JP2023150351A - Virus concentrator material - Google Patents

Abstract

To provide a virus concentrator material which can easily concentrate viruses to enhance detection sensitivity.SOLUTION: A virus concentrator material according to an aspect of the present invention comprises a cellulose membrane mainly composed of α-cellulose, and an antibody against a target virus immobilized on the cellulose membrane. The cellulose membrane has a pair of adsorption surfaces configured to allow the antibody to adsorb the target virus.SELECTED DRAWING: Figure 1

Description

The present invention relates to a virus concentration material capable of concentrating viruses in a liquid to be tested, a method for manufacturing the same, a virus concentration kit using the same, a virus test kit, a virus concentration method, and a virus test method.

In order to diagnose and treat infectious diseases caused by viruses, samples are collected from patients and tested for viruses. Furthermore, the detection of viruses that exist in natural environments such as in the air, water, and soil, as well as in artificial environments such as indoor air and hard surfaces of buildings, is also useful for identifying the source of infection by viral infections and Virus testing is important in predicting the risk of infectious diseases caused by viruses, and in this case, virus testing is performed using samples collected from each environment. Viral tests that can determine the presence or absence of infection and the presence of viruses include genetic tests that detect virus genes (nucleic acids) from specimens, and virus-specific proteins that are detected from specimens using virus-specific antibodies. Examples include antigen tests for detection.

On the other hand, each testing method has a detection limit, and it becomes difficult to detect a small amount of virus below the detection limit. As a way to increase the detection sensitivity of virus tests, attempts have been made to concentrate viruses in specimens. For example, Patent Document 1 describes a virus concentration material having an antibody against a target virus on its surface.

Japanese Patent Application Publication No. 2002-78485

In recent years, there has been an urgent need to prevent the spread of infectious diseases caused by viruses such as COVID-19. Along with this, there is a growing demand for highly sensitive virus detection from a large number of samples, including samples from early stages of infection and asymptomatic patients. Therefore, there is a need for a technology that can easily and quickly concentrate viruses at a higher concentration rate than the material for virus concentration described in Patent Document 1, and increase the detection sensitivity of viruses.

The present invention relates to a virus concentration material that can easily concentrate viruses and increase detection sensitivity, a method for producing the same, a virus concentration kit, a virus test kit, a virus concentration method, and a virus test method using the same. .

The virus concentration material according to one embodiment of the present invention is
A cellulose membrane whose main component is α-cellulose,
an antibody against the target virus immobilized on the cellulose membrane;
Equipped with
The cellulose membrane has a pair of adsorption surfaces each configured to be able to adsorb the target virus using the antibody.
Other features of the invention will be apparent from the claims and the following description.

According to the virus concentration material of the present invention, it is possible to easily concentrate viruses and increase detection sensitivity.

1 is a schematic plan view showing a virus concentration material according to an embodiment of the present invention, and an enlarged view enlarging a part thereof. It is a sectional view of the above-mentioned virus concentration material. FIG. 2 is a schematic diagram for explaining the principle of virus concentration using the virus concentration material of the present invention. FIG. 2 is a flow diagram showing a method for manufacturing a virus concentrate material according to an embodiment of the present invention. (A) is a flow diagram showing a virus concentration method according to one embodiment of the present invention, and (B) and (C) are flow diagrams each showing a virus concentration method according to other embodiments. It is a typical diagram for explaining filtration in the above-mentioned virus concentration method. (A) is a flow diagram showing a virus testing method according to one embodiment of the present invention, and (B) is a flow diagram showing a virus testing method according to another embodiment. FIG. 1 is a schematic diagram showing a virus concentration kit and a virus test kit including a virus concentration material according to an embodiment of the present invention. FIG. 3 is a schematic diagram showing a virus concentration kit and a virus testing kit including a virus concentration material according to another embodiment of the present invention. It is a graph showing the results according to Test Example 1 of the present invention, in which the amount of N protein derived from SARS-CoV-2 was detected by Western blotting before and after adding a virus concentration material to the SARS-CoV-2 solution and shaking it. It is a figure showing a result. It is a graph showing the results according to Test Example 2 of the present invention, in which the amount of N protein derived from SARS-CoV-2 was detected by Western blotting before and after filtering the SARS-CoV-2 solution using a virus concentration material. It is a figure showing the result.

In this specification, when numerical limits or ranges are described, the endpoints thereof are included. In addition, all values and subranges within a numerical limit or range shall be specifically stated, as if written out explicitly.
In this specification, nouns written in the singular shall include the meaning of "one or more."
Obviously, many modifications and variations of the present invention are possible in light of the following teachings. It is therefore understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
All patent documents and references mentioned herein are fully incorporated by reference into this specification, as if set forth at length.

[Virus concentrate material]
The virus concentration material of the present invention is a material that can easily concentrate a target virus in a liquid to be tested for virus testing and can increase detection sensitivity.
The virus test of the present invention includes an antigen test and a genetic test. Antigen tests are tests that detect proteins or protein fragments possessed by viruses. Genetic testing is a test that detects viral genes (nucleic acids).
In addition, in this specification, a "virus test" is also simply called a "test."
Furthermore, in this specification, the "target virus" is also simply referred to as "virus."
The "liquid to be tested" in the present invention is a liquid to be tested for viruses, and refers to a liquid sample collected from a test subject or a liquid containing a sample. The "inspection object" may be a part of a living body such as a human (subject) or a domestic animal, or a non-living body. Non-living objects to be tested include a wide range of objects that may be contaminated with viruses, such as doorknobs, desks, toilets, equipment operation panels, input devices connected to computers, switches, solid objects such as floors, walls, etc. This includes artificial objects, sewage, river water, soil, indoor or outdoor air, etc.
In the present invention, "concentration" refers to an operation for producing a virus-containing liquid with a higher virus concentration from a liquid to be tested. Specifically, it is an operation to collect viruses in the liquid to be tested into the virus concentration material of the present invention. and adsorption operations.
In the present invention, "adsorption" of a virus means selectively binding the virus and/or its fragments. Viral fragments include, for example, viral proteins.

For example, the virus concentration material of the present invention can be brought into contact with a liquid to be tested for virus testing to adsorb viruses. The method of contact between the virus concentration material and the liquid to be tested is not particularly limited. For example, the virus concentration material may be immersed in the liquid to be tested, or may be shaken while being immersed. Alternatively, the virus concentration material may be used as a filter for filtering the liquid to be tested. Alternatively, the target virus in the test liquid may be concentrated by wiping the detection target to which the test liquid has adhered with a virus concentrating material. A specific concentration method according to the present invention will be described later.
After contacting the virus concentration material with the liquid to be tested, the virus concentration material is collected. Many viruses can be adsorbed to the collected virus concentration material by the configuration described below. By collecting viruses from this virus concentration material, a highly concentrated virus-containing liquid can be obtained, and detection sensitivity in virus testing can be increased.

In one embodiment of the present invention, the virus concentration material 1 includes a cellulose membrane 2 and an antibody 3 immobilized on the cellulose membrane 2. This example is shown schematically in FIGS. 1 and 2.
Note that in FIG. 2, hatching schematically shows the cross-sectional shape of the cellulose membrane 2, and does not indicate the orientation of the fibers.

(cellulose membrane)
The cellulose membrane 2 is the base material of the virus concentration material 1. The cellulose membrane 2 has α-cellulose as its main component.
"The cellulose membrane 2 contains α-cellulose as a main component" means that the cellulose membrane 2 contains α-cellulose in an amount of 50% by mass or more when the entire mass of the cellulose membrane 2 is 100% by mass. say. The content of α-cellulose in the cellulose membrane 2 is preferably 80% by mass or more, more preferably 90% by mass or more. By increasing the content of α-cellulose in the cellulose membrane 2, steps such as chemical modification of α-cellulose can be omitted and production efficiency can be increased. Furthermore, by simplifying the composition of the cellulose membrane 2, the manufacturing cost of the cellulose membrane 2 can be reduced. In addition, as shown in Examples described later, such a cellulose membrane 2 with a high content of α-cellulose can stably immobilize the antibody 3 and obtain a high concentration rate.

As illustrated in FIG. 1, the cellulose membrane 2 preferably contains fibers F. Furthermore, the cellulose membrane 2 is preferably an aggregate of fibers F formed into a membrane shape.
The fibers F are preferably fibers containing α-cellulose as a main component. Fibers containing α-cellulose as a main component refer to fibers containing 50% by mass or more of α-cellulose when the mass of the entire fiber F is 100% by mass. The content of α-cellulose in fiber F is preferably 80% by mass or more, more preferably 90% by mass or more.
Note that in the example shown in FIG. 1, the antibody 3 is immobilized on the surface of the fiber F.

