JP2006204201A - Method for separation/concentration and recovery/sample preparation of virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for preparing a sample for measurement of viruses. <P>SOLUTION: This method for preparing the sample for the measurement of the viruses includes (1) a process for bringing a specimen which has a possibility of containing the viruses of a measurement object into contact with divalent metal ions, together with water-insoluble particles having an ampholytically charged substance which has a molecular weight of ≥70,000 on their surfaces, so as to form an aggregated material comprising the viruses of the measurement object, the divalent metal ions, and the water-insoluble particles having the ampholytically charged substance, (2) a process for separating a treated material of the process (1) into the aggregated material and a liquid, so as to recover the aggregated material, and (3) a process for disaggregating the aggregated material recovered in the process (2), by removing the metal ions, so as to recover the viruses which are concentrated. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for preparing a virus sample suitable for the detection or measurement method when detecting or measuring the virus by an immunological measurement method, a nucleic acid amplification reaction (NAT), or the like. Including the step of recovering.

  It is widely known that viruses cause various diseases and illnesses against humans, animals and plants. Therefore, confirmation of the causative virus is very important to establish prevention, diagnosis and treatment for diseases and diseases. 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.

  The most common conventional virus testing / diagnosis methods include immunological measurement of viral antigens and antiviral antibodies. However, these measurements require the presence of a certain amount of viral antigen or antibody. However, there are many cases where antigen tests cannot be used for diagnosis because the amount of virus is small and below the measurement sensitivity despite being in an infectious state. Virus propagation methods using cultured cells have been established for some viruses, but this method of separating and recovering viruses for use in measurement samples is often not applicable. Even in the case where the above method is established, special facilities, equipment, and techniques are required for separation and recovery, and it cannot be generally said that the method can be easily used.

  In recent years, a method for amplifying a gene has been developed, and a method for detecting even a trace amount of virus has been opened by amplifying a viral gene. However, even in this method, complex and sophisticated measurement techniques are required to measure a very small amount of virus, and it is extremely difficult to carry out easily in a general facility. Therefore, a technique for concentrating the virus in the specimen is used to solve the problem.

  A typical example of the conventional virus concentration method is an ultracentrifugation method. However, it requires an expensive instrument and a long time, and it is difficult to process a large number of specimens at the same time. Some viruses do not precipitate depending on the time of infection. Due to the property that hepatitis B virus surface antigen (HBs antigen) binds to heparin, a chromatographic method using heparin sepharose carrier has also been reported, but it is also difficult to process a large number of specimens at the same time and the recovery rate is poor. .

  In addition, ammonium sulfate, polyethylene glycol, a combination of a polyanion and a divalent ion, for example, a granular material having an acidic group (JP-B-6-22627) or a method using particles and a divalent metal (JP-A-62-2172536), There is a method of precipitating the virus by adding a water-soluble polymer substance having a cationic group and separating the virus (Japanese Patent Laid-Open No. 4-342536), etc., but a large amount mixed in the reagent to be mixed and the virus to be separated Due to problems such as the presence of NAT inhibition in proteins and the like, there has been a problem that sample purification after virus precipitation separation is necessary.

  In order to solve these problems, a virus concentration method using anionic magnetic particles and divalent metal ions (Japanese Patent Laid-Open No. 13-258551), and a virus concentration method using cationic magnetic particles (Japanese Patent Laid-Open No. 13-299337 and Japanese Patent Laid-Open No. 13-299337). 2003-274942) and the like, there is a method of concentrating virus together with magnetic particles, but when extracting virus nucleic acid from the condensate, if magnetic particles are mixed in the presence of a protein denaturant, it is Adsorbs and cannot be dissociated. In addition, there are problems such as destruction of the magnetic particles themselves, NAT inhibition due to the dissociation of free metal ions and proteins bound to the magnetic particles and mixing with viral nucleic acids, and depending on the virus type, differences between samples may occur. There was a problem that there was instability of concentration due to.

