JPH0933530A - Determination of virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine the ratio between nonimmune complex virus and immune complex virus in organism using carrier particles for adsorbing immune complex virus while discriminating from nonimmune complex virus. SOLUTION: Virus is touched to carrier particles provided with means for adsorbing immunoglobulin, e.g. aggregation of immunoglobulin of surface coating. It is preferable and convenient to touch a virus in serum or plasma to the carrier particles because the ratio between immune complex virus and nonimmune complex virus in blood is reflected directly. Only a virus forming a complex together with immunoglobulin is adsorbed to the carrier particles. Finally, the adsorbed virus and/or non-adsorbed virus are separated from each other by means of low speed centrifugal separation or a magnet and determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to a technical field relating to a method for quantifying a virus. More specifically, a virus that forms a complex with an immunoglobulin and / or a virus that does not form a complex are fractionated and quantified, thereby predicting the clinical state of a carrier of this virus, and It belongs to the technical field relating to a method for quantifying a virus that makes it possible to easily understand the action of the host immune system against the virus.

[0002]

2. Description of the Related Art Various viral diseases such as viral hepatitis and AIDS are currently known. For example, hepatitis C is
Non-A non-B viruses are known to be caused by hepatitis C virus isolated as a causative virus of chronic liver disease. When infected with this hepatitis C virus, infected persons frequently develop chronic liver disease after exhibiting liver damage, and also frequently develop liver cirrhosis and liver cancer.

When infected with hepatitis C virus, the immune system works in the body of the patient to increase the antibody level against hepatitis C virus. However, among the hepatitis C viruses whose structural proteins have been altered by very rapid mutation, some that survive the attack of the host immune system and remain in the patient's body without being completely eliminated appear. Is. The remaining hepatitis C virus is a cause of repeated new infections, resulting in repeated inflammation of the liver of hepatitis C patients. In a viral disease, such as this hepatitis C disease, which develops chronically and repeatedly, in the body of the patient, a virus that forms an immune complex with an antibody specific to the infecting virus, It has been revealed that viruses that do not form such immune complexes are mixed (M. Hjikata et a
I., J. Virology, Apr.1993, p1953-1958).

This is due to the presence of a virus that is attacked by the patient's immune system, as well as the above-mentioned hepatitis C virus, which can escape attacks from this immune system mainly due to frequent structural protein mutations. It is believed to reflect existence. That is, it is considered that a virus that will be attacked by the patient's immune system forms an immune complex, and a virus that does not form this immune complex is highly likely to escape the attack of the immune system. Thus, it has almost been determined that viruses that do not form immune complexes are a major factor in persistent infection and re-emergence (Y. Shimiz
u et al., J. Virology, May.1994, p1494-1500 / N.Kato e
t al., Viral Hepatis and Livir Disease, 1994, p329〜
333.

[0005]

In such a background, for example, a virus that forms the above-mentioned immune complex in blood (hereinafter referred to as an immune complex virus) and a virus that does not (hereinafter referred to as a non-immune complex virus) It will be possible to predict the clinical condition of the host of the virus and to easily understand the action of the host's immune system against the virus if the ratio of In other words, if the ratio of non-immune complex virus to all viruses in the body is high, the clinical symptoms are likely to deteriorate rapidly in the near future, and even patients with good clinical condition may be likely to deteriorate. Is considered to be high. Then, if such a state can be grasped promptly, it becomes possible to give an appropriate treatment before the clinical symptoms of the patient actually deteriorate. On the contrary, if the ratio of the above-mentioned immune complex virus is high, it is considered that the risk of worsening clinical symptoms in the near future is low.

Therefore, in recent years, various virus quantification methods have been proposed in order to obtain the ratio of non-immune complex virus and immune complex virus in the living body. For example, a method in which sodium chloride solution is added to serum and mixed and subjected to ultracentrifugation to compare the RNA amount of virus in the supernatant with that of the virus in the precipitate, or in serum at 15000 rp
After centrifuging at m for 15 minutes, anti-human immunoglobulin (IgA +
IgG + IgM) was added and left overnight at 4 ° C., then 6
Attempts have been made to compare the RNA amount of the virus in the supernatant after centrifugation at 80 rpm for 15 minutes and the amount of RNA in the precipitate.

