02), and HC11-7 (FERM BP-600.
A hybridoma cell line selected from the group consisting of 3) is provided. 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
It is a virus that forms a virus particle having a structure composed of a structural protein that wraps genomic RNA or DNA and a membrane protein or lipid membrane that surrounds it. Representative examples of the above viruses having RNA as the genome include hepatitis C virus (HCV) and HCV-related viruses.

HCV-related viruses include hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, hepatitis G virus, hand-foot-and-mouth disease virus, flaviviruses (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, canine system virus) Virus,
Newcastle disease virus, RS virus, Lindapest virus, 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, coxsackie virus, echovirus, bovine enterovirus,
Porcine enterovirus, simian enterovirus, mouse encephalomyelitis virus, human rhinovirus, bovine rhinovirus, horse 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, Lhasa virus, 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 (nowwalk virus), bunyavirus (renal symptomatic hemorrhagic fever virus), filovirus (Ebola) Virus,
Marburg virus) and the like.

Further, as a representative example of the above-mentioned virus having DNA as a genome, hepatitis B virus (HBV),
And HBV-related viruses. Examples of HBV related viruses include poxvirus (vaccinia virus, alastrium virus, cowpox virus, smallpox virus), parvovirus (human parvovirus, porcine parvovirus, bovine parvovirus, dog parvovirus,
Feline leukopenia virus, Mink Aleutian disease virus), papovavirus (papillomavirus, polyomavirus), adenovirus, herpesvirus (herpes simplex virus, cytomegalovirus, varicella zoster virus, EB virus, equine herpesvirus, feline herpes) Virus, Marek's disease virus), African swine fever virus and the like.

In addition to these, various pathogenic viruses are known and unidentified viruses still exist. The present invention can also be released into a state suitable for an immunoassay by the treatment method as long as it is a virus that forms a virus particle having a structure composed of a structural protein to be wrapped and a membrane protein or lipid membrane surrounding it. Is obvious.

Embodiments will be described below centering on the HCV core antigen. HCV has a blood concentration of 10 ² copies / ml to 10
^{Since 6} copies / ml is lower than that of HBV (10 ⁹ copies / ml), extremely high sensitivity is required to detect viral antigens. In general, in a detection method represented by an immunological method 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 that binds to an antigen. , For example, increasing the number of molecules of the antibody, III) a probe that defines the lower limit of detection sensitivity, for example, reducing non-specific reactions caused by the binding of a substance other than the antigen to the antibody, IV) a labeling substance used for detection A method of increasing the detection sensitivity is conceivable, and the sensitivity can be increased by combining these methods.

As a method for increasing the number of antigen molecules, I
-1) It is most easily conceivable to increase the amount of the sample, but in a generally used reaction system (for example, 96-well immunoplate), the maximum additionable volume is about 300 μl and the upper limit is naturally set. Because it is regulated
I-2) A method of increasing the number of molecules added to the reaction system by concentration is used.

II-1) In order to increase the number of probes for detection binding to an antigen, such as an antibody, II-1) increase the number of recognition epitopes by using a plurality of probes such as antibodies, II-2 It is a technique that can be easily conceived of by increasing 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 for improving the affinity between the antigen and the probe, for example, the antibody, a method of changing the composition of the buffer solution in the reaction system, a method of modifying the probe, or a combination thereof can be considered. II-3) It is possible to capture many antigens by widening the reaction area with a limited amount of antigen by binding a large amount of antibody to a large surface area carrier such as beads or magnetic particles. .

In the case of infectious diseases, it is expected that human antibodies having high affinity for binding to an antigen are present in a sample, and the epitope of these antibodies overlaps with a probe used for detection, for example, the epitope of the antibody. Since it is expected that a competitive reaction will occur and the number of antibodies used for detection will decrease, it is necessary to increase the number of antibody molecules for detection that bind to the antigen by removing the antibody that inhibits the reaction in this sample. Connect (II-3). Although it is difficult to generalize methods for reducing nonspecific reactions, III-
1) A non-specific reaction is reduced by increasing the affinity (affinity and avidity) of a probe, for example, an antibody, by changing the composition of a buffer solution. III-2) Removal of substances causing the non-specific reaction Measures such as doing are possible.

As a method for increasing the detection sensitivity of the labeled substance, IV-1) a labeled substance having high detection sensitivity (such as a radioisotope) is used, and IV-2) a signal is obtained by using an enzyme or a catalyst as the labeled substance. Amplification, IV-3) Modification of enzyme substrate into more sensitive substrate, IV-4) Amplification of signal of substrate of enzyme reaction or chemical reaction chemically, electrically or mechanically, IV-5) Methods such as increasing the number of labeled substances per antibody and increasing the sensitivity of the equipment used for detecting the IV-6) signal are conceivable.

When the steps of the pretreatment method of the disclosed HCV core antigen detection method are analyzed, the antigen is concentrated by collecting HCV as a precipitate by centrifugation after adding polyethylene glycol to the sample (I-2). At the same time, after performing a step (II-2) of removing a part of the serum component, the human antibody present in the sample is inactivated by resuspending it in a solution containing urea and an alkaline agent (II- 3) HC
It consists of 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 give a solution for reacting with the monoclonal antibody.

As already pointed out above, the centrifugal operation and the resuspension operation of the precipitate are operationally complicated processes and require skill. Therefore, the goal of the present invention is a core antigen detection system that solves these operational problems. H
Although the appearance of CV itself has not been clarified yet, HCV particles are identified as genome R from the genome structure, the structure of related virus particles, and information on general viruses.
E1, E2 / NS in which NA is packed with the core antigen and anchored to the lipid membrane so as to surround it.
It is presumed to exist 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 outer coat so that the probe used for detecting the core antigen, for example, an antibody can be bound. In addition, it has been reported that virus particles have a complex structure surrounded by LDL (low density lipoprotein) in blood, and since there are antibodies against the coat protein, the virus particles and anti-coating are also present. It is also expected to exist as an immune complex with protein antibodies. That is, in order to increase the number of antigen molecules to be detected, it is important to efficiently remove the envelopes and contaminants surrounding the virus particles and efficiently release the core antigen molecule.
Almost the same can be said for viruses other than HCV, and it is necessary to efficiently release viral antigens.

