JPH1169971A - Isolation, recovery, concentration and preparation of sample, of virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for isolation, recovery, concentration, and preparation of a sample, of a virus, by which a highly sensitive virus assay capable of being carried out even in an ordinary facility is provided to the study and diagnosis of the virus. SOLUTION: This method for isolation, recovery, concentration and preparation of a sample, of a virus comprises a step for allowing a lipoprotein- combining and precipitating material to act on a sample including a virus, and a step for solubilizing the precipitated material. A divalent metal ion is preferable as the lipoprotein-combining and precipitating material, and the addition of a polyvalent anionic material as a precipitation accelerator is preferable. As the result, a virus sample is prepared in a research organization or a medical facility without requiring a special equipment, and the method contributes to the study of the virus, and the diagnosis, treatment and prevention of a virus disease through an antigen examination and a gene examination.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating and concentrating a virus present in a material, recovering the virus, and preparing a sample.

[0002]

2. Description of the Related Art It is widely known that viruses cause various diseases and diseases on humans, animals and plants. Therefore, in order to establish a method for preventing, diagnosing, and treating diseases and diseases, the starting point is to confirm the presence of the causative virus, which is extremely important. However, as seen in the research history of AIDS and various viral hepatitis, it is not easy to search for and diagnose the causative virus.

[0003] The most common virus diagnostic method is the measurement of viral antigens as well as antiviral antibodies. However, these measurements require the presence of a certain amount or more of a virus antigen or virus antibody. However, in many cases, antigen testing cannot be used for diagnosis because the amount of virus is low and the sensitivity is lower than the measurement sensitivity despite the infection state. For viruses for which a virus propagation method in vitro using cultured cells has been established, after virus-infected cells are cultured, the virus must be separated and recovered from the cells by an appropriate method, and used as a sample for measurement. You can also
Very few viruses have established an in vitro culture method, and in many cases this method cannot be practically adopted. Further, even in the case where the above method is established, special facilities, equipment and technology are required for separation and recovery, and it cannot be said that the method can be generally used simply.

In recent years, methods for amplifying genes have been developed,
By amplifying the viral gene, the possibility of detecting and measuring even a small amount of virus has been opened. However, even in this method, measurement of an extremely small amount of virus requires complicated and sophisticated measurement techniques, is sensitive to contamination from the environment in the facility, and requires special pollution control measures. Furthermore, special precautions are required for the sample collection and preservation methods, and the technology has not yet reached a technical level that can be easily implemented in general facilities.

Since the situation of virus research and diagnosis is as described above, it has been desired to develop a highly sensitive virus measurement method that can be carried out in ordinary facilities. As a method for solving this problem, a method has been considered in which the virus is concentrated to a level that can be detected by ordinary measurement techniques.

One of the conventionally used virus concentration methods is a method utilizing ultracentrifugation. In this method, virus particles are simply precipitated by centrifugal force, and then a virus fraction is obtained by continuous sucrose density gradient equilibrium centrifugation.
Further, there are some which are purified by floating centrifugation. The method using the density gradient equilibrium centrifugation is a method of concentrating by utilizing the fact that when a substance is centrifuged in a carrier having a density gradient, an equilibrium state is established at the same carrier position as the substance density. Because each virus has a unique density, this method can be used to concentrate the virus. But,
In this method, sample preparation is complicated, long-time high-speed centrifugation is required, necessary equipment is expensive, and the density gradient carrier may need to be removed by dialysis or the like before measurement. It is not suitable for a sample preparation method such as a diagnostic test which requires processing of many samples at once.

On the other hand, Einarsson et al. Have reported a method for purifying HBs antigen by a chromatography method using a heparin sepharose carrier, utilizing the property that a hepatitis B virus surface antigen (HBs antigen) binds to heparin. However, although this method is effective for preparing a single sample, it requires a lot of labor and time to process a large number of samples at once, and it is necessary to prepare equipment for the number of samples. However, it is similarly difficult to use it as a sample preparation method for inspection in a general facility.