However, the above method requires labor and cost and has a problem in accuracy, and therefore it is difficult to simultaneously process a large number of specimens. In addition, in general, the quality of anti-human immunoglobulin often varies from lot to lot, and not only the quality of this immunoglobulin tends to affect the quantification result, but also the quantity of this immunoglobulin is large and large. Since it is necessary to add it to the type reaction system, the aggregate becomes huge, and this huge aggregate also incorporates the non-immune complex virus in the supernatant, and it is undeniable that the accuracy of the quantification decreases. Further, in many cases, the boundary between the supernatant and the precipitate after the final centrifugation is not clear, and from this point, it cannot be denied that the quantitative results obtained above are difficult to trust.

[0008] Large and heavy viruses such as HIV have relatively close mass ratios of non-immune complex virus and immune complex virus. Therefore, when such a virus is fractionated and quantified by the above-mentioned quantification method or quantification, the fractionation due to the mass difference between the non-immune complex virus and the immune complex virus becomes very difficult, and the accuracy of the quantification further decreases. There is also the drawback of being lost.

Thus, the abundance ratio of the non-immune complex virus and the immune complex virus in the living body obtained by the method for quantifying these viruses is practical as a means for predicting clinical symptoms of virus-infected patients. It has not come to be a thing. Therefore, the problem to be solved by the present invention is to establish a means for determining the in vivo ratio of the non-immune complex virus and the immune complex virus, which is extremely simple and accurate as compared with such conventional methods. It is to provide a means for predicting the clinical symptoms of an infected patient and easily grasping the action of the host immune system against the virus.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems. As a result, they have found that the above problems can be solved by providing the following virus quantification method. That is, the inventor of the present invention proposes that, in claim 1, the virus is brought into contact with carrier particles provided with means capable of adsorbing immunoglobulin, and only the virus forming a complex with immunoglobulin is used as the carrier particles. A method for quantifying a virus is provided, which comprises: adsorbing the virus onto a substrate, and then fractionating and quantifying the adsorbed virus and / or the virus not adsorbed.

According to a second aspect of the present invention, in the method for quantifying a virus according to the first aspect, carrier particles to which only a virus forming a complex with an immunoglobulin is adsorbed,
The reaction system is fractionated by low-speed centrifugation to quantify the virus forming a complex with the carrier particle fraction and the immunoglobulin and / or the virus not forming a complex with the supernatant fraction with the immunoglobulin. A method for quantifying a virus is provided.

According to a third aspect of the present invention, in the method for quantifying a virus according to the first aspect, the carrier particles provided with means capable of adsorbing the immunoglobulin are magnetic particles and form a complex with the immunoglobulin. The carrier particles having adsorbed only the virus contained therein are separated by a magnetic force in the reaction system to form a complex with the immunoglobulin of the carrier particle fraction and / or the immunoglobulin of the supernatant fraction. The present invention provides a method for quantifying a virus, which comprises quantifying a virus that does not form a complex.

[0013] In claim 4, the means capable of adsorbing the immunoglobulin is a coating of immunoglobulin agglutinin on the surface of the carrier particles. A method for quantifying the described viruses is provided.

In a fifth aspect, there is provided the method for quantifying a virus according to the fourth aspect, wherein the immunoglobulin agglutinin is protein G or protein A.

In a sixth aspect, there is provided the method for quantifying a virus according to any one of the first to fifth aspects, wherein the virus is hepatitis C virus.

The present invention will be described in detail below. A. The quantification method of the present invention is a method of quantifying a virus in which only carrier particles provided with means capable of adsorbing immunoglobulin are contacted with the virus to adsorb only the immune complex virus to the carrier particles. The carrier particles provided with means capable of adsorbing the immunoglobulin are particularly preferably those carrier particles capable of separately adsorbing the immune complex virus from the non-immunity complex virus by providing means for adsorbing the immunoglobulin. The material is not limited, and can be selected according to the treatment mode of the carrier particles to which the immune complex virus is adsorbed at the time of virus quantification described later.