Therefore, the present invention relates to a treatment method for bringing viral antigens in a sample (serum) into a state suitable for detection using a probe without concentrating by a complicated operation such as centrifugation. Further, as described above, a probe used for detection, such as a human antibody that competes with the antibody for binding, is present in the sample at a high titer, and an operation for removing this is important for increasing the sensitivity. Therefore, one aspect of the present invention relates to a treatment method in which a human antibody present in a specimen is also inactivated at the same time by using a treatment method of easily releasing a viral antigen in the specimen.

By using the treatment method presented by the present invention, the viral antigen present in the sample is released from the viral particles or immune complex in a state suitable for forming an immune complex with the 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 easily and with high sensitivity by an immunoassay method using a probe such as an antibody.

The probe used for detection, such as an antibody, specifically binds to a viral antigen, has a certain high affinity, and does not induce a nonspecific reaction when added to the reaction system. Although it may be present, as shown in Example 4, one of the probes used in the primary reaction preferably contains one capable of recognizing and binding to the C-terminal side of the HCV core antigen. Here, the C-terminal side of the HCV core antigen is
The 81st to 160th sequences 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 sequences shown in SEQ ID NO: 2 or a part thereof.

Here, the probe means a polyclonal antibody obtained by immunizing an experimental animal such as mouse, rabbit, chicken, goat, sheep or cow, and spleen cells are separated from the immunized individual and fused with myeloma cells. The monoclonal antibody produced by the hybridoma, or the monoclonal antibody produced by spleen cells or cells in which blood leukocytes have been immortalized with EB virus, H
Antibodies produced by humans or chimpanzees infected with CV; variable region gene fragments obtained from cDNA or chromosomal DNA of immunoglobulin of mouse, human, etc., or artificial with a part of cDNA or chromosomal DNA of immunoglobulin Variable region gene fragment constituted by combining with the sequence prepared in
A variable region gene fragment constructed using an artificial gene sequence or a recombinant antibody constructed by combining an immunoglobulin constant region gene fragment with a variable region gene fragment produced by a gene recombination technique using these as materials. Recombinant antibodies produced by cells transformed with the gene;

By combining the above variable region gene fragment with, for example, a bacteriophage structural protein, a phage antibody, or by combining the above variable hearing region gene fragment with other applicable gene fragments such as a part of myc gene. Recombinant antibodies produced by cells transformed with the recombinant antibody gene to be constructed, probes produced by artificially introducing variable regions into trypsin molecules, molecules that specifically bind to proteins such as receptors Any molecule can be used as long as it has a high specificity and affinity for the core antigen, such as a probe obtained by artificially modifying it, or a probe produced by other combinatorial chemistry techniques.

Furthermore, the present invention provides that a sample containing a viral antigen is released from a virus particle or an immune complex so as to be in a state suitable for forming an immune complex with the above virus core antigen and a probe such as an antibody. , A step of treating the sample with a treating agent that simultaneously inactivates human antibodies present in the sample that inhibit the detection reaction, and detecting and quantifying the released core antigen by an immunoassay using a probe such as an antibody An assay method and a test kit are provided.

Analyte treatment agents and treatments according to the present invention
Method 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 operations on the sample,
The most important requirement is to treat the core antigen in the sample in a state suitable for 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 the core antigen contained in virus particles and the like.

As already known by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE), most of the proteins are SDS.
It is denatured by heat treatment in the presence, and becomes a monomer except for the molecules bound by covalent bonds. That is, when a treatment agent containing an anionic surfactant such as SDS is added to the measurement sample, it is possible to destroy the virus and simultaneously denature the anti-core antibody in the sample and release the core antigen in the sample. is there. This means that as shown in Example 7, SDS
It was also confirmed that when the HCV core antigen in the sample treated with the treatment agent containing is subjected to molecular weight analysis using gel filtration, it is detected at the position of the monomer theoretically expected.

In addition, Kashiwa Kuma et al. (J. Immunological
al Methods 190 79-89, 199.
As reported by 6), when a sample consisting of a recombinant HCV-expressing cell extract was separated by SDS-PAGE and HCV core antigen was detected by Western blotting, the molecular weight was considered to be a monomer. Its immune activity is detected. It is easily conceivable for those belonging to this field that the antigen can be efficiently released and the number of antigen molecules can be increased by adding a denaturant containing SDS to the sample.

However, as is well known, SD
Since anionic surfactants such as S have a large protein denaturing action, if they are directly added to the reaction of forming an immune complex with a probe such as an antibody in the presence thereof, the antibody will also be denatured, its function will be lost and sensitivity will be reduced. Guide Further, it is known that the epitope structure is lost by treatment with an anionic surfactant such as SDS, and as a result, the binding of the antibody is weakened and the sensitivity is lowered. In order to eliminate these effects that lead to a decrease in sensitivity, it is necessary to weaken the denaturing effect by some method after SDS treatment.

Surfactants, including anionic surfactants, are:
For example, it is known that it can be removed by dialysis, ultrafiltration, gel filtration, electrophoresis, ion exchange, precipitation, membrane transfer, etc. That an antigen can be detected
It is shown that the antigen-antibody reaction can be carried out by adding 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 excessive amount of the reaction solution, it is possible to prevent the reaction from being affected by reducing the concentration to a level at which the denaturing action does not occur. However, in this method, the reaction solution increases and, for example, a microtiter well It is obviously not suitable for the purpose of the present invention because it cannot be applied to an immunoassay method that has a limitation on the amount of sample to be added such as a measurement method using. Therefore, the present inventor added a treatment agent containing an anionic surfactant, and further added some kind of additive to prevent the denaturing effect of the anionic surfactant from affecting the probe such as an antibody. It was investigated whether it could not be weakened, and at the same time, the action of releasing the HCV core antigen by the anionic surfactant could not be enhanced.