From the viewpoint of increasing the concentration rate, the median fiber diameter of the fibers F of the cellulose membrane 2 is preferably 100 μm or less, more preferably 50 μm or less. Thereby, the specific surface area of the cellulose membrane 2 can be increased, and many antibodies 3 can be immobilized on the cellulose membrane 2. Therefore, the concentration performance of the virus concentration material 1 can be improved.
Moreover, from the viewpoint of increasing the strength of the cellulose membrane 2 and maintaining the structure of the cellulose membrane 2, the median fiber diameter of the fibers F of the cellulose membrane 2 is preferably 10 μm or more, preferably 20 μm or more.
The "fiber diameter" in the present invention means a circular equivalent diameter. The fiber diameter of the fibers can be determined by arbitrarily selecting 500 fibers from a two-dimensional image obtained by scanning electron microscopy (SEM), excluding defects such as fiber clumps and fiber intersections, and measuring the fiber diameter by a line perpendicular to the longitudinal direction of the fibers. It can be measured by directly reading the length when the fiber diameter is pulled. The median fiber diameter can be determined from the distribution of the 500 measured fiber diameters.

From the viewpoint of increasing the concentration rate, the cellulose membrane 2 has a pair of adsorption surfaces 2s each configured to be able to adsorb viruses by the antibodies 3. For example, the antibody 3 is fixed at least to the surface of the fiber F that constitutes the pair of adsorption surfaces 2s. By using both sides of the cellulose membrane 2 as adsorption surfaces 2s, the amount of virus adsorption can be increased.
Note that the antibody 3 is preferably immobilized on the fibers F inside the cellulose membrane 2 in addition to the fibers F forming the pair of adsorption surfaces 2s.
Further, by configuring the base material of the virus concentrating material 1 in a membrane shape, a wide adsorption surface 2s in one virus concentrating material 1 can be ensured. Thereby, the amount of virus adsorbed per sheet of virus concentration material 1 can be increased, and concentration processing for one sample can be performed using one or several sheets of virus concentration material 1. Therefore, preparation of the virus concentration material 1 during virus concentration and collection of the virus concentration material 1 after concentration can be easily performed.
The cellulose membrane 2 may be a single layer or a laminate. In the example shown in FIG. 2, the cellulose membrane 2 is a single layer.

The pair of suction surfaces 2s typically have the same planar shape. The planar shape of the suction surface 2s has any planar shape selected from a circle, an ellipse, or a similar shape, a polygonal shape or a similar shape, or other shapes. The cellulose membrane 2 may be used in an expanded state or in a deformed state by bending or the like.
From the viewpoint of convenient concentration, the planar shape of the adsorption surface 2s is preferably circular. Thereby, in plan view, the dimension from the center of gravity of the cellulose membrane 2 to the peripheral edge becomes approximately constant. Therefore, even when pressure is applied from the liquid to be tested, such as during shaking or filtering, unintended changes in shape of the peripheral portion can be suppressed. Furthermore, by making the planar shape of the suction surface 2s circular, the shape becomes suitable for a cylindrical or conical inspection instrument, and the concentration process can be performed efficiently.

The area of each of the pair of suction surfaces 2s can be set as appropriate depending on the amount of the liquid to be tested and the size of the instrument used. From the viewpoint of efficiently concentrating a small amount of the test liquid of about 0.5 to 2 mL, the area is preferably 7 mm ² or more, more preferably 19 mm ² or more, and even more preferably 78.5 mm ^{2 or} more. , preferably 8000 mm ² or less, more preferably 2000 mm ² or less, even more preferably 800 mm ² or less, even more preferably 300 mm ^{2 or} less. By setting within the above range, a sufficient area of the pair of adsorption surfaces 2s can be ensured, and a sufficient concentration ratio can be obtained.
Further, when the planar shape of the pair of suction surfaces 2s is circular, the diameter of these surfaces is, from the same viewpoint, preferably 3 mm or more, more preferably 5 mm or more, and preferably 50 mm or less, more preferably It is 30 mm or less.

It is preferable that the cellulose membrane 2 has flexibility from the viewpoint of improving handling properties. The term "flexible cellulose membrane" as used herein refers to a soft cellulose membrane that can be easily bent by hand. Specifically, the "flexible cellulose membrane" is a membrane having a stiffness equal to or lower than that of paperboard, and preferably has a Taber stiffness (JIS P8125:2000) of 30 mN·m or less, for example. Examples of flexible cellulose membranes include paper-like cellulose membranes, and more specifically, paper filters described below.
This makes it easier to grasp the periphery of the cellulose membrane 2 with a tool such as tweezers, making it easier to remove it from a package containing a plurality of virus concentration materials 1 and to recover the virus concentration material 1 from the liquid to be tested. Furthermore, by making the cellulose membrane 2 thin enough to have flexibility, a large adsorption surface can be ensured relative to the volume, and the concentration rate can be increased. Moreover, by configuring the cellulose membrane 2 thinly, it becomes easier to immerse it in the entire liquid to be tested, and a sufficient concentration ratio can be obtained. Furthermore, by configuring the cellulose membrane 2 thinly, the volume of the virus concentration material 1 can be reduced, and the space required for storage can be reduced.

On the other hand, it is preferable that the cellulose membrane 2 has a certain degree of strength in order to suppress unintended changes in shape and tearing during concentration. From the viewpoint of obtaining a cellulose membrane 2 with a good balance between thinness and strength, the basis weight of the cellulose membrane 2 is preferably 70 g/m ² or more, more preferably 80 g/m ² or more, and preferably 200 g/m ² or less, More preferably, it is 1500 g/m ² or less.
Note that the basis weight of the cellulose membrane 2 is measured as follows.
The cellulose membrane 2 to be measured is cut into a predetermined size to obtain small pieces for measurement. The weight of these small pieces is measured, and the calculated weight is divided by the area of each small piece to calculate the basis weight of the small piece. The average value of the basis weights of the five small pieces is taken as the basis weight of the cellulose membrane 2.

From the same viewpoint as the basis weight, the thickness of the cellulose membrane 2 is preferably 0.15 mm or more, more preferably 0.16 mm or more, and preferably 0.4 mm or less, more preferably 0.3 mm or less.
Note that the thickness of the cellulose membrane 2 is measured as follows.
Three cellulose membranes 2 are prepared as samples for measurement. Using a laser displacement meter (ZSLD80 manufactured by Omron Corporation), the thickness at three arbitrary locations in the sample is measured. The average value of all the measured thicknesses is defined as the thickness of the cellulose membrane 2.

As an example of a structure that has a good balance between thinness and strength and can satisfy the above thickness and basis weight ranges, the cellulose membrane 2 is preferably structured in a paper shape. The paper-like cellulose membrane 2 is manufactured by sticking fibers F together. In addition, from the viewpoint of suppressing tearing and dissolution when brought into contact with the liquid to be tested, "paper" in this specification does not include water-disintegrable paper such as toilet paper.
Thereby, appropriate flexibility and strength can be imparted to the cellulose membrane 2. Furthermore, since the cellulose membrane 2 can be made thinner than in a cotton-like structure, the storage space can be reduced. Furthermore, it becomes easier to take out the cellulose membranes 2 one by one, and convenience in use can be improved.

From the viewpoint of increasing the concentration rate, it is preferable that the cellulose membrane 2 includes fibers F and is also configured in a filter shape.
For example, as shown in the enlarged view of FIG. 1, by forming a plurality of holes P between fibers F, the cellulose membrane 2 is formed into a filter shape.
Thereby, the liquid to be tested flows into the pores P of the cellulose membrane 2, causing a flow of the liquid to be tested, thereby increasing the chance of contact between the virus in the liquid to be tested and the antibody 3. Therefore, the amount of virus adsorbed onto the cellulose membrane 2 can be increased, and the concentration rate can be increased.
Furthermore, since the antibody 3 is immobilized on the fiber F in contact with the hole P, when the test liquid flows into the hole P, the virus is easily adsorbed to the antibody 3. Such an action can also increase the amount of viruses adsorbed onto the cellulose membrane 2.

More specifically, it is more preferable that the cellulose membrane 2 is configured, for example, as a paper filter. Thereby, although it is in the form of a filter, it is possible to take advantage of the above-mentioned advantages of paper, and it is possible to obtain the cellulose membrane 2 that has sufficient strength when it comes into contact with the liquid to be tested.
Furthermore, it is more preferable that the cellulose membrane 2 is a filter paper for qualitative analysis (qualitative filter paper). This allows a sufficient amount of antibody 3 to be immobilized, making it easier to obtain a high concentration rate, as explained in Examples below. Moreover, the cellulose membrane 2 can be produced easily and inexpensively, and manufacturing costs can be reduced.

When the cellulose membrane 2 is configured in the shape of a filter, it is preferable to set the pore diameter to a certain degree from the viewpoint of increasing the chance of contact between the cellulose membrane 2 and the virus in the liquid to be tested. On the other hand, by densely arranging the fibers F, the surface area of the fibers F for immobilizing the antibody 3 can be increased.
As described above, from the viewpoint of obtaining a cellulose membrane 2 with a good balance between the pore size of the cellulose membrane 2 and the density of the fibers F, the retained particle diameter (particle retention capacity) of the cellulose membrane 2 is preferably 1 μm or more, more preferably 1.0 μm or more. It is 5 μm or more, more preferably 2 μm or more, preferably 50 μm or less, more preferably 40 μm or less, and still more preferably 30 μm or less.
The retention particle size is a value determined from the leakage particle size when a suspension of barium sulfate, etc. is naturally filtered as specified in JIS P 3801, and specifically, it is the particle size at which the retention efficiency is 98%. be. In other words, the retained particle diameter is a value that reflects the average pore diameter of the cellulose membrane 2. As shown in the Examples described below, in the present invention, even when the retained particle size is very large compared to the size of the virus, a sufficient concentration effect can be obtained by the antibody 3.