As another method, a method of precipitating a virus using ammonium sulfate, a method of precipitating using a polyethylene glycol, and a method of separating and recovering a virus by a two-phase partitioning method have been reported. There is a problem that sample preparation is necessary, and it cannot be said to be a simple sample preparation method. In the case of a virus having an envelope, it is said that the precipitation method is unsuitable for separating the virus while maintaining the envelope because of its physicochemical properties.

[0009]

As described above, the conventional virus separation / recovery / concentration method is complicated in operation and unsuitable for processing a large number of samples at the same time. Furthermore, performing this requires special equipment such as culture equipment, special centrifugal separators, separation carriers, and many chromatography-related equipment. Could not be used. The present invention solves these problems and provides a virus separation / collection / concentration method and a sample preparation method which are simple enough to be used in ordinary facilities and do not adversely affect the results of virus measurement. Aim.

[0010] The present inventors have found that some viruses have the same density as lipoproteins, and that such viruses contain a large amount of lipoproteins in their outer coats.
From this, the present inventors have focused on the possibility that the chemical properties of lipoproteins may be used for the purpose of separating, collecting, and concentrating viruses, and continued their research. However, an attempt was made to apply a method of adding dextran, dextran sulfate or phosphotungstic acid to a sample, which has been conventionally used as a method for precipitating lipoproteins, but was unable to separate, collect and concentrate the virus. For this reason, the present inventors thought that the conditions for simply precipitating lipoproteins were not sufficient for the purpose of separating, collecting and concentrating the virus, and as a result of continuing various studies, the present invention could be completed.

One of the facts that the present inventors were able to elucidate is that a divalent metal cation salt is used and coexists with various polyanion molecules such as sulfate polysaccharide. Thus, the virus can be sufficiently precipitated by the ordinary centrifugation method. No special method such as ultracentrifugation was required for this precipitation.

Next, in order to achieve the object of the present invention,
It is necessary not only to separate and collect and concentrate low-concentration viruses, but also to make the separated and recovered and concentrated viruses usable for various measurement methods, that is, to solubilize the separated and recovered and concentrated viruses by precipitation. there were. Therefore, the present inventors studied conditions for solubilizing the precipitate. As a result, it was found that removing the metal ions from the precipitate or separating the lipoprotein from the lipoprotein precipitate solubilized the precipitate. That is, the precipitate is solubilized by allowing the substance to remove divalent metal ions from the precipitate, the substance having an action of separating lipoprotein from the lipoprotein precipitate, or both, on the precipitate,
It was found that a sample was obtained that could be used for subsequent measurements. For example, a metal ion chelating agent or another metal ion salt such as NaHCO ₃ is useful as a method of removing metal ions, and potassium bromide or potassium bromide is used as a substance having an action of separating lipoprotein from a lipoprotein precipitated substance. Sodium bromide has been found to be useful.

As described above, the present invention provides a method for collecting and concentrating a virus, focusing on the fact that a virus having a lipoprotein in its outer coat is precipitated by the action of divalent metal ions.
In order to increase the recovery / concentration efficiency, it is useful to use a polyvalent anion substance in combination. Furthermore, the concentrated virus recovered by the method can be prepared in a state that can be used for antigen measurement and gene measurement.

A virus generally has an envelope as its structure, but it is presumed that its components differ from virus to virus. However, it is known that the envelope of some viruses contains lipoproteins, and when the lipoproteins are contained in a certain percentage or more, the outer coat of the virus has physical and chemical properties as lipoproteins. May be strongly indicated. Hepatitis C virus (hereinafter abbreviated as “HCV”) and GB virus-C (hereinafter “GBV-
C "). In the present invention, examples for these HCV and GBV-C are described. However, if the coat contains a lipoprotein and the virus has a remarkable property, the principle of the present invention can be applied to these viruses. The same applies. The "virus" in the present invention means a virus containing lipoprotein in the envelope to such an extent that it can be sufficiently precipitated with a lipoprotein precipitant. Whether or not the target virus is the “virus” in the present invention can be easily confirmed by the method disclosed in the present invention.