Here, the present inventor weakens 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, and as a result, SDS It was found that the sensitivity can be increased as compared with the treatment agent containing only. Also, S
Similar effects are observed when a treatment agent containing an anionic surfactant such as DS is also added to the treatment agent containing other surfactants or agents that weaken hydrogen ion bonds such as urea. It was found that the release of the HCV core antigen is further enhanced by enhancing the release of the HCV core antigen from the virus particles and the inactivation of the anti-core antigen antibody in the sample. Further, it was found that the HCV core antigen can be detected with higher sensitivity by performing a heat treatment step after adding a treatment agent containing SDS and other surfactants, and completed the present invention.

Here, the anionic surfactant used for the treatment of the sample is not limited to SDS, but sodium cetyl sulfate, other alkylated sulfates, alkyl sulfonates such as sodium dodecyl sulfonate, alkyl allyl sulfonates, etc. It is also possible to use a salt or the like, and as a surfactant to be added in addition to the anionic surfactant, CHAPS (3-
[3-Colamidopropyl) dimethylammonio] -1-
Propanesulfonic acid), CHAPSO (3- [colamidopropyl) dimethylammonio] -2-hydroxy-1
-Propanesulfonic acid), zwitterionic surfactants such as dodecyl-N-betaine, polyoxyethylene isooctyl phenyl ethers such as Triton X100,
Polyoxyethylene nonyl phenyl ethers such as NP40, polyoxyethylene sorbitol esters such as Tween 80, polyoxyethylene dodecyl ethers such as Brij58, and nonionic surfactants such as octyl glucoside are suitable, and preferably C
Zwitterionic surfactants such as HAPS and Triton-
Includes nonionic surfactants such as X100. It is also effective to add a drug (protein denaturing agent) such as urea or thiourea having a function of destroying the higher-order structure of the protein.

The concentration during the treatment was 0.5% or more for SDS, 0.1% or more for CHAPS, 1 M or more for urea, and T
It is more preferable to use the RITON X100 in an amount of 0.1% or more and 0.75% or less. The sample processing temperature as described above is 4 ° C, which is a range usually used in general laboratories.
The temperature may be 100 ° C or lower, but in the case of adding a nonionic surfactant, attention must be paid to the cloud point. The temperature is preferably 37 ° C. or higher, and 50 to 60 ° C. treatment generally used for inactivating serum is effective.

By using the processing method of the present invention, H
From a sample containing virus particles with a structure similar to CV,
It is clear that the viral antigen can be released in a state suitable for a so-called immunoassay method using an antibody or the like as a probe. Here, a 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, a membrane protein that surrounds it, and a lipid membrane. The related viruses include flaviviruses, retroviruses such as human immunodeficiency virus (HIV), and the like. Furthermore, DNA as the genome
Those having the same structure are included.

Furthermore, HCV which is an RNA virus,
Both HBV, which is a DNA virus, have these genomic R
It is a virus that forms viral particles with a structure consisting of structural proteins that wrap NA and DNA, and membrane proteins and lipid membranes that surround them. In any of the embodiments, by using the treatment method of the present invention, not only HCV and HBV but also virus particles having a structure similar to these are destroyed to fully expose the antigen of the virus, It also provides for detecting or measuring the virus by detecting or measuring.

Removal of interference due to hemoglobin When serum or the like is used as a measurement sample, red blood cells contained in the sample are hemolyzed during the pretreatment to release hemoglobin, and the denatured hemoglobin interferes with the measurement. There are cases. Therefore, in the first aspect of the present invention, it is preferable to eliminate such measurement interference. 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, imidazolidine thione, imidazolidone, histamine and the like, and imidazole. To be

As the indole ring-containing compound,
Tryptophan, indole acrylic acid, indole,
Indole acetic acid, indole acetic acid hydrazide, indole acetic acid methyl ester, indole butyric acid, indole acetonitrile, indole carbinol, indole carboxaldehyde, indole carboxylic acid, indole ethanol, indole lactic acid, indole methanol, indole propionic acid, indole pyruvic acid,
Examples include indolyl methyl ketone, indomycin, indoleacetone, indomethacin, indoprofen, indolamine and the like.

A suitable concentration of urea is 0.5 M to 5 M, and indole acrylic acid is 5 mM to 50 mM.
Is appropriate, other additives are 0.05M ~
A concentration of 0.5M is suitable.

Exposure of Viral Antigens According to the second aspect of the present invention, there is provided a method for detecting viral antigens in a sample collected in a wind period, in which an antibody against the viral antigens has not yet been produced. It is sufficient to destroy the particles to expose the viral antigens, not the antibodies present in the sample. Therefore, the pretreatment of the sample described above is not necessary, and it suffices if the virus particle disrupting agent for exposing the virus antigen is present in the measurement reaction solution. The viral antigen described here means an antigen existing inside a virus particle, and the core antigen is a representative thereof.

It is considered that the virus particle has a structure in which a nucleic acid which is 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. Furthermore, it is considered to exist in blood by forming a complex with low-density lipoprotein (LDL) and an antibody against a virus. Therefore, the probe cannot recognize and bind to the virus antigen represented by the core antigen existing inside the virus particle when the virus particle existing in the blood remains as it is. Therefore, in order to detect the core antigen, it is necessary to remove the structures surrounding the core antigen so that the probe recognizes the core antigen.

That is, in the present invention, the reaction conditions for exposing the core antigen in the virus particles contained in the sample so that the probe for recognizing the core antigen can recognize it, the reaction method comprising the reaction system, and the reaction Also provided are reagents that include the system. The reaction system suitable for antigen detection in the system provided by the present invention is a virus particle that is a complex structure existing in a specimen, under mild conditions that do not lose the function of the antibody against the viral antigen epitope. Therefore, the system consists of conditions under which the region recognized by the antibody, which is a probe that recognizes the viral antigen, is sufficiently exposed.