From the same viewpoint as the retained particle size, the drainage time of the cellulose membrane 2 is preferably 20 seconds or more, more preferably 30 seconds or more, and preferably 2000 seconds or less, more preferably 1500 seconds or less.
Drainage time is measured by the method described in JIS P 3801. Specifically, the filtration time is determined by filtering 100 ml of distilled water at 20°C and a pressure of 0.98 kPa on a 10 cm ² filter surface (for example, 2 s of adsorption surface for cellulose membrane 2) using a Herzberg filtration rate tester. It's time to do it. In other words, if the drainage time is long, it becomes difficult for water to permeate through the cellulose membrane 2.
By setting the lower limit of the drainage time as described above, the time for the test liquid to pass through the cellulose membrane 2 becomes longer, and the chances of contact between the test target liquid and the cellulose membrane 2 can be increased. Furthermore, the fibers F can be arranged densely, and a sufficient amount of the antibody 3 can be immobilized on the cellulose membrane 2. By setting the upper limit value of the drainage time as described above, the liquid to be tested can easily pass through the cellulose membrane 2.

(antibody)
The antibody 3 of the present invention is immobilized on the cellulose membrane 2, for example, on the surface of the fiber F, as described above.
"Antibodies 3 are fixed to the cellulose membrane 2" means that many antibodies 3 are not detached from the cellulose membrane 2 during storage and distribution of the virus concentration material 1 and when the cellulose membrane 2 comes into contact with the test liquid. means a state in which it is fixed on the surface of the cellulose membrane 2. From the viewpoint of immobilizing many antibodies 3 having virus-binding activity to the cellulose membrane 2, the antibodies 3 are immobilized to the cellulose membrane 2 using the affinity between the molecules of the antibody 3 and the molecules contained in the cellulose membrane 2. It is preferable. Examples of methods for immobilizing the antibody 3 and the cellulose membrane 2 using the affinity include covalent bonds, hydrophobic bonds, hydrogen bonds, electrostatic bonds, and other intermolecular forces (van der Waals forces). Can be mentioned.

From the viewpoint of improving the virus concentration rate by the virus concentration material 1, it is preferable that many antibodies 3 are immobilized on the cellulose membrane 2. From this point of view, the content of antibody 3 per 1 g of cellulose membrane 2 on which antibody 3 is immobilized is preferably 1 mg or more, more preferably 1.5 mg or more, and still more preferably 3 mg or more. In addition, from the viewpoint of appropriately controlling the density of the antibody 3 on the surface of the cellulose membrane 2 and ensuring the virus-binding activity of the antibody 3 and from the viewpoint of manufacturing cost, it is necessary to The content of antibody 3 is preferably 60 mg or less, more preferably 30 mg or less, even more preferably 15 mg or less.
In addition, "the content of antibody 3 per 1 g of the cellulose membrane 2 on which the antibody 3 is immobilized" means the content of the antibody 3 per 1 g of the entire cellulose membrane 2 on which the antibody 3 is immobilized. shall be.
The content of antibody 3 can be measured as follows. It can be determined by eluting the antibody 3 from the cellulose membrane 2 with a known protein elution solution such as a surfactant solution and measuring the antibody concentration in the eluted antibody solution. The antibody concentration can be determined by a known protein concentration measurement method, but it is also possible to quantify the amount of the antibody 3 band by using an ELISA kit for antibody measurement, which is an antibody-specific measurement method, or by staining with a protein staining solution after electrophoresis. It is preferable to find it by doing.

Antibody 3 of the present invention typically has a structural domain that includes CDRs that can specifically bind to a portion of a protein of a target virus. CDR (Complementarity Determining Region) includes an antigen recognition site or random sequence region with variable sequence, and is also referred to as a hypervariable region. Binding to the target virus is controlled by the amino acid sequence of the CDR.
Examples of proteins that can be recognized by antibody 3 include the spike protein (S protein), nucleotide protein (N protein), envelope protein (E protein), and matrix protein of the target virus. In order to adsorb the virus itself, the antibody 3 preferably binds to the spike protein present on the surface of the virus, and more preferably binds to the S1 protein of the spike protein.

Specifically, the antibody 3 is preferably one or more selected from IgG antibodies, heavy chain antibodies, and fragment antibodies.
IgG antibodies consist of a heavy chain (VH) and a light chain (LH), and the heavy chain and light chain each have three CDRs, CDR1, CDR2, and CDR3.
A heavy chain antibody consists only of a heavy chain and has three CDRs: CDR1, CDR2, and CDR3.
Fragment antibodies are antibodies fragmented using enzymes or genetic engineering techniques, and include Fab, F(ab') ₂ , Fv, VHH antibodies, and the like.
In particular, by using a heavy chain antibody or a fragment antibody as the antibody 3, the molecular weight of the antibody 3 can be made smaller than that of an IgG antibody, and more antibodies 3 can be immobilized on the surface of the cellulose membrane 2. Therefore, the amount of virus adsorbed by the cellulose membrane 2 is improved, and the virus concentration rate is improved more reliably.

Furthermore, antibody 3 is more preferably a VHH antibody among fragment antibodies. VHH antibodies are fragment antibodies of heavy chain antibodies, and are single domain antibodies found in the serum of camelids. VHH antibodies have a structural domain that includes three CDRs: CDR1, CDR2, and CDR3. In the structural domain, three CDRs exist, for example, in the order of CDR1, CDR2, and CDR3 from the N-terminal side.
Furthermore, while IgG antibodies and the like must be produced using cultured animal cells, VHH antibodies can be produced using E. coli or yeast. Further, VHH antibodies are easily modified by genetic engineering, and tags (peptides) such as His tags can be easily introduced therein. Furthermore, VHH antibodies have high resistance to heat and the like, and can maintain stable binding activity. From these characteristics, by using the VHH antibody as the antibody 3, the virus concentration material 1 having stable concentration ability can be obtained with high productivity.

The target virus of Antibody 3 is preferably a pathogenic virus that has been reported to infect humans and livestock, and in particular coronaviruses, influenza viruses, noroviruses, rotaviruses, and adenoviruses that require virus testing. , herpesvirus, retrovirus, and sapovirus.
Further, the target virus of antibody 3 is preferably an enveloped virus having a spike protein. Examples of enveloped viruses include influenza viruses, herpes viruses, coronaviruses, and retroviruses.

In particular, the enveloped virus that is the target virus of the present invention is preferably SARS-CoV-2, which belongs to the coronavirus. SARS-CoV-2 is the virus that causes acute respiratory disease (COVID-19). By concentrating SARS-CoV-2 in a liquid to be tested easily and at a high concentration rate according to the present invention, the detection sensitivity of SARS-CoV-2 in virus testing is improved. This increases the possibility that COVID-19 will be diagnosed even in patients in the early stages of infection or in asymptomatic patients. Furthermore, as will be described later, the present invention enables concentration of the target virus very quickly and easily, increasing testing efficiency. Therefore, according to the present invention, measures to prevent infection of SARS-CoV-2 can be appropriately taken, and it can contribute to preventing the spread of COVID-19.

When the target virus of antibody 3 is SARS-CoV-2, antibody 3 preferably has one or more of the following structural domains 1), 2), or 3), including CDR1, CDR2, and CDR3.
The structural domain of 1) is a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one amino acid is substituted with another amino acid;
CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one amino acid is substituted with another amino acid;
It includes the amino acid sequence shown in SEQ ID NO: 3 or a CDR3 consisting of an amino acid sequence in which one amino acid is substituted with another amino acid.
Here, the amino acid sequence shown by SEQ ID NO: 1 is GSTFSDYVMA, the amino acid sequence shown by SEQ ID NO: 2 is TISRNGGTTT, and the amino acid sequence shown by SEQ ID NO: 3 is VGGDGDS. An example of an antibody having these CDR sequences is CoVHH1 (SEQ ID NO: 4).
The structural domain of 2) is a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence in which one amino acid is substituted with another amino acid;
CDR2 consisting of the amino acid sequence shown by SEQ ID NO: 6 or an amino acid sequence in which one amino acid is substituted with another amino acid;
It includes the amino acid sequence shown in SEQ ID NO: 7 or a CDR3 consisting of an amino acid sequence in which one amino acid is substituted with another amino acid.
Here, the amino acid sequence shown by SEQ ID NO: 5 is DQIFDNYNMA, the amino acid sequence shown by SEQ ID NO: 6 is ALSWGDSNTG, and the amino acid sequence shown by SEQ ID NO: 7 is VTWLRGDY. An example of an antibody having these CDRs is CoVHH-E5 (SEQ ID NO: 8).
The structural domain of 3) is a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence in which one amino acid is substituted with another amino acid;
CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence in which one amino acid is substituted with another amino acid;
CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence in which one amino acid is substituted with another amino acid.
Here, the amino acid sequence shown by SEQ ID NO: 9 is GTIFSTNAMS, the amino acid sequence shown by SEQ ID NO: 10 is AITSGGNTN, and the amino acid sequence shown by SEQ ID NO: 11 is PGVVTGSYDVRNY. An example of an antibody having these CDRs is CoVHH-E9 (SEQ ID NO: 12).