Japanese Patent Publication No. 6-22627 JP-A-6-2172536 JP-A-4-342536 Japanese Patent Laid-Open No. 13-258551 JP-A-13-299337 JP 2003-274492 A

  Accordingly, the present invention solves the problems of the virus concentration method described above, can easily process a large number of samples simultaneously by simple means, can easily cope with automation, It is an object of the present invention to provide a virus separation / concentration recovery / sample preparation method that does not adversely affect nucleic acid amplification tests. More specifically, the present invention forms a virus-containing aggregate in a high yield from a specimen containing the virus to be measured, and then uses this aggregate to perform subsequent viral amplification steps such as immunological measurement methods and NAT. Provided is a method for extracting and recovering viral nucleic acid with high yield without interfering contaminants.

  As a result of various studies to solve the above-mentioned problems, the present inventor has obtained a specimen that may contain the virus to be measured in the above-described virus aggregation step, a divalent metal ion, and a molecular weight of 50,000 to 100,000 on the surface. By bringing into contact with the water-insoluble particles having the above positively charged substance, aggregates of the virus to be measured, the divalent metal ions, and the water-insoluble particles having the positively charged substance can be formed efficiently. If there is a variation in the recovery of the virus to the aggregate depending on the specimen, such as hepatitis B virus, the antibody against the surface protein of the virus to be measured such as hepatitis B virus can coexist. We found that the virus can be recovered efficiently. It was also found that by coexisting with a monovalent metal ion, the effect of bilirubin is suppressed and the virus can be recovered.

  Further, the present inventor further recovered the virus nucleic acid from the above-mentioned aggregate by removing free metal ions and proteins by washing operation and performing virus-bound metal ion removal treatment. It has been found that a sample for measurement can be prepared without interfering with amplification of proteins and nucleic acids, such as biological measurement methods and NAT methods.

Therefore, the present invention provides a method for preparing a sample for virus measurement,
(1) By contacting a sample that may contain a virus to be measured with water-insoluble particles having a divalent metal ion and a positively charged substance having a molecular weight of 50,000 to 100,000 on the surface, Forming aggregates of divalent metal ions and water-insoluble particles with positively charged substances;
(2) Separating the processed product of the step (1) into the aggregate and liquid to collect the aggregate,
(3) The aggregate collected in the step (2) is freed of metal ions and proteins mixed in a free state by a washing operation; and (4) The aggregate collected in the step (3) is removed. Recovering concentrated virus by deaggregation by removal of metal ions;
A method comprising the steps.

  In the formation of aggregates containing viruses, it is particularly preferable for viruses such as hepatitis B virus that vary in the recovery of viruses to aggregates to further contain antibodies and monovalent metal ions against the target virus surface antigen. The positively charged substance is preferably a polyamino acid, more preferably polylysine. The divalent metal ion is preferably a zinc ion or a copper ion, and particularly preferably a zinc ion. The water-insoluble particles are preferably magnetic metal particles, and the separation / recovery step is preferably performed by magnetism or centrifugation, particularly preferably by magnetism.

  It is preferable to remove the free metal ions and proteins by a washing step. The removal of the metal ions is preferably performed by adding a metal chelating agent. A preferable virus to be measured in the present invention is, for example, hepatitis B virus (HBV). The method of the present invention is particularly preferred when preparing a viral nucleic acid for a template when the target viral nucleic acid is to be propagated by the nucleic acid amplification reaction method (NAT method).

Virus samples can be prepared in research institutions, laboratories and medical facilities without the need for special equipment, which can contribute to the diagnosis, treatment and prevention of viral diseases through virus research, antigen testing and genetic testing.
Next, the present invention will be described more specifically.

Viruses and specimens to which the present invention is applied The method of the present invention can be applied to various viruses, such as hepadnavirus (such as hepatitis B virus), adenovirus, flavivirus (such as Japanese encephalitis virus). ), Herpesvirus (herpes simplex virus, varicella-zoster virus, cytomegalovirus, EB virus, etc.), pox virus, parvovirus (adeno-related virus, etc.), orthomyxovirus (influenza virus, etc.), rhabdovirus (rabies virus) Etc.), retroviruses (acquired immunodeficiency syndrome virus, etc.), hepatitis C virus, etc.

However, the present invention is particularly useful when detecting or measuring multiple viruses simultaneously including hepatitis B virus (HBV) or hepatitis B, such as hepatitis B virus (HBV), hepatitis C virus (HCV) and human. It is particularly preferred when immunodeficiency virus (HIV) is detected or measured simultaneously.
The type of sample to which the method of the present invention is applied is not particularly limited, but is a blood sample, serum, plasma, and the like when screening a large number of samples such as a blood sample collected by blood donation and a clinical sample collected from a patient. Or other various body fluids are mentioned.