Virus particles already separated by the ultracentrifugation method (Takahashi et al., 1992, J. Am.
Gen. Virol, 73: 667-672)), by treating the HCV particles aggregated and precipitated with polyethylene glycol with a nonionic surfactant such as Tween 80 or Triton X100 (Kas.
hiwakuma et al. , 1996, J .; Im
Munological methods: 190: 79
-89), it has been shown that the core antigen can be detected, but the detection sensitivity is insufficient in the former, 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 itself of the surfactant is not mentioned.

In the present invention, first, the conditions are examined based on the surfactant, and the reaction solution is made to have a composition centered on the surfactant, so that the HCV antigen detection system already reported can be 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 the core antigen from the virus particles, suppress the mutual reaction with various substances in serum, and provide conditions under which the probe and the antigen can efficiently react. As an 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. Can be mentioned.

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

The above-mentioned nonionic surfactant is 12
Those having a hydrophilic / hydrophobic ratio between 14 and 14 are preferable, and polyoxyethylene isooctyl phenyl ethers such as Triton X100, Triton X114 and the like, or polyoxyethylene nonifanyl ethers such as Nonidet P40, Triton N1.
01, Nikkol NP and the like are preferable. In the present invention, the above-mentioned 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. Furthermore, 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 means HC
It is a gene fragment containing the core region of the structural protein gene of V, and is a DNA fragment having a base sequence encoding a polypeptide containing at least the 1st to 160th amino acid sequences at the N-terminus of HCV. Specifically, SEQ ID NO: 2
Is a gene fragment containing a nucleotide sequence encoding the amino acid sequence of.

The polypeptide having HCV antigen activity as used in the present invention means a fusion polypeptide or a polypeptide which immunologically reacts with an anti-HCV antibody, and is used for producing the hybridoma of the present invention and a monoclonal antibody obtained therefrom. It can be used as an antigen to utilize. Specifically, it is a fusion polypeptide having an HCV antigen activity containing the amino acid sequence of SEQ ID NO: 1 or a polypeptide having an HCV antigen activity containing a part of the amino acid sequence of SEQ ID NO: 1 at the N-terminal or C-terminal thereof. It may have an extra amino acid sequence added.

Monoclonal antibodies against the fusion polypeptide of the present invention and the polypeptides containing the amino acid sequences 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,
Intraperitoneally or intradermally in BALB / c mice or the like, the fusion polypeptide or the polypeptide (hereinafter referred to as the present antigen) alone or as an antigen in which BSA, KLH, etc. are combined, is mixed with an adjuvant such as a simple or Freund's complete adjuvant. And immunize regularly. When the antibody titer in the blood rises, this antigen is administered as a booster immunity into the tail vein, and the spleen is aseptically removed, followed by cell fusion with an appropriate mouse myeloma cell line to obtain a hybridoma. This method is based on the method of Koehler and Milstein (N
Nature 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 producing an antibody showing a specific reaction to the present antigen is selected and cloned. In addition to the limiting dilution method, soft agar method (Eu
r. J. Immunol. 6: 511-519, 197.
6) etc. can be used. Then, the produced monoclonal antibody is purified by a method such as column chromatography using protein A or the like. In addition to the above monoclonal antibodies, molecules to be used as probes can be prepared. For example, recombinant antibodies are described in detail in the review article by Hoogenboon (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, agglutination for detection and quantification of viral structural proteins. Assay reagents, or other well known immunoassay methods, can be used as test reagents. When a labeled antibody is used for detection, examples of labeling compounds include fluorescent substances, chemiluminescent substances, radioactive substances,
Enzymes, dyes, etc. are used.

For example, when a method based on the sandwich reaction system is used to detect a viral antigen in a sample (serum), the diagnostic kit to be used is coated on a solid support (for example, the inner wall of a microtiter well). One or more monoclonal antibodies of the present invention and one or more monoclonal antibodies or fragments thereof bound to a labeling substance. The combination of the monoclonal antibody immobilized on the solid support and the labeled monoclonal antibody is arbitrary, and a combination with high sensitivity can be selected.

Examples of solid supports that can be used include polystyrene, polycarbonate, polypropylene, polyvinyl microtiter plates, test tubes, capillaries, beads (latex particles, red blood cells, metal compounds, etc.), membranes (liposomes, etc.), filters, etc. Can be mentioned.

[0068]

EFFECTS OF THE INVENTION According to the method of the present invention, it becomes possible to easily release a viral antigen from virus particles in a state suitable for a so-called immunoassay method in which an antibody or the like is used as a probe for detection. Further, by treating a sample containing virus particles by the method shown by the present invention, a so-called immunoassay method of detecting an antigen using an antibody or the like as a probe can easily and sensitively detect and quantify a viral antigen. Becomes Further, it becomes possible to prepare a kit for discriminating the presence or absence of a virus in a sample, a kit for quantifying, and a diagnostic agent using the immunoassay method 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. Example 1. 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 Patent Laid-Open No. 6-38765) was designated as pUC11.
The plasmid pUC.C11-C obtained by incorporating it into
21 and pUC.C10-E12 DNA (1 μg) was added to a restriction enzyme reaction solution (20 μl) [50 mM Tris-HCl.
(PH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 100 mM NaCl, 15 units 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] respectively
After digestion for 1 hour at 7 ° C., 0.8% agarose gel electrophoresis was performed to purify the EcoRI-ClaI fragment of about 380 bp and the ClaI-KpnI fragment of about 920 bp.