A CDR consisting of an amino acid sequence in which one amino acid is substituted with another amino acid in each of the amino acid sequences shown in SEQ ID NOs: 1 to 3, SEQ ID NOs: 5 to 7, and SEQ ID NOs: 9 to 11 above is SEQ ID NOS: 1 to 3. , SEQ ID NOs: 5 to 7, and SEQ ID NOs: 9 to 11. Furthermore, even if mutations are introduced into CDR1, CDR2, and CDR3, they are included in the CDRs of the antibody of the present invention as long as they have the ability to bind to SARS-CoV-2.
Here, preferred substitutions include substitutions with amino acids similar in polarity and size. Examples of the above preferred substitutions include substitution of a threonine residue with an asparagine residue, isoleucine residue or methionine residue, substitution of a phenylalanine residue with an isoleucine residue, substitution of a serine residue with an arginine residue or an asparagine residue. Substitution of alanine residue with threonine residue, substitution of arginine residue with serine residue, substitution of asparagine residue with tyrosine residue, substitution of glycine residue with serine residue, valine residue Examples include substitution of phenylalanine residue or isoleucine residue, and substitution of aspartic acid residue with glycine residue or valine residue.
The position of the amino acid to be substituted and the type of amino acid after substitution are not particularly limited, but preferred examples are listed below.
In the structural domain of 1), preferred positions for amino acid substitution include positions 3, 4, 5, or 10 in CDR1, and positions 3, 4, 5, 6, 7, 8, 9, or 10 in CDR2. and in CDR3, the 1st, 2nd, 3rd, or 4th position.
In the structural domain 1), the following combinations are particularly preferred for the position of the substituted amino acid and the type of amino acid after substitution.
In CDR1, the 3rd threonine residue is replaced with an asparagine residue, the 4th phenylalanine residue is replaced with an isoleucine residue, the 5th serine residue is replaced with an arginine residue, and the 10th alanine is replaced. Substitution of the residue with a threonine residue is preferred.
In CDR2, the third serine residue is replaced with an asparagine residue, the fourth arginine residue is replaced with a serine residue, the fifth asparagine residue is replaced with a tyrosine residue, and the sixth glycine residue is substituted with a tyrosine residue. Substitution of the residue with a serine residue, substitution of the 7th glycine residue with a serine residue, substitution of the 8th threonine residue with a methionine residue, substitution of the 9th threonine residue with an isoleucine residue. Substitution, substitution of the 10th threonine residue with a methionine residue, is preferred.
In CDR3, the first valine residue is replaced with a phenylalanine residue or isoleucine residue, the second glycine residue is replaced with an arginine residue, the third glycine residue is replaced with a valine residue, Preferably, the fourth aspartic acid residue is replaced with a glycine residue or a valine residue.

Furthermore, it is more preferable that antibody 3 has one or more structural domains of 3) above, including CDR1, CDR2, and CDR3. Such antibodies 3 are immobilized on the cellulose membrane 2 and can stably adsorb viruses, as will be explained in Examples below.

Furthermore, it is more preferable that antibody 3 is a VHH antibody having a structural domain comprising CDR1, CDR2, and CDR3 of the above amino acid sequence. Thereby, antibody 3 can be obtained which has the above-mentioned advantages of VHH antibodies while having specific binding activity to SARS-CoV-2. Therefore, the virus concentration material 1 having adsorption ability for SARS-CoV-2 can be obtained with high production efficiency.

Further, the antibody 3 may have an amino acid sequence corresponding to a tag peptide such as a FLAG tag, a His tag, an HA tag, and a GST tag to facilitate purification and the like. For example, antibody 3 has a His tag having the amino acid sequence of SEQ ID NO: 13, which increases its affinity for metal ions. Utilizing this property makes it easy to purify and detect antibody 3. Note that the amino acid sequence shown by SEQ ID NO: 13 is HHHHHH.
Furthermore, the tag peptide is preferably bound to the C-terminus of the antibody 3 via a hinge peptide. More specifically, the hinge peptide is not particularly limited, but preferably contains one or more amino acid sequences selected from the amino acid sequences shown in SEQ ID NOs: 14 to 16. Among these, when the tag peptide is a His tag, the hinge peptide more preferably has the sequence shown in SEQ ID NO: 16. Here, the amino acid sequence shown by SEQ ID NO: 14 is EPKTPKPQS. The amino acid sequence shown by SEQ ID NO: 15 is GGG. The amino acid sequence shown by SEQ ID NO: 16 is GGGS.

(Concentration principle and effects using virus concentration material)
With reference to FIG. 3, the concentration principle and typical effects of the virus concentration material 1 having the above configuration in virus testing will be explained.
FIG. 3(A) shows a state in which the virus V is dispersed in the liquid L to be tested. The test target liquid L may be collected in a container or the like, or may be attached to the surface of the test target.
In FIG. 3(B), the cellulose membrane 2 is brought into contact with the liquid L to be tested. Since an antibody (not shown in FIG. 3) is immobilized on the cellulose membrane 2, the virus V in the test liquid L is selectively adsorbed to the adsorption surface 2s of the cellulose membrane 2 via the antibody. As a result, the viruses V in the liquid to be tested L are gathered on the cellulose membrane 2. That is, the virus V in the test liquid L is concentrated by the cellulose membrane 2.
Then, as shown in FIG. 3(C), the cellulose membrane 2 is separated from the test liquid L and collected. Thereby, the concentrated virus V is separated from the liquid to be tested L, and the concentration process is completed.

The virus V adsorbed on the cellulose membrane 2 is separated from the cellulose membrane 2 using, for example, a virus extract and used as a sample for virus testing.
As the cellulose membrane 2 selectively adsorbs and concentrates the virus V, the concentration of the virus V in the concentrated virus-containing liquid such as the virus extract is increased.

From the viewpoint of increasing the concentration rate of the virus V, in the virus concentrating material 1 of the present invention, the cellulose membrane 2 has a pair of adsorption surfaces 2s each configured to be able to adsorb viruses by the antibodies 3. Thereby, the cellulose membrane 2 can adsorb the virus V using the two adsorption surfaces 2s. Furthermore, in one embodiment, the antibody 3 is immobilized on the surface of the fiber F, and the virus is adsorbed on the surface of the fiber F. Therefore, the area in the cellulose membrane 2 that can adsorb the virus V increases, and the amount of the virus V adsorbed per unit mass of the cellulose membrane 2 is increased.
From this, as shown in the following specific example, the concentration rate of virus V can be increased.
For example, even if the mass (size) is constant, a cellulose membrane 2 that can adsorb a higher amount of virus V per unit mass can be formed. As a result, more viruses V can be transferred from the test liquid L to the virus extract, and the concentration of virus V in the virus extract is increased. This achieves a high concentration of virus V.
Furthermore, for example, it is also possible to form a cellulose membrane 2 having a small mass (size) while maintaining the amount of virus V that can be adsorbed to the cellulose membrane 2. Thereby, the volume of the virus extract in which the cellulose membrane 2 can be immersed can be reduced. If the volume of the virus extract for extracting the same amount of virus V is 1/10, the concentration rate will be 10 times. Therefore, from this point of view as well, it is possible to increase the concentration of virus V.

By increasing the concentration of virus V, the detection sensitivity of the virus test is increased and the reliability of the virus test is increased. Furthermore, it may be possible to detect the virus in specimens from patients in the early stages of infection or asymptomatic patients, who carry only a small amount of virus. As a result, infection prevention measures can be taken more appropriately based on the test results, and the spread of viral infections can be prevented.

By using the virus concentration material 1 of the present invention, the virus V can be concentrated in a simple step mainly consisting of bringing the cellulose membrane 2 into contact with the test liquid L. First, virus V can be concentrated without the need for equipment such as a centrifuge. For example, since the virus concentration material 1 is based on the cellulose membrane 2, it is easy to take out and use one sheet at a time, and steps such as measuring during use can be easily omitted. In addition, the cellulose membrane 2 can be managed relatively easily compared to special materials such as magnetic materials.
As described above, according to the virus concentration material 1 of the present invention, techniques and special equipment for concentrating virus V are not required, and virus V can be concentrated in various places and by various workers (users). processing becomes possible. Furthermore, the concentration of virus V is completed in a short time. Therefore, according to the virus concentration material 1 of the present invention, appropriate virus test results can be obtained quickly and in large quantities, and can greatly contribute to improving the efficiency of virus tests. As a result, infection prevention measures can be taken more appropriately and the spread of viral infections can be prevented.