Water-insoluble particles In the present invention, as the water-insoluble particles serving as the nucleus of virus aggregate formation, polymer materials such as various particles formed from polymer materials and metal particles can be used. In order to easily collect the particles by magnetism, magnetic particles are preferable. For example, various ferrites such as triiron tetroxide, (Fe ₃ O ₄ ), γ-heavy ferric oxide (γ-Fe ₂ O ₃ ), etc., metals such as iron, manganese, cobalt, chromium, or alloys of these metals Etc. can be used. The particle size of the particles is usually 0.08 μm to 300 μm, preferably 0.1 μm to 100 μm. If the particle size is less than 0.8 μm, it is necessary to apply strong magnetism for a long time when the aggregate is separated from blood or body fluid by magnetism, and the aggregate cannot be recovered sufficiently. is there. When separating the aggregates by centrifugation, there is a problem in operability, such as requiring treatment for several minutes at a high rotation speed (5,000 rotations) of the centrifuge. On the other hand, when the particle diameter exceeds 300 μm, there is a problem that the surface area per particle volume becomes small and the trapping efficiency of virus aggregates decreases.

The positively charged substance attached to the surface of the positively charged substance particles is preferably a polyamino acid, and examples of the polyamino acid include polylysine, polyarginine, polyhistidine, and the like, and polylysine is particularly preferable. The molecular weight of the polyamino acid needs to be 50,000 to 100,000 or more, and if it is outside this range, it is difficult to sufficiently capture and aggregate the virus. In order to bind a polyamino acid such as polylysine to the particle surface, a carboxyl group is provided on the surface of the water-insoluble particle, for example, a magnetic metal or metal oxide particle or a polymer particle, and between the terminal amino group of the polyamino acid. An amide bond is formed on the particle surface, or an amino group is provided on the particle surface, and an amide bond is formed between it and the terminal carboxyl group of the polyamino acid. Here, the molecular weight of the polylysine and the carboxyl group are particularly affected. The number of particles per particle is important, and methods such as 12-30 per particle are optimal. The formation of the amide bond may be performed according to a conventional method of organic chemistry.

Examples of the divalent metal ion for aggregating the divalent metal ion magnetic particles and the virus include zinc ion (Zn ++) and copper ion (Cu ++), with zinc ion being preferred. This is because the optimum type of divalent metal ion differs depending on the virus, but zinc ion and copper ion can be commonly used for various viruses. The concentration of divalent ions in the reaction mixture for forming the virus-containing aggregate is 20 mM to 100 mM, preferably about 25 mM to 35 mM. This divalent metal ion may be added to the specimen simultaneously with the water-insoluble particle for virus supplementation, but it is preferable to first add the water-insoluble particle for virus supplementation and then add the divalent metal ion.
This is because the capture efficiency increases .

Antiviral antibody treatment and monovalent metal ion treatment In the presence of divalent metal ions, when viruses are aggregated and captured on the surface of particles having positively charged substances on the surface, many viruses depend on the amount of virus in the sample. However, certain viruses, such as hepatitis B virus (HBV), may cause variations in the amount of virus that is aggregated and captured, which is an obstacle to the measurement of viral load. It was. As a result of pursuing this cause, the present inventor has clarified that bilirubin and milk cake present in the specimen inhibit virus aggregation. For bilirubin, HBV-containing specimens that do not contain bilirubin were artificially added with various concentrations of bilirubin to prepare a plurality of specimens having different bilirubin concentrations, and these were aggregated under the same conditions. It was confirmed that the amount of virus aggregation / capture decreased as the concentration of bilirubin increased.