The two DNA fragments and pUC119 were cloned into E
The vector digested with coRI and KpnI was added to the buffer for ligase [660 mM Tris-HCl (pH 7.
5), 66 mM MgCl ₂ , 100 mM dithiothreitol, 1 mM ATP] 5 μl, T4 ligase 1 μl (3
Water was added to 50 units / μl) to make 50 μl, and the mixture was incubated at 16 ° C. overnight to carry out a ligation reaction. Escherichia coli JM109 was transformed with this plasmid to obtain plasmid pUC.
-C21-E12 was obtained.

This plasmid pUC.C21-E12
1 ng of DNA is combined with 2 primers (5'-GAATTCA
TGGGCACGAATCCTAAA-3 '(SEQ ID NO: 7), 5'-TTAGTCCTCCAGAACCC
PCR was performed using GGAC-3 '(SEQ ID NO: 8)). PCR is GeneAmpTM (DNA Amplification Reagent
Kit, manufactured by Perkin Elmer Cetus) under the conditions of denaturing DNA 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 is subjected to 0.8% agarose gel electrophoresis. It separated by electrophoresis and refine | 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 treated with 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
Water was added to 50 units / μl) to make 50 μl, and the mixture was incubated at 16 ° C. overnight to carry out a ligation reaction. Escherichia coli JM109 was transformed with this plasmid to obtain plasmid pUC.
l9.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.
Digestion reaction was carried out for 1 hour, and then 0.8% agarose gel electrophoresis was carried out to obtain about 490 bp EcoRI-Bam.
The HI fragment was isolated and purified by the glass powder method.

Next, the expression vectors Trp and TrpE
1 μg of DNA of 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.
Digest for 1 hour, add 39 μl of water to the reaction mixture, and add 70
After heat treatment at 5 ° C for 5 minutes, 1 µl of bacterial alkaline phosphatase (BAP) (250 units / µl) was added and the mixture was kept at 37 ° C for 1 hour.

Phenol was added to this reaction solution for phenol extraction, and the obtained aqueous layer was precipitated with ethanol and the precipitate was dried. 1 μg of the obtained EcoRI-BamHI-treated vector DNA and the above-mentioned core 140 fragment were treated 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
Water was added to 50 units / μl) to make 50 μl, and the mixture was incubated at 16 ° C. overnight to carry out a ligation reaction.

E. coli HB was prepared by using 10 μl of this reaction solution.
The 101 strain was transformed. The susceptible E. coli strains used for transformation were the calcium chloride method [Mandel, M. and Higa, A., J. M.
ol. Biol., 53, 159-162 (1970)]. Transformed E. coli with LB containing 25 μg / ml ampicillin
Plate (1% tryptone, 0.5% NaCl, 1.5
% Agar) and kept at 37 ° C. overnight. Take 1 platinum loop of the bacterial colony on the plate, 25 μg / ml
Was transferred to LB medium containing ampicillin and cultured overnight at 37 ° C. 1.5 ml of the bacterial culture was centrifuged to collect the cells, and the plasmid DNA was miniprepared by the alkaline method [Mann
iatis et al., Molecular Cloning: A Laboratory Manual,
(1982)].

1 μg of the obtained plasmid DNA was added to 20 μl of a restriction enzyme reaction solution [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].
It was digested at 7 ° C for 1 hour and subjected to agarose gel electrophoresis to select a Trp / TrpE core 160 expression plasmid which produces an EooRI-BamHI fragment of about 490 bp.

(B) Expression and Purification of Polypeptide Encoded by Clone Core 160 Escherichia coli HB101 strain having the expression plasmid Trp / TrpE core 160 contained 50 μg / ml of ampicillin 3.
2 ml of 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) is inoculated and cultured at 37 ° C. for 9 hours. 1 ml of this culture solution 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) was subcultured and cultured at 37 ° C. When OD600 = 0.3, indole acrylic 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 bacterial cells.

20 ml of buffer A [50 mM Tri was added to the cells.
s-HCl (pH 8.0), 1 mM EDTA, 30 mM N
aCl] was added to suspend the cells, and the cells were centrifuged again to obtain 2.6 g of the expressing bacterial cells. 10 ml of the buffer solution A obtained
The cells were suspended in a sonicator, and the Escherichia coli membrane was disrupted by ultrasonic disruption, followed by centrifugation 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.

Example 2. Method for producing hybridoma Fusion polypeptide (TrpC1 prepared by the above method
1) Dissolved 6M urea and then added 0.15M NaCl 1
The final concentration of 0.2 mM to 1 mM was added to 0 mM phosphate buffer (pH 7.3).
It was diluted to 0 mg / ml and mixed with an equal amount of adjuvant (Titermax) to give a TrpC11 suspension. The suspension prepared to have a TrpC11 concentration of 0.1 to 0.5 mg / ml was intraperitoneally administered to 4 to 6 week old BALB / c mice. Similar immunization was performed every 2 weeks, and after about 2 weeks, TrpC11 dissolved in physiological saline was added.
10 μg was administered into the tail vein.

On day 3 after the final booster immunization, the spleen was aseptically removed from the immunized animal, and the spleen was dissected into individual cells using scissors as a section and a mesh, and RPMI-
It was washed 3 times with 1640 medium. After the mouse myeloma cell line SP2 / 0Ag14 in the logarithmic growth phase was washed 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 xg for 5 minutes, the supernatant was removed, and 1 ml of RPMI-1640 medium containing 50% polyethylene glycol (PEG) 4000 (manufactured by Merck & Co., Inc.) kept 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 by culturing in PMI-1640 medium for about 10 days. Then, a clone producing the desired antibody was searched by the ELISA method to obtain a hybridoma producing the monoclonal antibody of the present invention having a desired reaction specificity.

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 respectively transferred to the Institute of Industrial Science and Technology, Institute of Industrial Science and Technology on July 4, 1997 by FERM BP.
-6006, FERM BP-6004, FERM B
Deposited as P-6002 and FERM BP-6003.

Example 3. Method for producing monoclonal antibody The hybridoma obtained by the method described in Example 2 was transplanted into the abdominal cavity of a mouse treated with pristane or the like to obtain a monoclonal antibody produced in ascites. For purification of the monoclonal antibody, an IgG fraction was separated by a Sepharose column to which protein A was bound.