[Method for manufacturing virus concentrate material]
In one embodiment, the virus concentrate material of the present invention is manufactured as follows.
The method for producing the virus concentrate material of the present invention includes:
a step of preparing a cellulose membrane (S11);
Immobilizing the antibody on the cellulose membrane by bringing the cellulose membrane into contact with the antibody of the target virus (S12);
including. An example of this manufacturing method is shown in FIG.

(Preparation of cellulose membrane (S11))
First, a cellulose membrane 2 is prepared. The material of the cellulose membrane 2 may be commercially available paper, paper filter, or other commercially available cellulose sheet materials, or may be a cellulose sheet material produced by a known method.

An example of a method for producing a paper filter-like cellulose membrane 2 will be described.
As a raw material for the cellulose membrane 2, purified α-cellulose and/or naturally derived cellulose raw material is prepared. The naturally derived cellulose fibers may be one or more types selected from cotton yarn, hemp, coconut, pulp, and other plant materials.
These cellulose raw materials are mixed with water to swell the cellulose raw materials. The swollen cellulose raw material is beaten using a beating machine or the like. After that, store and remove dust as necessary. Subsequently, using a paper machine, the swollen cellulose raw material is spread thinly on a mesh, water is removed from the cellulose raw material, and the cellulose raw material is dried. The dried sheet is wound up using a roll or the like, and finishing processing is performed as necessary. Then, the cellulose membrane 2 is produced by cutting this sheet material into a predetermined shape.

As a commercially available sheet material used for the cellulose membrane 2, for example, a paper filter (filter paper) is preferable, and a qualitative filter paper is more preferable. Qualitative filter paper is suitable as a material for the cellulose membrane 2 because it is available at a relatively low cost and has a good balance between thinness and strength.
Furthermore, when selecting a commercially available sheet material suitable for the cellulose membrane 2, it is preferable to select one having a thickness, basis weight, retained particle size, and/or drainage time within the above-mentioned ranges.
When using a commercially available sheet material, the cellulose membrane 2 is produced by cutting the sheet material into a predetermined shape.

(Antibody immobilization treatment (S12))
Subsequently, the antibody 3 is immobilized on the cellulose membrane 2 by bringing the cellulose membrane 2 into contact with the antibody 3.
As a method for immobilizing the antibody 3 on the cellulose membrane 2, a known method can be used. For example, the cellulose membrane 2 may be immersed in an antibody-containing liquid containing antibodies. Alternatively, the cellulose membrane 2 may be brought into contact with the antibody by, for example, spraying an antibody-containing liquid onto the cellulose membrane 2.

The antibody-containing solution is preferably a solution containing water or a water-soluble liquid medium and the antibody 3, and can be, for example, a solution in which the antibody 3 is dispersed in a buffer solution.
The antibody 3 is immobilized on the cellulose membrane 2 by, for example, a covalent bond, a hydrophobic bond, a hydrogen bond, an electrostatic bond, or other intermolecular force (van der Waals force) bond.
The antibody-containing liquid may contain, in addition to the antibody 3, one or more selected from a surfactant, ethanol, and a buffer, from the viewpoint of increasing the affinity between the antibody 3 and the cellulose membrane 2. As the surfactant, nonionic surfactants are preferred from the viewpoint of not reducing antibody function, and examples include alkyl glycosides and polyoxyethylene alkyl ethers.
The concentration of antibody 3 in the antibody-containing solution is preferably 0.01 mg/L or more, more preferably 0.1 mg/L or more, from the viewpoint of immobilizing a sufficient amount of antibody 3 on cellulose membrane 2.
Further, from the viewpoint of reducing costs, the concentration is preferably 10 mg/L or less, more preferably 1 mg/L or less.

The immersion time when the cellulose membrane 2 is immersed in the antibody-containing solution is preferably 15 minutes or more, more preferably 1 hour or more, and even more preferably The time period is 3 hours or more, more preferably 6 hours or more.
Moreover, from the viewpoint of increasing productivity, the immersion time is preferably 36 hours or less, more preferably 24 hours or less, still more preferably 18 hours or less, even more preferably 12 hours or less.
The temperature of the antibody-containing solution in this step is preferably 4° C. or more and 60° C. or less from the viewpoint of efficiently fixing the antibody 3 to the cellulose membrane 2 and preventing denaturation of the antibody 3 and the cellulose membrane 2.

The antibody 3 against the target virus may be a commercially available product or may be produced by a known method. For example, VHH antibodies can be produced by a combination of peptide solid-phase synthesis and native chemical ligation (NCL) methods, or by genetic engineering. A preferred method is to incorporate a nucleic acid encoding antibody 3 into a suitable vector, introduce this into a host cell, and produce it as a recombinant antibody.

Host cells used in production as recombinant antibodies include, for example, E. coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus/insect cells, or yeast cells. As expression vectors for expressing antibodies, vectors suitable for various host cells can be used. Expression vectors include, for example, vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common with Escherichia coli and Bacillus subtilis such as pHY300PLK; Yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified therewith can be used.
These expression vectors have replication origins, selection markers, and promoters suitable for each vector, and as necessary, enhancers, transcription assembly sequences (terminators), ribosome binding sites, polyadenylation signals, etc. may have.
Further, as described above, the expression vector may contain a base sequence inserted therein for expression by fusing FLAG tag, His tag, HA tag, GST tag, etc.

When extracting the expressed antibody 3 of the present invention from cultured bacterial cells or cultured cells, after culturing, the bacterial cells or cultured cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonication, After destroying the bacterial bodies or cells using lysozyme and/or freezing and thawing, a soluble extract is obtained by centrifugation or filtration. From the obtained extract, the antibody of interest can be obtained by appropriately combining known separation and purification methods. Furthermore, when a host that secretes and produces antibody 3 outside the cells is used, a culture supernatant is obtained from the culture solution by centrifugation or filtration. The desired antibody can be obtained from the obtained culture supernatant by appropriately combining known separation and purification methods.
Known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, methods that primarily utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration, SDS-PAGE, and ion Methods that utilize differences in charge such as exchange chromatography, methods that utilize specific affinity such as affinity chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, or isoelectric focusing A method that utilizes the difference in isoelectric points, etc., is used.

Note that the cellulose membrane 2 brought into contact with the antibody-containing liquid may be dried or dehydrated as necessary. As the drying method, for example, one or more known drying methods selected from air drying, drying by heating, natural drying, etc. can be used. Among these, air drying is preferably used from the viewpoint of increasing productivity while preventing denaturation of the antibody 3 and cellulose membrane 2. From the viewpoint of preventing denaturation of the antibody 3 and cellulose membrane 2, the drying temperature is preferably 4° C. or higher and 60° C. or lower.

[Virus concentration method]
The virus concentration method of the present invention includes the step of preparing the virus concentration material 1 of the present invention (S20) and the step of concentrating the target virus in the test liquid by bringing the cellulose membrane 2 into contact with the test liquid (S21). ) and including. An example of this is shown in FIG. 5(A).
In the virus concentration method of the present invention, the test liquid may contain a specimen derived from a living body or a specimen derived from a non-living body. Examples of specimens derived from non-living organisms will be described later.
Examples of the above-mentioned biological samples include tracheal swabs, nasal swabs, throat swabs, nasal washings, nasal aspirates, nasal discharge, saliva, sputum, blood, serum, urine, feces, tissues, cells, Examples include crushed tissue or cells.
When the sample contains solid matter, the liquid to be tested may be a liquid in which the solid matter is suspended in an appropriate specimen processing liquid.
The liquid to be tested is one or more biological body fluids selected from tracheal swabs, nasal swabs, throat swabs, nasal washings, nasal aspirates, nasal discharge, nose blows, saliva, sputum, blood, serum, urine, etc. Preferably, it contains saliva, and more preferably, it contains saliva, as will be described in detail later. In this case, from the viewpoint of further increasing the concentration of the virus in the liquid to be tested, it is even more preferable that the liquid to be tested is saliva itself.

The virus concentration method of the present invention preferably includes:
(1) A method of concentrating the virus concentration material 1 by immersing it in the test target liquid collected in a container,
(2) A method of filtering and concentrating the test liquid collected in a container using the virus concentration material 1;
(3) A method of wiping the test target with a cellulose membrane 2 and concentrating the virus in the test target liquid,
Contains at least one of the following.
Hereinafter, (1) will be described as Embodiment 1, (2) as Embodiment 2, and (3) as Embodiment 3, respectively.

(1) Embodiment 1
The virus concentration method of the present embodiment includes, for example, a step of preparing the virus concentration material 1 of the present invention (S20), a step of collecting a test target liquid in a container (S22), and a concentration step (S21). . An example of this is shown in FIG. 5(B).