  Next, as a result of investigating a method for eliminating the inhibition of virus aggregation / capture by bilirubin or chyle as described above, an antibody against the surface antigen of the virus to be measured, for example, HBV is detected and measured. It was experimentally confirmed that by adding an antibody against the surface protein in the aggregation concentration step, the inhibition of aggregation / capture due to bilirubin or milk cake was eliminated. It was also confirmed that the inhibition of aggregation and capture by bilirubin was eliminated by the coexistence of monovalent metal ions. Therefore, when the method of the present invention is applied to detection and measurement of HBV virus, or simultaneous detection and measurement of a plurality of viruses including HBV, for example, simultaneous detection and measurement of three types of viruses of HBV, HCV and HIV. It is highly preferable to add an antibody against the surface protein of HBV in the aggregation / capture step. The preparation of an antibody against the HBV surface protein (HBs antibody) can be carried out according to a conventional method and does not constitute a feature of the present invention. Since it is difficult to obtain a sufficient amount of human HBs antibody for use as a reagent, it is preferable to use a non-human HBs antibody, such as a horse HBs antibody.

Washing of concentrated and recovered virus aggregates When viruses are aggregated and captured on the surface of particles with positively charged substances in the presence of divalent metal ions, many viruses depend on the amount of virus in the sample. However, depending on the specimen, there may be some variation in the measurement results of the viral load. As a result of pursuing this problem, it has been found that the problem arises as a result of free metal ions, proteins and the like being mixed into the aggregate. In addition, it was experimentally confirmed that the amount of protein and metal ion inhibited nucleic acid extraction in the NAT method.

  Next, as a result of examining a method for eliminating viral nucleic acid extraction inhibition by free metal ions and proteins as described above, the virus aggregates were washed, and excess metal ions and proteins were washed away to inhibit extraction. It became possible to eliminate. Therefore, when the method of the present invention is applied to simultaneous detection and measurement of a plurality of viruses containing HBV, for example, three viruses, HIV.HCV and HIV, the washing treatment of the aggregates is very difficult in the capture / extraction process. preferable.

Virus aggregation / capture treatment The virus aggregation / capture step can be performed as follows. When 1 mL to 5 mL of a specimen is prepared and an antibody against the surface protein of the detection / measurement target virus is added thereto, 25 to 100 μL of this antibody (for example, horse purified HBs antibody) is first added and mixed, and then 20 to 20 Let stand for 30 minutes. Next, particles having positively charged substances such as poly-L-lysine bound to the surface, such as magnetic particles, are added to a concentration of 1 mg to 5 mg / mL, and then a solution of divalent metal ions such as zinc ions at a concentration of 1.1 M is added. Add 30-150 μL, mix well, and allow the mixture to stand for about 5 minutes to form aggregates.

Separation of Aggregate Next, the mixture containing the aggregate formed as described above is separated into an aggregate and other liquid, and the aggregate is recovered. Thereby, blood components such as serum and plasma contained in the liquid and other various contaminants present in the specimen are removed. When the particles used are magnetic particles, the mixture containing the aggregates is allowed to settle on the magnetic stand by magnetically attracting the aggregates containing the magnetic particles. Can be done. Moreover, even if the particles are magnetic particles or non-magnetic particles, the aggregate and other liquid can be separated by centrifugation.

Washing the aggregate Next, the aggregate obtained as described above contains free metal ions and proteins, and other contaminants in addition to the target virus. It becomes an obstacle to extraction of certain viral nucleic acids. For this reason, it is necessary to remove excess metal ions and proteins from the aggregate. At this time, depending on the cleaning solution, dissociation of viruses and the like, collapse of magnetic particles, and the like occur, resulting in a decrease in concentration. For this reason, in the present invention, it is necessary to wash the aggregate with a washing solution that does not cause virus dissociation.

  As the washing solution, physiological saline, Tris buffer solution, and phosphate buffer solution are used, but phosphate buffer solution is preferable from the viewpoint of effect, and phosphate buffer solution having a phosphate concentration of 2 mM is particularly preferable.

Separation of viral nucleic acid from aggregate Next, the aggregate obtained as described above contains particles that became the core of the aggregate, and other contaminants, in addition to the target virus, These may interfere with nucleic acid amplification, typically the polymerase chain reaction (PCR), for the next step, detection and measurement of viral nucleic acids. For this reason, it is necessary to extract and separate the viral nucleic acid from the aggregate. At this time, if too strong extraction conditions are used, the magnetic particles may collapse, and this collapsed product may interfere with the nucleic acid amplification reaction. For this reason, in the present invention, the aggregate is disrupted or loosened by deaggregating by removing the divalent ions added to promote the formation of the aggregate in the initial stage.