For example, when the carrier particles to which the immune complex virus is adsorbed are separated by low-speed centrifugation, sepharose particles, polystyrene latex, polybead latex, particles having a uniform particle size into which amino groups and succinyl groups are introduced. (For example, Eupergit
It) C: resin particles such as manufactured by Funakoshi Co., Ltd. are preferably selected. In addition, when performing magnetic adsorption, magnetic particles should be selected inevitably.

Note that it is convenient for the sepharose particles or resin particles to have a large surface area and be uniform in order to adsorb a large amount of immunoglobulin agglutinin, which is a typical means for adsorbing immunoglobulin. Then, it is preferable to select the above-mentioned Sepharose or Eupergit C as the carrier particles, and for the above-mentioned magnetic particles, magnetic porous glass beads (for example, MPG (Magnetic Porus Glass): manufactured by Funakoshi Co., Ltd.) and the like are used. Preference is given to choosing as carrier particles.

The means for adsorbing immunoglobulin on these carrier particles is not particularly limited, but usually, the surface of the carrier particles is coated with immunoglobulin agglutinin capable of specifically agglomerating immunoglobulin. Can be mentioned. As an immunoglobulin agglutinin,
For example, protein G, protein A, anti-human IgG monoclonal antibody, anti-human IgG polyclonal antibody, anti-human immunoglobulin polyclonal antibody, etc. can be mentioned. Among these, protein G and protein A are stable among lots and stable. It is particularly preferable to pay attention to the fact that it can be supplied afterwards.

Carrier particles coated with these immunoglobulin agglutinins on the surface are commercially available products such as UltraLink.
(Trademark) Protein A, G, A / G series (manufactured by Funakoshi), Protain Gsepharose 4FF (manufactured by Pharmacia)
Etc. (Currently, these commercial products are used for preparation of affinity chromatography in which immunoglobulin is bound to immunoglobulin agglutinin on carrier particles or for separation of polyclonal or monoclonal antibody in serum) Of course it can be used,
Depending on the desired form of the immunoglobulin agglutinin-coated carrier particles, it can be appropriately prepared by a generally known method.

That is, for example, a linker corresponding to the molecular structure of the desired coupling site of the immunoglobulin agglutinin, which is a ligand, is previously bound to the surface of any carrier particle, and the immunoglobulin agglutinin is bound via this linker. Can be coated. For example, for a porous glass for affinity chromatography as carrier particles, as a linker, for example, an aminopropyl group,
A carboxyl group, a long-chain aminoalkyl group, an aminoallyl group, a hydrazide group, etc. are introduced, and the desired immunoglobulin agglutinin is brought into contact with the carrier particles having the linker introduced therein to perform coating. Incidentally, carrier particles in which various linkers have been introduced in advance are commercially available (for example,
In addition to the above-described porous glass for affinity chromatography in which a linker is introduced, an immobilizing carbonyldiimidazole activated carrier, an bis-acrylamide / azalactone copolymer activated carrier, allyl glycidyl ether, methacrylamide, N -Activation carrier composed of copolymer of methylenebismethacrylamide, etc.) It is also possible to coat these commercially available products with a desired immunoglobulin agglutinin.

Further, by taking advantage of the property that avidin and biotin bind to each other very specifically, the biotin-bound antibody agglutinin is brought into contact with the carrier particles into which avidin has been introduced in advance to obtain the desired coated carrier particles. Can also be prepared. Further, the desired coated carrier particles can be prepared by reacting the carrier particles with the crosslinking reagent and the antibody agglutinin to crosslink the functional groups on the surface of the carrier particles with the functional groups in the antibody agglutinin. When magnetically separating non-immune complex virus and immune complex virus from each other, for example, by using the same coating means as the above carrier particles on magnetic porous glass beads for separation, etc. Can be coated.