(Sample collection step (S22))
In this step, a liquid to be tested is collected into a container using a known method.
The container has a size that is capable of containing a sufficient amount of the liquid to be tested and that allows insertion and removal of the cellulose membrane. The capacity of the container is preferably 1 mL or more, more preferably 5 mL or more, and preferably 50 mL or less, more preferably 15 mL or less. Specifically, the container is composed of a centrifuge tube, a cup, and the like. As the container, preferably, a container included in the virus concentration kit of the present invention can be used.
An appropriate method for collecting the liquid to be tested can be selected depending on the type of specimen.
For example, if the liquid to be tested includes body fluids such as saliva and sputum, the body fluids may be collected directly into a container.
Alternatively, a sample collected using a specimen collection device such as a cotton swab, swab, or syringe may be placed in a container. In this case, if the collected material is a liquid, the collected material may be used as the liquid to be tested, or a suitable processing liquid may be added to the collected material to prepare the liquid to be tested.

In this embodiment, it is more preferable that the liquid to be tested includes saliva as a biological fluid. This allows the specimen to be collected without causing any pain to the test subject. In addition, it becomes possible for the subject to be tested to collect the specimen himself, thereby suppressing infection of medical workers during specimen collection. Furthermore, the possibility of droplets flying during sample collection is reduced, which also reduces infection to the surrounding area.
In the present embodiment, even if the liquid to be tested contains saliva, the virus is sufficiently concentrated in the concentration step described below, so that a virus test with good detection sensitivity can be performed.

From the viewpoint of sufficient contact between the cellulose membrane and the liquid to be tested, the amount of the liquid to be tested is preferably 0.1 mL or more, more preferably 0.5 mL or more, and preferably 10 mL or less, more preferably 5 mL or less. be.

Furthermore, when the test target liquid contains biological body fluids such as saliva, it is preferable to add a chelating agent to the test target liquid before the concentration step (S21). Thereby, the concentration rate in the concentration step can be stably increased. The chelating agent is not particularly limited, and for example, one or more selected from EDTA, NTA, DTPA, HEDTA, GEDTA, TTHA, HIDA, DHEG, citric acid, MGDA, GLDA, etc. can be used. For example, the chelating agent is preferably an aminocarboxylic acid-based chelating agent, and more preferably EDTA.
The concentration of the chelating agent in the test liquid is preferably 0.0005 mol/L or more, more preferably 0.005 mol/L or more, and even more preferably 0.05 mol/L or more, from the viewpoint of sufficiently obtaining the above effects. It is. Further, the upper limit of the concentration is not particularly limited, but is preferably 1 mol/L or less, more preferably 0.5 mol/L or less.

(Concentration step (S21))
In the concentration step of this embodiment, the target virus in the test liquid is concentrated by immersing the cellulose membrane 2 in the test liquid collected in a container.
In this step, for example, the user of the virus concentration material 1 may hold the cellulose membrane 2 with tweezers or the like and immerse the cellulose membrane 2 in the liquid to be tested.
By immersing the cellulose membrane 2 in the liquid to be tested, the virus in the liquid to be tested binds to the antibody 3 immobilized on the cellulose membrane 2, as explained using FIG. Thereby, the virus in the liquid to be tested is collected on the cellulose membrane 2, and the virus is concentrated.
From the viewpoint of increasing the concentration rate, the cellulose membrane 2 may be stirred or shaken in the liquid to be tested. Thereby, the chance of contact between the virus in the liquid to be tested and the cellulose membrane 2 is increased, and more viruses can be adsorbed onto the cellulose membrane 2.
The immersion time is preferably 60 seconds or more, more preferably 300 seconds or more, and preferably 900 seconds, from the viewpoint of sufficiently contacting the cellulose membrane 2 with the virus in the test liquid and efficiently performing the concentration step. The time is preferably 600 seconds or less.
After the cellulose membrane 2 is immersed, the cellulose membrane 2 is separated from the liquid to be tested. Thereby, the virus collected on the cellulose membrane 2 is also separated from the liquid to be tested, making it possible to concentrate the virus.

In the virus concentration method of the above embodiment, by using the virus concentration material 1 of the present invention, the amount of virus adsorbed per unit mass of the cellulose membrane 2 increases, and the virus concentration rate improves. Therefore, by using the virus concentrated in the above concentration step for virus testing, the virus concentration in the test sample is improved, and the detection sensitivity of the virus test is improved.
Moreover, in the above virus concentration method, the virus is concentrated by a very simple operation such as immersing the cellulose membrane 2 in the liquid to be tested. As a result, appropriate virus test results can be obtained quickly and in large quantities, making a significant contribution to improving the efficiency of virus tests. As a result, infection prevention measures can be taken more appropriately and the spread of viral infections can be prevented.

(2) Embodiment 2
In the concentration step (S21) of this embodiment, the target virus in the test liquid is concentrated by filtering the test liquid using the virus concentration material 1.
"Filtration" refers to a process in which a liquid to be tested is passed through a virus concentrating material 1 configured in a filter shape. An example of filtration is shown in FIG.
In the example shown in FIG. 6, first, the virus concentration material 1 is attached to the housing 11 of the filter unit 10. The housing 11 includes a first end 11a and a second end 11b, and a flow path is formed between the first end 11a and the second end 11b. The virus concentrating material 1 is arranged in the housing 11, for example, so that the pair of adsorption surfaces 2s are substantially perpendicular to the flow direction of the liquid (the direction of the white arrow in FIG. 6).
Then, the syringe 12 containing the liquid to be tested L is attached to the first end 11a of the housing 11.
Subsequently, the plunger 12a of the syringe 12 is pressed. As a result, the liquid to be tested L is pressurized and passes through the virus concentration material 1 . As a result, the virus V in the test liquid L is adsorbed to the virus concentration material 1.
After passing through the liquid, the virus concentration material 1 is collected from the filter unit 10. Since the cellulose membrane 2 adsorbs the virus V in the liquid to be tested, the virus V can be concentrated.

(3) Embodiment 3
In the concentration step of this embodiment, the target virus in the test liquid is concentrated by wiping the test object to which the test liquid has adhered with the cellulose membrane 2 (S21). An example of this is shown in FIGS. 5A and 5C.
"Test subject" means a source from which a specimen to be tested is collected, and includes parts of living bodies such as humans (subjects) and domestic animals, as well as non-living bodies.
In this step, for example, the user of the virus concentration material 1 may hold the cellulose membrane 2 with tweezers or the like and wipe the test object with the cellulose membrane 2.
When the test object is a living body, for example, the cellulose membrane 2 is used to wipe the test object to which body fluid, which is the test object fluid, has adhered. Examples of the test object to which the body fluid is attached include the nasal cavity and the pharynx. Thereby, the virus in the body fluid is adsorbed to the cellulose membrane 2, and the virus is concentrated.

On the other hand, in this embodiment, the object to be inspected may be a non-living solid. Examples of non-living solids include doorknobs, desks, toilets, operation panels such as keyboards, input devices such as mice, switches, floors, walls, sewage, water from rivers, soil, indoor or outdoor air, etc. It will be done.
In this case, before the concentration step, it further includes a step (S23) of wetting the cellulose membrane 2 or the surface of the solid with the medium of the test liquid, and in the concentration step, the surface of the solid may be wiped with the cellulose membrane 2. good. An example of this is shown in FIG. 5(C).
The medium of the test target liquid is a liquid contained in the test target liquid, and is a liquid that allows the virus on the test target to bind to the antibody 3 immobilized on the cellulose membrane 2. The medium contains one or more components selected from, for example, a buffer such as PBS (Phosphate-buffered saline), a surfactant such as Tween 20 or Tween 80, or an organic solvent such as ethanol.
Examples of the method of wetting the cellulose membrane 2 with the medium include a method of immersing the cellulose membrane 2 in the medium, a method of spraying the medium onto the cellulose membrane 2, and the like.
Examples of the method of wetting the surface of the solid with the medium include a method of spraying the medium onto the surface of the solid, a method of pouring the medium onto the surface of the solid, and the like.
By wetting the cellulose membrane 2 or the surface of the solid with the medium and then wiping the surface of the solid with the cellulose membrane 2, viruses can be adsorbed to the cellulose membrane 2 wet with the medium. The liquid containing the virus and medium attached to the cellulose membrane 2 is treated as the liquid to be tested.

In the virus concentration method of this embodiment, by wiping the test object with the cellulose membrane 2 of the virus concentration material 1 of the present invention, more viruses can be collected compared to conventional sample collection methods. can. This allows the virus to be efficiently concentrated and increases the detection sensitivity of the virus test.
Furthermore, non-living solid objects such as doorknobs, desks, and operation panels that many people may come into contact with can be tested for viruses easily and with high sensitivity. Therefore, the present invention is also useful for epidemiological investigations such as identifying the source of infection, and can greatly contribute to preventing the spread of infection.