Monoclonal antibodies C11-14 and C1 produced from the above 5 hybridomas, respectively.
The isotypes of 1-10, C11-7 and C11-3 are the rabbit anti-mouse Ig isotype antibodies (Zyme).
C11-1 by an immunoassay using a
0, C11-7 is IgG2a, C11-14, C11-
It was revealed that 3 was IgG1. Obtained 4
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 HCV / core region, Table 1
It was found to be a monoclonal antibody that specifically recognizes a part of the core region as shown in.

[0086]

[Table 1]

Example 4. Examination of sample processing conditions 1) Examination of SDS concentration 100 μl of serum of healthy subjects and HCV-RNA positive serum
And SDS dissolved in various concentrations and 0.6% CHAPS
100 μl of the treatment liquid containing the above was added. Put in a heat insulation box set to 56 ° C and perform treatment for 30 minutes.
0 μl was used as a measurement sample. The results of the measurement methods described below are shown in FIG. 1 with the horizontal axis representing the SDS concentration during the treatment reaction.

2) Examination of CHAPS concentration 100 μl of serum from healthy subjects and HCV-RNA positive serum
To this, 100 μl of a treatment solution containing CHAPS and 5% SDS dissolved in various concentrations was added. Put it in a heat insulation box set at 56 ° C and perform treatment for 30 minutes.
1 was used as a measurement sample. The results of the measurement method described below are
The CHAPS concentration during the treatment reaction is plotted on the horizontal axis and is shown in FIG.

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

4) Examination of Triton X100 Concentration 100 μl of normal human serum and HCV-RNA positive serum
To this, 100 μl of a treatment solution (5% SDS, 0.6% CHAPS, 6M urea) containing Triton X100 dissolved in various concentrations was added. The sample was placed in a heat insulation box set at 56 ° C. and treated for 30 minutes, 80 μl of which was used as a measurement sample. The results of the measurement methods described below are shown in FIG. 4, in which the horizontal axis represents the Triton X100 concentration during the treatment reaction.

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

Measurement Method The samples obtained in the examination of the serum treatment method were evaluated using the following measurement methods. That is, anti-HCV core antigen monoclonal antibody (equal amounts of antibodies C11-3 and C11-7 mixed)
Was diluted with 0.1 M carbonate buffer (pH 9.6) to a final concentration of 6 μg / ml, and 100 μl was dispensed per well of a 96-well microplate (Nunc). After standing overnight at 4 ° C, containing 0.15M NaCl 1
Washing twice with 0.35 ml of 0 mM sodium phosphate buffer (pH 7.3) containing 0.5% casein-Na 10
0.35 ml of mM sodium phosphate buffer (pH 7.35) (hereinafter, 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% Tween20 and the measurement sample obtained by each serum treatment method were added to each well, reacted for 2 hours at room temperature, and washed 5 times with 300 μl of washing solution, Furthermore, peroxidase (POD) labeled monoclonal antibody (C
100 μl of an equal mixture of 11-10 and C11-14) was added and reacted at room temperature for 30 minutes. After the reaction, the substrate was washed 5 times with 300 μl of the above washing solution, 100 μl of a substrate (orthophenylenediamine, hereinafter OPD) solution was added and reacted at room temperature for 30 minutes, then 100 μl of a 2N sulfuric acid solution was added, and the absorbance at a wavelength of 630 nm was used as a control. Absorbance at 492 nm (OD492) was measured.

From FIGS. 1 to 4, optimization of each treatment condition was performed, but it was difficult to detect the core antigen in the untreated specimen. It became possible to detect the core antigen. In particular, the SDS concentration during the treatment reaction was 0.5% or more, and the CHAPS concentration was 0.
Above 1%, urea concentration above 1M, TritonX1
00 concentration is 0.1 to 0.75%, 4 ℃
It was shown that the core antigen can be satisfactorily detected in the range of 70 to 70 ° C.

Example 5. Structural region core antigen detection and
Assay method (1) 100 μl of serum was treated with treatment solution (5% SDS, 0.6% CH
APS, 6M urea, 0.75% TritonX100)
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 thereof was used as a measurement sample. Anti-HCV core antigen monoclonal antibody (C
11-3 and C11-7 equivalent amount) to give a final concentration of 6 μg /
The mixture was diluted with 0.1 M carbonate buffer (pH 9.6) to a final volume of 100 ml, and 100 μl was dispensed per well of a 96-well microplate (Nunc). After standing overnight at 4 ° C,
It was washed twice with 0.35 ml of 10 nM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl, 0.35 ml of 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-mentioned treatment method were added to each well and reacted at room temperature for 2 hours. Cleaning solution 300μ
1 times, and then washed with peroxidase (POD) -labeled monoclonal antibody (C11-10 and C11-1).
(4: Equal volume mixture) 100 μl was added and reacted at room temperature for 30 minutes. The substrate (OP
D) 100 μl of the solution was added and reacted at room temperature for 45 minutes, then 100 μl of a 2N sulfuric acid solution was added, and the absorbance at the wavelength of 492 nm (OD) was used with the absorbance at the wavelength of 630 nm as a control.
492) was measured. In addition, as a standard serum, 1 U / ml of panel serum 50 was serially diluted with 10 mM sodium phosphate buffer (pH 7.3) containing 1% BSA, and the same treatment was performed and measured.

FIG. 6 shows the dilution line of the panel serum 50 used as the 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 treatment method of the present invention and a monoclonal antibody,
It was revealed that the HCV core antigen can be detected or quantified.