The polyvalent anion molecule used in the present invention may be any polyvalent anion molecule that binds to lipoprotein. For example, sulfated polysaccharides, especially dextran sulfate, amylopectin sulfate, heparin.

The lipoprotein-binding / precipitating substance used in the present invention includes a divalent metal cation.
²⁺ and Zn ²⁺ .

The method of recovering and concentrating the insoluble conjugate of the virus and the polyvalent anion molecule formed in the presence of the metal cation includes the method of physically separating and recovering the insoluble conjugate of the virus, and the method of collecting particles. There is a method of collecting. As a physical separation and recovery method, there is a commonly used centrifugation method. The insoluble conjugate can be recovered as a precipitate by centrifugation at a rotational speed of preferably about 12,000 rpm for tens of minutes or more.

The virus recovered and concentrated by the above method forms an insoluble conjugate. For this reason, in this state, it is not appropriate and sometimes impossible to measure a virus antigen or a virus gene. This is because when the virus is in an insoluble binding state, the binding between the virus antigen and the antibody to the antigen is physically inhibited, so that it is impossible to measure the virus antigen using the reaction between the antigen and the antibody. Heparin and dextran sulfate inhibit the polymerase chain reaction method (hereinafter abbreviated as “PCR”), which is a method for measuring a gene with high sensitivity. Therefore, in order to achieve the object of the present invention, the recovered and concentrated virus is dissociated from the insoluble conjugate, the polyvalent anion molecule bound to the virus is removed from the virus, and the coexisting inhibitory substance is removed. -It is necessary to have a step of making the virus measurable by reducing it.

As a method for dissociating and solubilizing a conjugate comprising a polyvalent anion molecule and a virus, a salt is allowed to act,
There is a method of dissociating the bond between the polyvalent anion molecule and the lipoprotein. For example, potassium bromide at a saturated concentration or 1.5
There is a method in which M sodium chloride and 1 mM phosphotungstic acid are allowed to act. Other salts having the same action can be used. There is a method in which a precipitated substance is removed by adding a substance that binds or chelates to a divalent metal cation forming an insoluble conjugate, and then dissociated by adding the above-described salt having a dissociating action to solubilize. For example, sodium bicarbonate or citric acid is allowed to act on the insoluble conjugate to remove Mn ions, and potassium bromide is added thereto to solubilize the virus. Other than those having the same action, those other than sodium hydrogen carbonate and citric acid can be used.

Next, when the virus separated / recovered / concentrated by the above-mentioned method is to be used for PCR, the inhibitory effect of polyvalent anion molecules may be a problem. One way to address this problem is to eliminate inhibitors directly. For example, a substance that binds to the inhibitor and forms a precipitate may be added, and the inhibitor may be precipitated to separate the solubilized virus. For example, 10% NaH was added to the virus separated and collected and concentrated using dextran sulfate-manganese chloride.
There is a method of forming Mn (HCO ₃ ) ₂ by adding CO ₃ . There is also a method in which dextran sulfate is precipitated by adding saturated potassium bromide after chelating Mn.

[0021]

According to the present invention, virus separation, recovery, concentration, and sample preparation that required special equipment and skilled techniques and long operation time can be performed in a short time and without any special equipment or techniques. it can. This makes it possible to measure viral antigens or viral genes that could not be measured conventionally or could not achieve sufficient sensitivity, thereby contributing to the diagnosis, treatment and prevention of viral diseases.

[0022]

EXAMPLES Examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.