A virus is brought into contact with carrier particles provided with means capable of adsorbing the immunoglobulin thus prepared, and the immune complex virus is adsorbed to the carrier particles. The virus to be brought into contact with the carrier particles needs to reflect the ratio of the immune complex virus and the non-immunological complex virus in the living body as directly as possible. Specifically, it is preferable and convenient to contact the virus in serum or plasma that directly reflects the ratio of the immune complex virus and the non-immunity complex virus in blood.

The serum referred to here is obtained by the traditional method,
For example, it is of course possible to use serum prepared through heparin blood collection, but other generally known serum preparation methods,
For example, instead of heparin, serum prepared using a preparation method using an anticoagulant such as EDTA or sodium citrate can be used in the quantification method of the present invention.

Further, as will be described later, it is possible to adopt a method of utilizing a viral nucleic acid by the PCR method as a means for quantifying the virus, but heparin remarkably inhibits the activity of the thermostable DNA polymerase used in the PCR method. Therefore, in such a case, it is unsuitable for the serum as a sample to be prepared through blood collection with heparin.
It is necessary to use serum prepared by substituting heparin with an anticoagulant that does not affect the thermostable DNA polymerase activity such as sodium citrate.

The manner of contacting the carrier particles with the virus does not require any special operation, and it is sufficient, for example, to directly mix the carrier particles with serum and incubate. In addition, the mixing ratio of carrier particles and serum is about equal to or more than the volume ratio of carrier particles to carrier particles, and preferably 2 or more.
More than double. The above-mentioned incubation does not need to be performed under special low temperature conditions, and is usually performed at a temperature of about 37 ° C. Also, this incubation time is
Although it is possible from 15 minutes or more, it is usually preferably about 1 hour in consideration of the accuracy and efficiency of the quantification. In this manner, the immune complex virus is adsorbed on the carrier particles, and the non-immunized virus not adsorbed on the carrier particles and the immune complex virus can be separated.

The viruses to which the quantification method of the present invention can be applied include
The virus is not particularly limited as long as it is a virus capable of forming an immune complex in vivo, but the non-immune complex is obtained by applying the quantitative assay method of the present invention to a virus having a high mutation rate of a structural protein and a strong tendency of becoming chronic in infected persons. Calculating the abundance ratio of virus and immune complex virus has great clinical significance. Specifically, HCV (hepatitis C virus), HIV
(AIDS virus), HBV (hepatitis B virus), G
BV (hepatitis GB virus) and the like can be mentioned as viruses to which the quantification method of the present invention is preferably applied for the above purpose. The viruses listed here are merely examples, and the viruses to which the quantification method of the present invention can be applied are not limited thereto.

B. In the quantification method of the present invention, the immunocomplex virus adsorbed on the carrier particles and / or the non-immunocomplex virus not adsorbed on the carrier particles are fractionated and quantified. This separation / quantification means can be appropriately selected according to the adsorption method selected above. First, as a means for separating the immune complex virus from the non-immune complex virus, centrifugation is performed to separate the carrier that has adsorbed the immune complex virus into the precipitate fraction, and the non-immune complex virus into the supernatant fraction. The means for doing so can be mentioned.

As described above, in the quantification method of the present invention, low-speed centrifugation, that is, about 100%, is performed in the quantification method of the present invention, as compared with the prior art in which the immune complex virus was fractionated by ultracentrifugation.
Centrifugation at 0 to 2000 rpm (low speed centrifugation as defined in the present invention is 2000 rpm or less) and for a short time of 1 to 2 minutes, the immunocomplex virus fraction is sufficiently precipitated to form a non-immunity complex virus fraction. It becomes possible to separate from the fraction (supernatant).

It should be noted that the rotation speed of the centrifugal separation shown above is not limited to the above range in the present invention, and for example, by extending the centrifugation time, the quantification method of the present invention at a rotation speed of less than 1000 rpm. Can be carried out, and conversely, even if centrifugation is performed at a rotation speed of more than 2000 rpm, the non-immune complex virus does not precipitate (approximately 15
If it is 000 rpm or less), it does not actually cause any harm in separating viruses.