Example 6. Detection of HCV structural region core antigen
And quantification (2) Method using alkaline phosphatase-labeled monoclonal antibody 96-well black microplate (Nunc) as a solid phase carrier, alkaline phosphatase-labeled monoclonal antibody as a labeled antibody, and CDPst as a substrate
ar (Emerald II as sensitizer) was used. The dilution straight line of the panel serum 50 used as the standard serum is shown in FIG. 7, and the core antigen in the sample was measured in a concentration-dependent manner, and it was possible to detect about 0.5 mU / ml. It has been clarified that the HCV core antigen can be detected or quantified by using the assay method using this alkaline phosphatase-labeled monoclonal antibody.

Example 7. Recognized in serum processing and assay
Analysis of molecular form 0.25 ml of panel serum 13 was treated by each serum treatment method, and gelloca column (Superdex200HR, 1
x30), and the anti-core immunoreactivity in these fractions was measured, and the results are shown in FIG. Molecular weight about 2
It is considered to recognize a molecule of 0 to 30 kDa, and it is shown that the core antigen in the virus is released by the above-mentioned pretreatment due to virus destruction and inactivation of anti-core antibody present in serum. It was

Example 8. HCV structural region in serum samples
(A) Assay method of antigen Using an Amplicor HCV monitor kit (Roche) which is a PCR method, the amount of HCV-RNA is 10 ³ to 10
^Using the serum measured at ⁷ copies / ml and the serum of a healthy person, the HCV core antigen in the serum was quantified by the method described above. As standard serum, panel 50 serum (1 U / ml)
Table 2 shows the measurement results, which was used in the same manner as above, but was diluted stepwise with a 10 mM sodium phosphate buffer (pH 7.3) containing 1% BSA and treated in the same manner. Of the samples measured this time, all healthy subjects are below the detection limit,
All of the PCR method-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 high correlation.

[0101]

[Table 2]

Example 9. Suppresses sensitivity deterioration due to hemolyzed serum
Examination of additives for the purpose of studying the effect of serum components on the sensitivity, it was confirmed that the addition of hemoglobin significantly reduced the sensitivity. Pretreatment with a pretreatment agent containing SDS, CHAPS, or Triton X100 denatured hemoglobin, and it was possible that heme was released. Therefore, an additive that can reduce the influence of modified hemoglobin was examined.

HCV core antigen positive serum (panel serum No.
To 3), a high-concentration hemoglobin (manufactured by International Reagents: interference check) was added to prepare a model sample, urea was added to the above-mentioned pretreatment agent, and core antigen measurement was performed according to Example 6 to obtain urea. The effect of addition of was investigated. Table 3 shows the core antigen activity amount of the 430 mg / dl hemoglobin-added group when the core antigen activity amount of the control non-hemoglobin-added group was 100%. The amount of core antigen activity in the hemoglobin-added group when urea was not added decreased 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 of amino groups and carboxyl groups were also considered. Therefore, various amino acids were added and their effects were investigated. The results are shown in Table 4.

[0106]

[Table 4]

The ones most recognized for the effect of suppressing interference were tryptophan and histidine. The results of examining the concentration dependence of these interference suppression effects are shown in Table 5.

[0108]

[Table 5]

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

[0110]

[Table 6]

When indole and indole acrylic acid were added to the reaction solution, the effect of suppressing hemoglobin interference in a concentration-dependent manner was observed as in the case of adding amino acids. From this, a substance containing an imidazole ring in the reaction solution,
It was found that the addition of a substance containing a histidine or indole ring, for example, tryptophan, enables the core antigen to be detected with high sensitivity even in a specimen containing hemoglobin. The effect of combining the above various additives was examined. The results are shown in Table 7. The combination of histidine and tryptophan recovered 90% or more, and the combination of urea further increased the detection sensitivity.

[0112]

[Table 7]

Example 10. Hepatitis B virus (HBV)
Detection of Core Antigens It was examined whether the treatment methods described in the various examples above can be applied to the detection of structural proteins in other viruses. HBV
Monoclonal antibody against core antigen (Special Immune Research Laboratories) was adjusted to 3 μg / ml with 0.1 M carbonate buffer (pH).
9.6) and add 1 to 96-well microplate.
Dispense by 00 μl each. After allowing to stand overnight at 4 ° C., the plate was washed with a phosphate buffer solution, and 350 μl each of 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 was positive, and anti-H
Five patient sera that were negative for Be antibody and 10 healthy human sera were used as samples. A processing reagent (7.5% SDS, 0.75% CHAPS, 0.15%) was added to 100 μl of the sample.
Triton X-100) (50 μl) was added, and the mixture was treated at 56 ° C. for 30 minutes. After the treatment, 50 μl thereof was added to the well filled with the reaction solution, and the mixture was reacted at room temperature for 90 minutes.

As a comparison (without pretreatment), each sample 1
Diluted to 00 μl with 50 μl of purified water and used 50 μl for the reaction. After washing 5 times with the washing solution, biotin-labeled anti-HBV
Core monoclonal antibody (HBc-2, HBc-5, H
(Bc-14 equivalent mixture) was added and reacted at room temperature for 30 minutes. After washing 5 times with a washing solution, avidin-labeled alkaline phosphatase was added, and the reaction was carried out at room temperature for 30 minutes. After washing 5 times with a washing solution, CDPstar (using emerald II as a sensitizer) was added and 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 measured amount of core antigen in the sample is shown in Table 8. The detection limit is 21 ng / ml, and the cutoff value for distinguishing between positive and negative core antigen is 60 ng / ml.
As a result, the serum of healthy person was pretreated in all 10 cases.
The core antigen was negative in both cases without pretreatment, and was not detected in hepatitis B patient serum without pretreatment, but the core antigen was determined to be positive in all cases by pretreatment.

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

[0118]

[Table 8]

Example 11. Efficient without pretreatment of antigen
Method for quantitatively detecting HCV-containing specimens was diluted with a reaction solution containing a surfactant, and the detection efficiency of HCV core antigen was examined. The detection of HCV core antigen was performed by sandwich enzyme immunoassay (EIA) using a monoclonal antibody against HCV core antigen. Among the monoclonal antibodies obtained in Example 3, C11-3 and C11-7 were used as antibodies that complement the core antigen, and C11-10 and C11-14 were used.
Was used as an antibody to detect the captured core antigen.