[Virus testing method]
The present invention further provides a virus testing method.
The virus testing method of the present invention includes the step of preparing the virus concentration material 1 of the present invention (S20) and the step of concentrating the target virus in the test liquid by bringing the cellulose membrane 2 into contact with the test liquid (S21). ) and a step (S31) of detecting the concentrated target virus. An example of this is shown in FIG. 7(A).
Furthermore, the virus testing method of the present invention may include, before the detection step, a step (S32) of extracting the target virus from the cellulose membrane 2 brought into contact with the test target liquid. An example of this is shown in FIG. 7(B).
The virus concentration step described above is the same as the concentration step explained in the section of the above-mentioned "Virus concentration method," so the explanation will be omitted.

(Extraction step (S32))
In this step, viruses can be extracted from the cellulose membrane 2 after the concentration step using a known virus extraction method.
For example, the virus is separated from the cellulose membrane 2 and extracted by immersing the cellulose membrane 2 after the concentration process in a known virus extract solution. The virus extract contains, for example, a surfactant, a buffer, and the like. In this step, stirring of the cellulose membrane 2 in the virus extract, heating of the virus extract, etc. may be performed as necessary.
The virus extract after the extraction step may be used as it is in the virus detection step, or may be further prepared with a diluted solution or the like.
By including the above extraction step in the virus testing method of the present invention, the concentrated virus is separated from the cellulose membrane 2, making it easier to handle in the detection step.

(Detection step (S31))
In this step, viruses can be detected from the cellulose membrane 2 after the concentration step using a known virus detection method.
The virus detection method in this step includes a virus protein detection method using an antigen test or a nucleic acid detection method using a genetic test.
Antigen tests include methods that utilize antigen-antibody reactions, such as ELISA and immunochromatography.
Examples of genetic testing include PCR method, real-time PCR method, and LAMP method.
When detecting a viral protein by an antigen test, for example, the virus can be detected by reacting a concentrated virus extract with an antibody capable of binding to the protein of the virus.
When detecting a virus by genetic testing, for example, a nucleic acid extraction process is performed on the concentrated virus extract or a cellulose membrane to which the virus has been adsorbed, and the extracted nucleic acid is used to perform a nucleic acid amplification process. The nucleic acid of the virus can be detected.

In the virus testing method of the present invention, by using the virus concentrating material 1 of the present invention, the virus concentration rate is improved as described above, and the sensitivity of virus testing is increased. Furthermore, by quickly concentrating the virus with a simple operation, it becomes possible to test a large number of samples for the virus, and more appropriate infection prevention measures can be taken. Therefore, the present invention can greatly contribute to expanding infection prevention.

[Virus concentration kit]
The virus concentration material of the present invention may be used in a virus concentration kit for concentrating viruses in a liquid to be tested in a virus test.
In one embodiment, the virus concentration kit 4 of the present invention preferably includes a virus concentration material 1 and a container 5.
The virus concentration material 1 has the configuration described in the section of "Virus concentration material" above, and specifically includes a cellulose membrane 2 whose main component is α-cellulose, and an antibody 3 immobilized on the cellulose membrane 2. and has. An example of this is shown in FIGS. 8 and 9.
In addition, in FIG. 8, for convenience of illustration, the size of the virus concentration material 1 with respect to the container 5 is shown to be larger than the actual size.

The container 5 is a device capable of holding therein the virus concentration material 1 and a test liquid for testing a target virus.
In the present invention, the "container" is not limited to a structure capable of storing a liquid to be tested, but also includes a structure capable of temporarily holding a liquid to be tested as a flow path, like the housing 11 described in FIG. 6. Furthermore, "the liquid to be tested is held inside the container 5" does not mean that all of the liquid to be tested for one sample is stored inside the container 5 at once; 5.
Furthermore, "the virus concentration material 1 is held inside the container 5" means that the virus concentration material 1 only needs to be placed inside the container 5, and that the virus concentration material 1 is fixed to the container 5. does not require
The container 5 is preferably sterilized and is made of, for example, sterilizable plastic, glass, or the like.

The container 5 is preferably a sample collection container for collecting a liquid to be tested such as saliva, or a container capable of storing a sample processing liquid in which a sample collected by a sample collection device is processed. More preferably, it is a container. An example of this is shown in FIG.
In the example shown in FIG. 8, the container 5 has a size that allows insertion and removal of the cellulose membrane 2, and its capacity is preferably 1 mL or more, more preferably 5 mL or more, and realistically 50 mL or less. Or 15 mL or less. Specifically, the container 5 is composed of a centrifuge tube, a cup, and the like. In the container 5, "the cellulose membrane 2 can be inserted and taken out" means that the cellulose membrane 2 can be inserted into the container 5 without impairing the ability to concentrate viruses, and the cellulose membrane 2 can be inserted into the container 5. It means to be taken out from.

Alternatively, the container 5 may be configured as the housing 11 of the filter unit 10 when performing filtration using the virus concentration material 1, as described using FIG. That is, by setting the virus concentration material 1 in the container 5, the filter unit 10 may be configured. An example of this is shown in FIG.
The container 5 (housing 11) includes a first end 11a and a second end 11b. The first end portion 11a is configured such that a syringe or the like containing the liquid to be tested can be attached thereto. The filtered flow-through flows out of the second end 11b.
In this example, the virus concentrate material 1 may be set inside the container 5 (housing 11) as shown in FIG. Alternatively, the virus concentrate material 1 may be distributed separately from the container 5 (housing 11) and set in the container 5 by the user.
Further, the container 5 only needs to have a cross-sectional shape that allows the virus concentration material 1 to be placed therein. The cross-sectional shape of the container 5 is preferably a cross-sectional shape that corresponds to the planar shape of the cellulose membrane 2, and is preferably circular, for example. When the cross-sectional shape of the container 5 is circular, the inner diameter can be, for example, 5 mm or more and 100 mm or less.
In addition, in this example, the virus concentration kit 4 may further include a container for collecting and/or storing the test target liquid shown in FIG. 8, in addition to the container 5 serving as the housing of the filter unit. . Further, the virus concentration kit 4 may further include a syringe shown in FIG. 6.
Such a virus concentration kit 4 also allows virus concentration through filtration.

According to the virus concentration kit 4 having the above configuration, the virus concentration material 1 and the container 5 are provided. Thereby, the virus concentration process can be performed more easily, and the virus concentration rate in the concentration process can be increased.

[Virus test kit]
The virus concentration material of the present invention may be used in a virus test kit for virus testing.
The virus test kit 6 of the present invention preferably includes a virus concentration material 1, a container 5, and a test reagent 7. An example of this is shown in FIGS. 8 and 9.
The virus concentration material 1 and the container 5 have the configurations described in the "Virus concentration material" and "Virus concentration kit" sections, so their description will be omitted.

The virus test kit 6 of the present invention is a kit for performing a virus test capable of detecting viruses. Virus tests to which the virus test kit 6 of the present invention is applicable include antigen tests and genetic tests.
Antigen tests include methods that utilize antigen-antibody reactions, such as ELISA and immunochromatography.
Examples of genetic testing include PCR method, real-time PCR method, and LAMP method.

The test reagent 7 is a test reagent for detecting a target virus using the test liquid adhered to the cellulose membrane 2. Although FIGS. 8 and 9 show an example in which the virus test kit 6 includes one test reagent 7, the virus test kit 6 may include a plurality of test reagents.
The test reagent 7 may contain, for example, a virus extract for separating the virus adsorbed onto the cellulose membrane 2 from the cellulose membrane 2. The virus extract contains, for example, a surfactant, a buffer, and the like. By separating the virus from the cellulose membrane 2 using the virus extract, a sample for virus testing can be easily prepared using the concentrated virus.
For example, the test reagent 7 for ELISA may contain one or more reagents selected from the above-mentioned virus extract, enzyme-labeled antibody-containing solution, substrate solution, washing solution, dilution solution, and the like.
For example, the test reagent 7 intended for immunochromatography may contain the above virus extract.
The test reagent 7 intended for genetic testing may contain one or more reagents selected from, for example, the above virus extract, a nucleic acid extract for extracting nucleic acids from viruses, a reagent for nucleic acid amplification, and the like.

Furthermore, the virus test kit 6 of the present invention may include test instruments used for virus testing, if necessary. Examples of the test equipment include a nitrocellulose membrane containing antibodies used in immunochromatography, a chip for dropping a test sample, and a test plate.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and it goes without saying that various changes can be made without departing from the gist of the present invention.
For example, the shape of the cellulose membrane 2 is not limited to that shown in FIG. 1, and can take various shapes. For example, the cellulose membrane 2 may have a rectangular shape or may have another shape.
In addition to the cellulose membrane and the antibody, the virus concentration material 1 may also include, for example, a rod-shaped holding member that holds the cellulose membrane. This allows the user to hold the holding member and perform the virus concentration process, thereby increasing convenience.
Further, the virus concentration kit 4 and the virus testing kit 6 are not limited to the examples shown in FIGS. 8 and 9, and may take various forms included in the present invention.

<Test Example 1: Examination of concentration ability of virus concentration material by immersion in test target liquid>
As Test Example 1, a cellulose membrane on which antibodies against a target virus were immobilized was prepared, and it was investigated whether the virus could be concentrated by immersing it in a test liquid.