That is, the rotation speed of the centrifugation shown above is the rotation speed normally used in a centrifugal separator capable of simultaneously processing a large number of commonly used specimens, and 1
Centrifugation as short as ˜2 minutes is sufficient for fractionating the desired virus, which shows a great significance of using this one-sorting means in the quantification method of the present invention.

Furthermore, in the ultracentrifugation which has been used as a means for separating viruses in the prior art, as described above, the immunocomplex virus fraction and the non-immunocomplex fraction are clearly precipitated as in the present invention. Since the fractions and the supernatant fractions are not fractionated, but both fractions are fractionated by a continuous gradient, the deficiency in the quantification accuracy cannot be avoided. On the other hand, in this one-sorting means in the quantification method of the present invention, the immunocomplex virus fraction and the non-immunocomplex fraction are formed into a form of precipitate and supernatant by the centrifugation at a low speed and for a short time as described above. Clearly, it is possible to clearly separate, and it is clear that not only the convenience of the separation operation but also the accuracy of the separation is extremely excellent.

As another means for differentiating the immune complex virus, the carrier containing the immune complex virus adsorbed by magnetic separation using a magnet and magnetic particles as the carrier is used as a precipitation fraction, and the non-immunity complex virus is used. Can be mentioned as a supernatant fraction. This means is a means for separating the immune complex virus and the non-immune complex virus by separating the carrier adsorbing the immune complex virus by a magnetic force as a precipitate fraction without centrifugation. This separating means can separate a desired virus extremely efficiently and highly accurately without the need for centrifugation.

The amount of the non-immune complex virus and / or the immune complex virus in the reaction system thus separated from the immune complex virus fraction was quantified, and as a result, The abundance ratio of the immune complex virus can be calculated. In the present invention, the term “abundance ratio of non-immune complex virus and immune complex virus” means not only the literal existence ratio of non-immune complex virus and immune complex virus, but also non-immune complex virus and total It also means the abundance ratio of virus and the abundance ratio of immune complex virus and total virus.

For this quantification method, it is also possible to adopt a direct method for quantifying the virus amount in each virus fraction. The total virus amount before fractionation is quantified, and the virus amount in any virus fraction is then determined. It is also possible to use an indirect method in which the virus amount of the other fraction is calculated by subtracting

In general, the latter indirect method is preferably used in consideration of its simplicity and accuracy. In this case, in general, quantifying the amount of virus in the supernatant is simpler and more accurate than quantifying the amount of virus that has precipitated, so the amount of virus in the supernatant, which is the non-immune complex virus fraction, is quantified. It is preferable to subtract this from the total amount of virus before fractionation to determine the amount of immune complex virus to calculate the abundance ratio of the non-immune complex virus and the immune complex virus in the desired reaction system.

As the virus quantification means that can be used in the present invention, generally known means can be widely selected. For example, when quantifying a virus based on the nucleic acid of the virus, multicyclic (multicyclic)
) RT-PCR method (International Hepatology Commun
ications, 1993; 1: p72-79) is preferable in that the required sample amount can be minimized and excellent reproducibility can be obtained. Specific operations of this multi-cyclic RT-PCR method will be described in Examples.

Further, when quantifying the virus based on the virus structural protein, the mantle membrane protein of the virus is treated with a dilute detergent or a dilute alkali to expose the core protein of the virus,
Quantification is preferably performed by a detection method represented by an enzyme immunoassay (EIA) or a radioimmunoassay (RIA) using a monoclonal antibody specific to this core protein.

C. As described above, by using the quantification method of the present invention, the abundance ratio of the non-immune complex virus and the immune complex virus in a patient with a viral disease can be easily determined. This abundance ratio is extremely important data that can predict the future clinical symptoms of virus-infected patients.

That is, even if the clinical symptoms are good at the present time, if the non-immune complex virus abundance ratio in the body is high, there is a strong possibility that the clinical symptoms will worsen if left unattended. Can be expected. And
On the other hand, by taking preventive measures against this exacerbated clinical condition, it becomes easy to control the morbidity of the virus-infected patient. On the contrary, even if the clinical symptoms are not good at present, it is possible to predict that the clinical symptoms are likely to improve in the near future when the abundance ratio of the non-immune complex virus in the body is low.