[0023]

Example 1 Separation / recovery / concentration of hepatitis C virus using heparin-manganese chloride, solubilization of concentrated virus (1) Separation / recovery / concentration of HCV from plasma 1 ml of human plasma confirmed to have HCV Heparin solution (159 / usunits / mg in 0.15M)
10 μl of a solution dissolved in 5 ml of sodium chloride solution) was added, and 75 μl of 1 M MnCl ₂ was further added, followed by reaction at room temperature for 20 minutes. After the reaction, 15000 rpm
Separation was carried out for 0 minute with a cooling microcentrifuge, and the supernatant was discarded to obtain a precipitate. (2) Solubilization of separated / collected / concentrated virus 50 μl of saturated potassium bromide was added to the precipitate obtained in the above operation to solubilize the precipitate (separation / collection fractionation). Final volume is about 5
0 μl. (3) Confirmation of Separation / Recovery Rate A dilution series was prepared from the plasma used for separation / recovery using human plasma which was previously confirmed to be HCV-RNA negative, and this was used as a measurement sample. RNA was extracted from this sample using a conventional method. This extracted RNA
HCV-RNA amount by PCR method, which is generally implemented as a target 5'UTR region of the HCV gene for was 10 ^5. Then place the adjusted HCV-RNA amount in the same manner as described above diluted sample takes 2μl than separated and recovered fraction was measured, HCV-RNA amount of collected fractions was 10 ^5. From the above results, it was found that the virus recovery rate in this example was almost 100%, and the concentration rate was 25 times (Table 1).

[0024]

[Table 1]

[0025]

Example 2 Separation, collection and concentration of GBV-C from human plasma using dextran sulfate and manganese chloride, and measurement of GBV-C gene using the sample (1) Separation of GBV-C from plasma Collection and concentration Human plasma 1 for which the presence of GBV-C virus has been confirmed
Dextran sulfate was added to a final concentration of 0.25% per ml. Further, 50 M of MnCl ₂ was added.
-75 µl was added and reacted at room temperature for 20 minutes. After completion of the reaction, the mixture was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was discarded to obtain a precipitate. (2) Removal and solubilization of dextran sulfate from separated / collected / concentrated virus. 5% of 10% NaHCO ₃ was added to the separated, collected and concentrated virus.
0-75 μl was added. After the concentrated virus was solubilized by this operation, an appropriate amount of saturated potassium bromide was added to precipitate dextran sulfate. The sample was centrifuged under appropriate conditions, the supernatant was collected, and the solubilized virus was recovered and separated. The volume of the supernatant was 120 μl. (3) Confirmation of separation / recovery In the same manner as in Example 1, the RNA concentration of the plasma sample used for separation / recovery / concentration before separation / recovery was determined, and the total virus amount was calculated. Next, GBV-C-RNA measurement was performed using the total amount of the solubilized virus from which the dextran sulfate was removed, and the recovery rate was determined.
The amount of GBV-C-RNA in the sample before separation and recovery was determined from 100 μl of plasma, and the GBV of the sample after separation and recovery.
The measurement of the amount of -C-RNA was performed by preparing a dilution series from the total amount of the collected 120 µl. As a result, the recovery rate was almost 10
0%, and the enrichment rate was confirmed to be about 8 times (Table 2).

[0026]

[Table 2]

[0027]

Example 3 Separation and collection and concentration of HCV from human plasma using dextran sulfate and manganese chloride, and measurement of HCV gene using the sample (1) Separation and collection and concentration of HCV from plasma. Dextran sulfate was added to 1 ml of human plasma in which the presence of HCV was confirmed so as to have a final concentration of 0.25%, and 50 to 75 μl of 1 M MnCl ₂ was added, followed by reaction at room temperature for 20 minutes. 15000r after completion of reaction
After centrifugation at 10 pm for 10 minutes, the supernatant was discarded to obtain a precipitate. (2) Removal and solubilization of dextran sulfate from the separated and concentrated virus. 50-75 μl of 10% NaHCO ₃ was added to the separated, collected and concentrated virus. After the concentrated virus was solubilized by this operation, an appropriate amount of saturated potassium bromide was added to precipitate dextran sulfate. The sample was centrifuged under appropriate conditions, the supernatant was collected, and the solubilized virus was recovered and separated. The volume of the supernatant was 120 μl. (3) Confirmation of separation / recovery In the same manner as in Example 1, the RNA concentration of the plasma sample used for separation / recovery / concentration before separation / recovery was determined, and the total virus amount was calculated. Next, HCV-RNA was measured using the total amount of the solubilized virus from which separation, collection, concentration, and dextran sulfate had been removed, and the recovery rate was determined. HCV-RNA content of the sample before separation and recovery was measured using 100 μM plasma.
For the measurement of the amount of HCV-RNA in the sample after separation and recovery, a dilution series was prepared from the entire 120 μl obtained. As a result, the recovery was almost 100%, about 8%.
The enrichment was found to be twice as high (Table 3).