The EIA was basically performed under the following conditions.
A solution obtained by diluting each of the monoclonal antibodies C11-3 and C11-7 in an acetate buffer at a concentration of 4 μg / ml was added to a microtiter plate and kept at 4 ° C. overnight. A blocking operation was performed by washing with a phosphate buffer and adding a phosphate buffer containing 1% BSA. Reaction liquid 10 there
Add 0 μl and 100 μl of the sample, stir, and then 1.5 at room temperature.
Reacted for hours. Monoclonal antibody C11 labeled with alkaline phosphatase after removing unreacted substances by washing with a phosphate buffer containing a low concentration of surfactant
-10 and C11-14 were added, and the mixture was reacted at room temperature for 30 minutes.

After completion of the reaction, the unreacted material was removed by washing with a phosphate buffer containing a low concentration of a surfactant, and a substrate solution (CDP-Star / emeraldll) was added, followed by reaction at room temperature for 20 minutes. It was measured. Various surfactants were added to the reaction solution and the effect was examined. The HCV antigen-positive serum, which is considered to contain almost no antibody against HCV and whose titer of the antibody against HCV is less than the detection sensitivity, was used to determine the core antigen activity depending on the amount of luminescence and the luminescence of healthy human serum to 1.0 And expressed as the reaction ratio. 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 12 to 14 caused the HCV antigen-positive serum to emit more light than the healthy human serum. Was found to increase, and the detection sensitivity increased. Similarly, dodecyl-N-
As represented by sodium sarcosinate and dodecyltrimethylammonium, the addition of a straight chain alkyl group having 10 or more carbon atoms and a surfactant having a secondary, tertiary or quaternary amine in its structure at the same time causes the HCV antigen It was also found that the detection sensitivity in positive serum increased. Carbon number 8
The surfactants having the following alkyl groups did not show such a sensitivity increasing effect. Further, by adding these two kinds of surfactants (mixing 2% sodium dodecyl-N-sarcosinate and 2% Triton X100 in Table 8), the detection sensitivity in HCV antigen-positive serum is further increased. Was also found.

Example 12. 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. ) Is added to 2% Trit in the primary reaction solution.
on X100 and 2% sodium dodecyl N-sarcosinate were added, and the measurement was carried out according to Example 11. The PHV905 panel used here showed positive conversion in the anti-HCV antibody test (Ortho EIA.3.0) on the 21st day (serum No.PHV905-7) after the start of observation, and the antibody titer was cut. Off index (S / CO)
It is represented by, and 1.0 or more is determined to be positive. The HCV core antigen activity (amount of luminescence) was expressed as a ratio (S / N) with respect to the amount of luminescence 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 solidified them. It was confirmed that it could be detected by reacting with the monoclonal antibody.

[0127]

[Table 11]

[0128]

[Sequence list] SEQUENCE LISTING <110> Tonen Corporation <120> Method for Detection or Measure element of Hepatitis C Virus <160> 8 <210> 1 <211> 177 <212> PRT <213> Hepatitis Virus <400> 1 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 Gly Val Arg Val Leu Glu                 165 170 175 Asp

[0129] <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

[0130] <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> <400> 3 Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu                   5 10 15 Leu Pro Arg Arg              20

[0131] <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

[0132] <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

[0133] <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

[0134] <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> Probe <230> Synthetic DNA <400> 7 gaattcatgg gcacgaatcc taaa 24

[0135] <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> Probe <230> Synthetic DNA <400> 8 ttagtcctcc agaacccgga c 21

[Brief description of drawings]

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

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

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

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

FIG. 5 is a diagram showing the results of examining the effect of temperature during sample processing. Healthy human serum (normal) and HC
V-RNA positive panel serum 13,44,50 was 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 a sample obtained by serially diluting a panel serum of standard serum with 50 as 1 U / ml and subjecting the sample to sample processing. .

FIG. 7 is a diagram showing dilution calibration curves and detection sensitivities when a sample treated with a sample serum, which was serially diluted with panel serum 50 of standard serum at 1 U / ml, was measured by a sandwich immunoassay reaction system. A luminescent substance is used as the substrate.

FIG. 8 shows a panel serum 13 which was subjected to sample treatment and fractionated using a gelloca column, and the core antigen immunoreactivity in the fraction was measured. The molecular weight is IgG
Is about 150 kD and albumin is about 68 kD.

FIG. 9 is a value 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 the correlation with the amount of HCV-RNA calculated | required by the monitor (PCR method).

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

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12P 21/08 C12Q 1/70 C12Q 1/70 C12N 15/00 ZNAA (72) Inventor Tatsuharu Kimura Nishitsuruga, Oi-cho, Iruma-gun, Saitama Prefecture Oka 1-33-1 Tonen Co., Ltd. Research Institute (72) Inventor Shintaro Yagi Nishitsurugaoka 1-3-3-1, Oi-cho, Iruma-gun, Saitama Tonen Co. Research Institute (56) References International Publication 96 / 041164 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/569 G01N 33/53 G01N 33/576 C12N 15/09 C12P 21/08 C12Q 1/70

Claims (4)

(57) [Claims] 1. A method for measuring hepatitis C virus, comprising:
Alkyl group having 10 or more carbon atoms and second, third or fourth
Hepatitis C virus antigen is measured by binding to its probe in the presence of a surfactant having a primary amine in the same molecule and a nonionic surfactant having a hydrophilic-hydrophobic ratio (HLB) of 12-14. A method characterized by the following. 2. The method according to claim 1, wherein the nonionic surfactant is polyoxyethylene isooctyl phenyl ether or polyoxyethylene nonyl phenyl ether. 3. The method according to claim 1, wherein the probe for the viral antigen is an antibody against the viral antigen. 4. The method according to claim 1, wherein the viral antigen is a core antigen.