The specific numerical value of the abundance ratio of the non-immune complex virus and the immune complex virus, which may be a problem, should be specifically determined individually depending on the type of virus and the individuality of the virus-infected patient. Although not constant, for example, in the case of hepatitis C patients, the F / T ratio of all hepatitis C virus (described later) is approximately 1.
If between 0 and 10, at least attention should be paid to the worsening of clinical symptoms in this patient. In particular, when this F / T ratio tends to increase, there is a strong possibility that the clinical symptoms of hepatitis C will worsen, and the administration of hepatoprotective agents represented by glycyrrhizin preparations such as potent minophagen C should be considered. In many cases.

As described above, by continuously monitoring the abundance ratio of this immune complex virus in virus-infected patients, it becomes possible to accurately predict and understand the disease state that should occur, and the result of the quantification method of the present invention. Therefore, it becomes very easy to give an appropriate treatment to a virus-infected patient. In addition, the quantification method of the present invention makes it possible to easily understand the action of the host immune system against viruses.

[0043]

EXAMPLES The present invention will be described more specifically by way of examples. However, the technical scope of the present invention should not be limitedly interpreted by this embodiment. [Example 1] Separation of hepatitis C virus (1) Protein G sepharose which is a carrier coated with protein G (protein G) as a substance for adsorbing human immunoglobulin on sepharose
se) (2mg protein G / midrained gel) (trade name:
5 ml of Protain Gsepharose 4FF (Pharmacia) was washed 3 times with 3 times the volume of phosphate buffered saline (PBS), equilibrated with PBS and diluted to 5.5 times. The diluted protein G sepharose is
550 μl was dispensed to each 1.5 ml microtube (100 μl of protein G sepharose to each microtube). Serum of one patient who developed chronic hepatitis C after transfusion for 5 years, following natural course (Blood collection interval: 1-2 months, Blood collection period: 5 years, 40 specimens in total) 5
0 μl was added to each of the tubes to which the protein G sepharose was dispensed and gently mixed by inversion at 37 ° C. for 1 hour to adsorb the immune complex hepatitis C virus to the protein G sepharose. After the adsorption reaction, 1000rp
Centrifugation was performed at m 2 for 4 minutes at 4 ° C. to obtain a supernatant containing non-immune complex hepatitis C virus. This supernatant 100
Using 1 μl for multicyclic RT-PCR, C
The amount of hepatitis virus RNA was determined (see (2)). On the other hand, 50 μl of patient serum before separation (untreated) was added to P
In addition to 450 μl of BS, gently invert and mix at 37 ° C. for 1 hour as in the case of separating the immune complex hepatitis C virus, and then centrifuge at 1000 rpm for 2 minutes at 4 ° C.
Total viral load was also determined by multicyclic RT-PCR using 0 μl (see (2)).

(2) Multicyclic RT-PCR
Quantitation with 50 μl of a sample was added to a guanidine thiocyanate RNA extract (5M guanidium thiocyanate, 25 mM sodium citr
ate, 0.5% sodium N dodecanoyl salcosinate,
0.2 μM β-mercaptoethanol) 500 μl, 4M sodium acetate aqueous solution (pH 4.0) 60 μl were added, and after stirring, water-saturated phenol 600 μl, chloroform 12
0 μl was added, and the mixture was vigorously stirred again. 10 reaction systems on ice
After standing for 4 minutes, centrifugation was carried out at 10,000 rpm for 20 minutes at 4 ° C., and 500 μl of the supernatant obtained by separation was recovered. To the recovered supernatant, 20 μg / μl of glycogen (2 μl) was added as a coprecipitant, and further 50 parts of isopropanol was added.
After addition of 0 μl, the reaction was cooled at −20 ° C. for 1 hour. After that, centrifugation at 15,000 rpm for 20 minutes at 4 ° C. is performed to hepatitis C virus RNA (HCV-RNA).
Was recovered as a precipitate. The obtained HCV-RNA is 75
After washing with% ethanol, it was dried and used for the reverse transcription reaction.