[0028]

[Table 3]

[0029]

Example 4 Measurement of HCV envelope antigen using HCV separated and recovered and concentrated from plasma using heparin-manganese chloride. (1) Preparation of anti-HCV envelope antibody-immobilized plate. Anti-HC prepared using HCV envelope antigen as immunogen
The V envelope antibody was immobilized on the wall surface of an ordinary polystyrene 96-well titer plate. That is, 280 nm
0.2M Carbona so that the absorbance becomes 0.01
The antibody solution diluted with the te buffer solution was added to the plate wells in an amount of 50 μl each, and the plate was shake-reacted at room temperature for 1 hour to be physically adsorbed on the wall surface, and then washed with a phosphate buffer solution containing 2% Tween 80. 2% skim milk (TB
(S buffer) and block for 1 hour at room temperature. After the reaction was completed, the plate was sufficiently washed with the above-mentioned washing solution to prepare an antibody-immobilized plate. (2) Measurement A sample diluent (2% NP-40, 0.4% skim milk, TBS) was placed in an Eppendorf tube having a capacity of 2 ml.
Buffer). To this tube, 50 μl of a test sample (plasma) solubilized and adjusted after separation / recovery / concentration by the method described in Example 1 was added, and reacted at room temperature for 1 hour. After completion of the reaction, the plate was sufficiently washed with the above-mentioned washing solution, and allowed to react with horseradish peroxidase-labeled anti-HCV envelope antibody at room temperature for 30 minutes. After completion of the reaction, the plate was washed with the above-mentioned washing solution, a reaction substrate was added to develop a color, and the measurement was performed after adding a stop solution. As a control, a healthy person was measured (Table 4).

[0030]

[Table 4]

[0031]

According to the present invention, it has become possible to easily carry out the separation, recovery and concentration of a virus, which has conventionally been troublesome and troublesome. According to the method of the present invention, a virus sample can be prepared in a research institution or a medical facility without requiring special equipment, thereby greatly contributing to the diagnosis, treatment and prevention of viral diseases through virus research and antigen testing and genetic testing. it can.

Claims (11)

[Claims] 1. A method for separating and recovering a virus, comprising: a step of causing a lipoprotein-binding / precipitating substance to act on a material containing a virus; and a step of solubilizing the precipitated substance.
Concentration and sample preparation methods. 2. The method according to claim 1, wherein a precipitation promoting substance is added. 3. The method according to claim 1, wherein a divalent metal ion is used as the lipoprotein-binding / precipitating substance. 4. The method according to claim 1, wherein a polyanionic substance is used as the precipitation promoting substance. 5. The method according to claim 1, wherein a polysaccharide sulfate is used as the precipitation promoting substance. 6. The method according to claim 1, wherein heparin is used as a precipitation promoting substance. 7. The method according to claim 1, wherein dextran sulfate is used as a precipitation promoting substance. 8. The method according to claim 1, wherein the divalent metal ionic substance is removed for solubilization. 9. The method according to claim 1, wherein the compound is solubilized by the action of potassium bromide or sodium bromide. 10. The method according to claim 1, wherein NaHCO ₃ is allowed to act to solubilize. 11. The method according to claim 1, wherein the solubilization is effected by the action of a citric acid solution.