  For this purpose, metal chelating agents, citrates, potassium bromide, sodium bromide and the like are used as divalent ion removing agents, and metal chelates are preferred from the viewpoint of effects. As the metal chelating agent, ethylenediaminetetraacetic acid (EDTA), EGTA, and the like can be used. The amount of the metal chelating agent used in this case is an amount sufficient to supplement the amount transferred to the aggregate among the initially added divalent ions. The above method (starting from 1 to 5 mL of the sample) In the method), 100 μL of a 0.1M to 0.2M concentration metal chelating agent solution may be added. Next, the aggregate and the metal chelator solution are mixed well.

Next, when the first used particles are magnetic particles, the above mixture can be left on a magnetic stand for 5 to 10 minutes to precipitate particles by magnetic suction, thereby obtaining a supernatant sample containing virus. .
In order to extract viral nucleic acid from the above supernatant, the virus can be digested with a proteolytic enzyme such as proteinase K in the presence of SDS, and then the nucleic acid can be precipitated and recovered by ethanol precipitation. The particle separation and the nucleic acid extraction may be performed in the reverse order.

Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1 . Preparation of magnetic particles coated with polylysine (1) Use of carrier COOH magnetic beads
125 mg (2.5 mL) of COOH magnetic beads were collected, and the particles were collected with a magnet and washed with shaking in 5 mL of 0.1 M 2-morpholinoethanesulfone (?) (MES) solution (PH5.0) for 10 minutes. The beads are suspended in a coupling buffer (1.2 mL distilled water, 5 mL 100 mM MES (PH5.0) solution, 50 μL 100 mg / mL poly-L-lysine solution) at a concentration of 25 mg beads / mL, This suspension was mixed by inversion for 15 minutes at room temperature.

  To this, 1.25 mL of 1-ethyl-3- (3-dimethyl-aminopropyl) -carbodiimide (EDC) aqueous solution was added and mixed by inversion at 10 ° C. for 20 hours. The upper solution was replaced with 1M monoethanolamine solution and blocked at 4 ° C. overnight. The beads were washed 5 times with phosphate buffer (PBS), suspended in PBS to 50 mg beads / mL, and stored at 4 ° C.

(2) Use of carrier M-270 epoxy magnetic beads
600 mg of washed beads are suspended in coupling buffer (12 mL 0.2 M Na ₂ CO ₃ (pH 10), 12 mL 3 M ammonium sulfate, 11.64 mL water, 360 μL 100 mg / mL poly-L-lysine), 35 Inverted at 24 ° C. for 24 hours. The beads were collected with a magnet, suspended in 18 mL of 1 M ethanolamine solution (pH 8.0), and blocked at 10 ° C. overnight. The mixture was mixed by inverting at 35 ° C. for 1 hour and washed 5 times with PBS. The suspension was suspended in PBS to 50 mg beads / mL and stored at 4 ° C.

Example 2 . Virus concentration method using 1 ml of specimen 25 μL of purified equine HBs antibody was added to 1 ml of specimen and mixed for 20 minutes at 10 minute intervals. Next, the poly-L-lysine-coated magnetic particles prepared in (1) of Experimental Example 1 were added at a concentration of 1 mg / mL. To this was added 30 μL of 1.1 M zinc acetate solution, mixed and allowed to stand for 5 minutes to form aggregates containing virus and magnetic particles. By allowing this mixture to stand on a magnetic stand for 5 minutes, aggregates containing magnetic particles and viruses were precipitated, and liquids containing plasma and serum components were removed. 1 mL of 2 mM PBS was added to the obtained aggregate, vortexed and washed, and allowed to stand on a magnetic stand after washing for 5 minutes to precipitate virus aggregates and remove the washing solution. Further, 1 mL of PBS was added on the magnetic stand and washed again. 50 μL of 0.4 M EDTA solution was added to the obtained aggregate and mixed.

EX.R & D Extraction Next, the treated product was allowed to stand on a magnetic stand, the supernatant was collected, and 500 μL of a mixture of a sample diluent 480 μL, an enzyme solution 20 μL, and a coprecipitation agent 5 μL was added for extraction.