That is, the above HCV-RNA had a final concentration of [dNTP (1 mM), Tris-HCl (pH 8.
3) (50 mM), KCl (50 mM), MgCl ₂ (8 m
M), dithiothreitol (DTT) (4 mM), Rnasin
(20 units), HCV13 antisense primer (5'-AACAC TACTC GGCTA GCAG
T-3 ': SEQ ID NO: 1) (100 pmole)]
V-XL reverse transcriptase (4 units) was added and incubated at 42 ° C. for 1 hour to carry out a reverse transcription reaction to prepare cDNA using the above HCV-RNA as a template.

Next, this cDNA was amplified by PCR reaction. That is, HCV14-3 sense primer (5'-ACTCC ACCATAGATC ACTCC
-3 ': SEQ ID NO: 2) (100pmole) is a commercially available thermostable DNA polymerase (trade name AmpliTag DNA polymer
ase: manufactured by Roche) was added to a reaction solution (80 μl) according to the prescription in the package insert, and this was added to the cDNA solution in which the reverse transcription reaction was completed, and the total reaction solution volume was 100 μl.
PCR reaction was carried out.

In this PCR reaction, the denaturation reaction is 94 ° C.
Min, annealing at 55 ° C for 1 minute, extension reaction at 72
This is a reaction in which the temperature cycle of 1 minute at 1 ° C. is one cycle, and in this example, 45 cycles of this temperature cycle were performed. At the time when this temperature cycle was repeated 30 cycles, 50 μl of the PCR reaction solution was withdrawn,
This was used as a PCR product for 30 cycles.

In the above series of PCR reactions, HCV
A 10-fold diluted sequence (109 copies-10 copies) of HCV-RNA synthesized in vitro from cDNA clone pIK37m using T7 RNA polymerase was used as a known amount of HCV.
-Used as an RNA standard, the same treatment as the sample was performed at the same time.

The PCR product thus obtained was dot-blotted on a nylon membrane, and the internal probe HCV13.14.3 (5'-CCGGC AGAGC) labeled at the 5'end with polynucleotide kinase.
CATAG TGGTCTGCGG AACCG G
-3 ': SEQ ID NO: 3) was used to carry out a hybridization reaction according to a conventional method, and a signal was measured using a BAS1000 system (manufactured by FUJIFILM Corporation). B
A calibration curve is prepared based on the signal of the standard (10-fold diluted sequence of known amount of HCV-RNA) obtained by AS1000 system, and the signal of the sample is measured based on this calibration curve, and the amount of HCV-RNA is determined. And

(2) Calculation of abundance ratio of non-immune complex virus in patient serum The amount of non-immune complex hepatitis C virus contained in a unit volume (10 μl) of a test sample was determined by the above multicyclic RT-PCR. According to the formula F / T ratio = non-immune complex virus amount / serum total virus amount × 100, the non-immune complex hepatitis C virus abundance ratio (F
/ T ratio: immunological free virus vition / total vi
rus vition ratio) was calculated.

(3) As shown above, in a series serum of patients who developed hepatitis C after blood transfusion and became chronic, the F serum was measured using the quantitative assay method of the present invention for 5 years.
The results of calculating the / T ratio are shown below. FIG. 1 is a graph showing changes over time in F / T ratio and blood total HCV-RNA amount, and FIG. 2 shows F / T ratio and GP.
It is a graph which showed the time-dependent change of T value in 5 years. 1 and 2 show the GPT showing the inflammatory state of the liver.
The changes in the values and the total amount of HCV-RNA in blood were almost in synchronism. However, the F / T ratio increased before the GPT value (FIG. 2), and liver inflammation was observed 1 to 3 months after the appearance of the peak.