Example 3 . Virus concentration method using 5 mL sample 100 μL of purified equine HBs antibody was added to 5 mL sample and mixed for 20 minutes at 10-minute intervals. Next, the poly-L-lysine coated magnetic particles prepared in (2) of Example 1 were added at a concentration of 5 mg / mL. To this, 150 μL of 1.1 M zinc acetate solution was added, mixed and allowed to stand for 5 minutes to form aggregates containing virus and magnetic particles. By allowing this mixture to stand on a magnetic stand for 5 minutes, aggregates containing magnetic particles and viruses were allowed to settle, and liquids containing plasma and serum components were removed. 1 mL of 2 mM PBS was added to the obtained aggregate, vortexed and washed, and after washing, the virus aggregate was allowed to settle by allowing it to stand on a magnetic stand for 5 minutes, and the washing solution was removed. Further, 1 mL of PBS was added on the magnetic stand and washed again. 50 μL of 0.4 M EDTA solution was added to the obtained aggregate and mixed.

EX.R & D Extraction Next, the treated product was allowed to stand on a magnetic stand, the supernatant was recovered, and extraction was performed by adding 500 μL of a sample dilution solution of 465 μL, an enzyme solution of 30 μL, and a coprecipitation agent of 5 μL.

Example 4 . Concentration of HBV, HCV and NIV Samples prepared in three stages of x1 , x10 and x100 were artificially prepared, and the method described in Example 2 (however, horse HBs antibody was not added) After concentration, the obtained viral nucleic acid sample was amplified by PCR. The results are shown in FIG. In this figure, the black bars indicate the results of concentration by the method of the present invention, and the white bars indicate the results of samples that have not been concentrated. For any virus, the detection sensitivity was improved about 10 times by the concentration method of the present invention. However, as shown in Example 5 and later, HBV has a problem in reproducibility.

Example 5 Reproducibility of concentration
For HBV and HCV, the enrichment method of Example 4 was repeated 5 times. The results are shown in FIG. As is clear from this figure, HCV concentration was highly reproducible, but HBV concentration was poorly reproducible.

Example 6 . Aggregation rate Therefore, in order to investigate the cause of the low reproducibility in the concentration of HBV, in the method described in Example 2 (however, no horse HBs antibody was added), the incubation time for the formation of the aggregate was changed. The virus capture rate was observed. The results are shown in FIG. HCV was captured almost instantaneously, whereas HBV was found to have a lower capture rate. The specimen used was a virus dispersed in 6% bovine serum albumin in phosphate buffer.

Example 7 . Inhibition of aggregation by bilirubin Further, in order to investigate the cause of low reproducibility in concentration of HBV, in the method described in Example 2, bilirubin F, bilirubin C, hemolyzed hemoglobin, or milk cake was added in the aggregation process, The capture rate of the virus was observed by changing the incubation time for aggregate formation. The result is shown in FIG. As is clear from this result, it has been clarified that bilirubins inhibit aggregation most greatly. In this experiment, 6% bovine serum albumin in phosphate buffer added with virus and inhibitor was used.
Example : Recovery of inhibition of extraction by washing method In the method described in Example 1.2, the type and concentration of the washing liquid were further examined and measured for the case of washing and the case of no washing. Table 00 shows the cleaning effect. As is clear from this table, it was found that PBS washing is good when washing, and 2 mM PBS washing can best avoid the inhibition of extraction, and can be eliminated by washing the extraction inhibition.

Example 8 . When anti-HBs antibody that inhibits aggregation by bilirubin was added and when horse HBs antibody was not added, bilirubin was added at various concentrations, and the amount of virus aggregation / capture was measured. The horse HBs antibody concentration was 4 μ / ml. FIG. 5 shows the relationship between the relative concentration of bilirubin and the relative amount of virus aggregation / capture. As is apparent from this figure, when anti-horse HBs antibody was added, aggregation inhibition did not occur regardless of the concentration of bilirubin, but when anti-horse HBs antibody was not added, it was dependent on the amount of bilirubin added. Aggregation inhibition occurred. As a result, it was clarified that the aggregation / capture inhibition in the virus concentration step is caused by bilirubin in the specimen, and the aggregation / capture inhibition by bilirubin can be eliminated by adding an anti-HBs antibody.

Example 9 . Stabilization of aggregation / capture The method of Example 8 was performed, but three types of specimens containing HBV were concentrated in the presence and absence of anti-horse HBs antibody. The results are shown in FIG. As is apparent from this figure, aggregation / capture was stable in the presence of anti-horse HBs antibody, and there was no variation.

Example 10 . Example of results of Example 2 (1)
For specimens (x1, x10, or x100) containing HBV or HCV at three concentrations, virus was concentrated by the method described in Example 2, and the concentrated virus was amplified by PCR. The results are shown in FIG. In this figure, the black bar shows the result of PCR amplification of a 0.1 ml sample obtained by concentrating 1 ml of the specimen 10 times, and the white bar shows the result of PCR amplification of the 0.1 ml specimen in the same manner.
As is clear from FIG. 7, not only HCV but also HBV, the virus detection sensitivity increased by the concentration method of the present invention.

Example 11 Example of results of Example 2 (2)
Four types of specimens (A, B, C, and D) containing HBV are diluted to three kinds of concentrations (dilution multiples x10, x100, and x1000), and 1 ml of these diluted specimens is used in the method of Example 2 And 0.1 ml of the concentrate was PCR amplified. As a control, 0.1 ml of an unconcentrated diluted sample was concentrated by PCR. The results are shown in Table 1 below.

表１から明らかな通り，本発明の濃縮方法により、HBVが約１０倍の感度で検出・測定できた。

As is clear from Table 1, HBV could be detected and measured with a sensitivity of about 10 times by the concentration method of the present invention.

Example 12 . Example results for Example 3
Four types of specimens (A, B, C, and D) containing HBV are diluted to three kinds of concentrations (dilution multiples x10, x100, and x1000), and 5 ml of these diluted specimens are used in the method of Example 3 And 0.1 ml of the concentrate was PCR amplified. As a control, 0.1 ml of unconcentrated diluted specimen was PCR amplified. The results are shown in Table 2 below.

表2から明らかな通り、本発明の濃縮方法により、HBVが約50倍の感度で検出・測定できた。

As is apparent from Table 2, the concentration method of the present invention was able to detect and measure HBV with about 50 times the sensitivity.

FIG. 1 is a graph showing the results of concentrating HBV, HCV and HIV by the method of the present invention. In this figure, HBV is also concentrated, but as shown in FIG. 2, there is variation in the concentration of HBV. FIG. 2 is a graph showing that the capture of HBV is unstable. FIG. 3 is a graph showing that the capture speed and capture rate of HBV are low. FIG. 4 is a graph showing that the inhibitor against HBV supplementation is bilirubin. FIG. 5 is a graph showing that inhibition of HBV capture by bilirubin is released by anti-HBs antibody. FIG. 6 is a graph showing the effect when a 1 ml sample is concentrated to 0.1 ml. FIG. 7 is a graph showing the relationship between the dilution rate and the PCR count.

Claims (11)

In the method for preparing a sample for virus measurement,
(1) By contacting a specimen that may contain a virus to be measured with water-insoluble particles having a divalent metal ion and a positively charged substance having a molecular weight of 50,000 to 100,000 on the surface, Forming aggregates of divalent metal ions and water-insoluble particles with positively charged substances;
(2) Separating the processed product of the step (1) into the aggregate and liquid to recover the aggregate; and (3) freeing the metal ion by washing the aggregate recovered in the step (2). And remove protein, etc.
(4) The aggregate collected in the step (3) is deaggregated by removing metal ions,
Recovering concentrated virus;
A method comprising steps. In the step (1), an antibody against the virus surface antigen to be measured is further present.
The method of claim 1.   The method according to claim 1, wherein the positively charged substance is a polyamino acid.   4. The method of claim 3, wherein the polyamino acid is polylysine.   The method according to any one of claims 1 to 4, wherein the divalent metal ion is a zinc ion or a copper ion.   The method according to claim 1, wherein the water-insoluble particles are magnetic metal particles.   The method according to claim 1, wherein the separation / recovery step is performed by magnetism or by centrifugation.   The method according to any one of claims 1 to 7, wherein the removal of the free metal ions and proteins is performed by adding a washing solution.   The method according to claim 1, wherein the removal of the metal ions is performed by adding a metal chelating agent.   The method according to any one of claims 1 to 9, wherein the virus to be measured is hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV).   The method according to any one of claims 1 to 9, wherein the measurement is a polymerase chain reaction method (PCR method) or a nucleic acid amplification reaction (NAT) and an immunological measurement method (EIA method or the like).

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