According to this example, an increase in the F / T ratio in hepatitis C virus, that is, an increase in the ratio of non-immune complex hepatitis C virus in blood, indicates that GP is an inflammatory condition of the liver.
It was revealed that the F / T ratio can be a very excellent marker for predicting the clinical symptoms of hepatitis C patients since it occurs prior to the increase of T value. The quantification method of the present invention can calculate this F / T ratio more easily, more quickly and accurately than in the past, and predicts clinical symptoms of hepatitis C patients by actually using this F / T ratio. It can be seen that this is a quantification method that realizes the significance as a marker that

The existing markers for hepatitis C are:
H using several antibody detection systems for measuring host antibodies to hepatitis C structural proteins and non-structural proteins and multicyclic RT-PCR as in this example.
There are CV-RNA detection quantification systems, all of which respond to the inflammation of the liver of the patient at the same time or with a delay,
The clinical presentation of hepatitis C was not predictable.

In addition, although the present example relates to hepatitis C, other chronic disease viruses have the same immune complex virus and non-immune complex virus in the blood for the same reason as in hepatitis C (internal body). It is clear that they coexist due to the immune reaction of (1) and competition with structural protein mutations of the virus). Therefore, it is clear that the F / T ratio can be a very excellent marker in predicting the clinical condition of a patient even in viruses other than hepatitis C virus (specifically, the above-mentioned examples), It is clear that the quantification method of the present invention, which is necessary for actually using this marker, is a very significant invention.

[0055]

INDUSTRIAL APPLICABILITY The present invention provides a very simple and accurate virus quantification means essential for determining the in vivo ratio of a non-immunocomplex virus to an immunocomplex virus. Means are provided to allow for the prediction of the clinical symptoms of infected patients and the easy understanding of the action of the host's immune system against the virus.

[0056]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin Hepatitis C virus cDNA sequence AACACTACTC GGCTAGCAGT 20

SEQ ID NO: 2 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Origin Hepatitis C Virus cDNA Sequence ACTCCACCAT AGATCACTCC 20

SEQ ID NO: 3 Sequence Length: 31 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Origin Hepatitis C Virus cDNA Sequence CCGGCAGAGC CATAGTGGTC TGCGGAACCG G 31

[Brief description of drawings]

FIG. 1 is a drawing showing changes over time in the amount of non-immune complex hepatitis C virus and the total amount of hepatitis C virus.

FIG. 2 is a drawing showing changes with time in non-immune complex hepatitis C virus load and GPT value.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Fuminori Hoshino 1361 Matoba, Kawagoe-shi, Saitama 1 BML Research Institute, Inc. (72) Inventor Takao Ando 1361 Matoba, Kawagoe-shi, Saitama BML Co., Ltd. (72) Inventor Akira Otani 1361 Matoba, Kawagoe City, Saitama Prefecture 1 BML Co., Ltd.

Claims (6)

[Claims] 1. A virus is brought into contact with carrier particles provided with means capable of adsorbing immunoglobulin, and only the virus forming a complex with immunoglobulin is adsorbed on the carrier particles, and then this adsorption is carried out. And / or virus that has not been adsorbed is separately quantified and quantified. 2. The method for quantifying viruses according to claim 1, wherein the carrier particles adsorbing only the virus forming a complex with an immunoglobulin are separated in the reaction system by low speed centrifugation, and the carrier particles are obtained. A method for quantifying a virus, which comprises quantifying a virus forming a complex with an immunoglobulin in a fraction and / or a virus not forming a complex with an immunoglobulin in a supernatant fraction. 3. The virus quantification method according to claim 1, wherein the carrier particles provided with means capable of adsorbing immunoglobulin are magnetic particles and only a virus forming a complex with immunoglobulin. The carrier particles having adsorbed are separated by a magnetic force in the reaction system to form a complex of the carrier particle fraction with the immunoglobulin and / or the supernatant fraction with the immunoglobulin. A method for quantifying a virus, which comprises quantifying a virus that has not formed. 4. The virus according to any one of claims 1 to 3, wherein the means capable of adsorbing the immunoglobulin is a coating of immunoglobulin agglutinin on the surface of carrier particles. Quantitation method. 5. The method for quantifying a virus according to claim 4, wherein the immunoglobulin agglutinin is protein G or protein A. 6. The method for quantifying a virus according to any one of claims 1 to 5, wherein the virus is hepatitis C virus.