[Preparation of virus concentration material]
(Preparation of cellulose membrane)
As a material for the cellulose membrane, Whatman (registered trademark) Qualitative Filter Paper No. I prepared 1. This was cut into a circle with a diameter of 10 mm to obtain a cellulose membrane. This cellulose membrane contains 90% by mass or more of α-cellulose.
The thickness of this cellulose membrane was 180 μm, and the basis weight was 87 g/m ² .
The retained particle size of this cellulose membrane was 11 μm, and the drainage time was 150 seconds.

(Preparation of antibodies)
VHH antibody, which is an anti-SARS-CoV-2 antibody, was produced. The VHH antibody was screened using cDNA display using SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His Tag) (His-tagged S1 protein, Sino Biological) as a target molecule. be.
The produced VHH antibodies are referred to as CoVHH1, CoVHH-E5, and CoVHH-E9. Among these, CoVHH-E9 was used in this test.

(artificial gene synthesis)
A base encoding His-tagged CoVHH-E9 was artificially synthesized. The amino acid sequence of CoVHH-E9 to be synthesized is SEQ ID NO: 17, in which a His tag sequence (SEQ ID NO: 13) is added to the C-terminal side of SEQ ID NO: 12 via the hinge sequence of SEQ ID NO: 16. Therefore, the artificially synthesized gene for expressing CoVHH-E9 is SEQ ID NO: 18, which is the nucleotide sequence encoding SEQ ID NO: 17 with a stop codon added to the 3' end. Furthermore, as a sequence for inserting an artificially synthesized gene for CoVHH-E9 expression into a plasmid vector, GCAGCTCTTGCAGCA (SEQ ID NO: 19) is at the 5' end and TCTATTAAAACTAGTT (SEQ ID NO: 20) is at the 3' end of SEQ ID NO: 18. The one with the end added was artificially synthesized by Eurofins and used for experiments.

(Construction of His-tagged VHH expression plasmid)
Using the recombinant plasmid pHY-S237 (Japanese Unexamined Patent Publication No. 2014-158430) prepared based on pHY300PLK as a template, 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 21) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG- 3' (SEQ ID NO: 22) Plasmid sequences were amplified by PCR using the primer set and PrimeSTAR Max DNA polymerase (TaKaRa). sp by PCR using the genome of strain 168 as a template and using the primer set 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 23) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTTCCC-3' (SEQ ID NO: 24). Promoter DNA derived from the oVG gene was amplified. . The obtained promoter DNA was incorporated into a plasmid sequence using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter. The plasmid sequence was amplified by PCR using a primer set of 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 25) and 5'-TCTATTAAAACTAGTTATAGGG-3' (SEQ ID NO: 26) and PrimeSTAR Max DNA polymerase (TaKaRa). The DNA containing the artificially synthesized gene was inserted into the obtained PCR fragment using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid containing the artificially synthesized VHH gene. This His-tagged CoVHH expression plasmid is called CoVHH-E9-His-pHY.

(Creation of recombinant Bacillus subtilis)
Deleting extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr and aprE) from Bacillus subtilis 158 strain according to the method described in paragraphs 0043 to 0048 of JP 2006-174707A, Furthermore, the sigF gene involved in sporulation was deleted according to the method described in paragraphs 0026 to 0029 of Patent No. 4336082. The obtained extracellular protease multiple deletion strain is designated as Dpr8ΔsigF. Plasmid introduction into Bacillus subtilis strain Dpr8ΔsigF was performed by the protoplast method described below. Bacillus subtilis stocked with glycerol was inoculated into 1 mL of LB liquid medium, and cultured with shaking at 30° C. and 210 rpm overnight. The next day, 10 μL of this culture solution was inoculated into a new 1 mL LB liquid medium, and cultured with shaking at 37° C. and 210 rpm for about 2 hours. The culture solution was collected into a 1.5 mL tube, centrifuged at 1,2000 rpm for 5 minutes, the supernatant was removed, and the pellet was mixed with SMMP (0.5 M sucrose, 20 mM disodium maleate) containing 4 mg/mL of Lysozyme (SIGMA). , 20mM magnesium chloride hexahydrate, 35% (w/v) Antibiotic medium 3 (Difco)) and incubated at 37°C for 1 hour. Next, the pellet was centrifuged at 3,500 rpm for 10 minutes, the supernatant was removed, and the pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with the plasmid, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this solution, mixed by inversion, and shaken at 30° C. and 210 rpm for 1 hour. The entire amount was applied onto a DM3 agar plate and incubated at 30° C. for 2 to 3 days.

(VHH production)
The prepared recombinant Bacillus subtilis was inoculated into 1 mL of LB medium containing 50 ppm tetracycline, and shaken back and forth overnight at 32° C. to prepare a preculture solution. 1% of the preculture solution was inoculated into 20 mL of 2×L-mal medium in a pleated Erlenmeyer flask, and cultured with shaking at 30° C. for 72 hours. At the end of the culture, the culture solution was centrifuged in a microtube at 4°C, 15,000 rpm, for 5 minutes, and the supernatant was collected. The VHH antibody in the supernatant was purified using Ni-NTA agarose beads (Fuji Film Wako Pure Chemical Industries, Ltd.) according to the kit protocol. PBS (30mM imidazole) was used as the eluate. In addition, the target protein was detected by SDS-PAGE.
As a result, it was confirmed that CoVHH-E9 was synthesized.

[Antibody fixation treatment]
The produced VHH antibody was immobilized on the produced cellulose membrane.
A CoVHH-E9 solution was added to PBS to prepare a solution containing CoVHH-E9 at a concentration of 1.17 mg/mL. Note that the antibody concentration was determined using a Bio-Rad Protein Assay Kit (BIO-RAD). 0.1 mL of the CoVHH-E9-containing solution was added to each cellulose membrane cut into a circular shape with a diameter of 10 mm, and the mixture was allowed to stand at room temperature for 30 minutes.
Thereafter, it was washed five times with HBST (20mM HEPES) and once with distilled water. Thereafter, it was air-dried at 50°C for 10 minutes.
As a result, the sample of Example 1, which was a cellulose membrane on which CoVHH-E9 was immobilized, was produced.
Note that a cellulose membrane on which CoVHH-E9 was not immobilized was used as a sample of Comparative Example 1.

[Immersion in virus solution and shaking]
PBS was added to the SARS-CoV-2 solution (Wuhan strain) to prepare a 2.7 x 10^8 copies/mL SARS-CoV-2 solution.
0.5 mL of SARS-CoV-2 solution was added to each of multiple 2 mL tubes (Protein LoBind tubes, Eppendorf). Two samples each of Example 1 and Comparative Example 1 were prepared, and one sample was placed in one tube and mixed by inversion (shaking). The supernatant of each solution was collected 30 minutes after starting the inversion mixing.

[Evaluation of concentration ability]
(Western blotting)
To measure the amount of SARS-CoV-2 N protein in the supernatant, Western blotting was performed using an anti-N protein antibody.
91 μL of the collected supernatant, 14 μL of 10× Sample Reducing Agent (Invitrogen), and 35 μL of 4× LDS Sample Buffer (Invitrogen) were mixed and heated at 100° C. for 5 minutes.
10 μL of the heat-treated sample was applied to 10% Bis-Tris, 1.0 mm, Mini Protein Gel (Invitrogen), and electrophoresed at 200 V for 40 minutes. After the electrophoresis was completed, the gel was taken out and the protein was transferred to a PVDF membrane using iBlot2 (Invitrogen). Subsequently, the membrane was taken out and an antigen-antibody reaction was performed using iBind (Invitrogen). The operation followed the attached protocol. Anti-SARS-CoV-2 Nucleocapsid Protein Rabbit Mab Antibody (SinoBiological, 40143-R019) diluted 1000 times was used as the primary antibody, and Anti-Rabbit I diluted 1000 times was used as the secondary antibody. gG HRP-linked Antibody (CST, 7074S) was used.
After the antigen-antibody reaction was completed, the membrane was washed twice with ion-exchanged water, 2 mL of ECL Prime Western Blotting Detection Reagent (Cytiva) was dropped onto the membrane, and the membrane was incubated for 5 minutes at room temperature. Excess solution was removed from the membrane, and chemiluminescence was detected using an imager with a CCD camera (Light capture II, ATTO).

(result)
FIG. 10 is a diagram showing images of bands obtained by Western blotting.
"Initial" indicates the result of Western blotting on the virus solution before contacting with the virus concentration material.
The amount of SARS-CoV-2 N protein in the supernatant of the virus solution contacted with the sample of Example 1 was significantly reduced compared to the supernatant of the virus solution contacted with the sample of Comparative Example 1. Almost no N protein was detected in the supernatant. This result showed that the virus concentration material of Example 1 had a high ability to adsorb SARS-CoV-2 and was able to concentrate SARS-CoV-2.

<Test Example 2: Examination of concentration ability of virus concentration material by filtration of test liquid>
As Test Example 2, we investigated whether it was possible to concentrate the virus by using a cellulose membrane on which antibodies against the target virus were immobilized for filtration of the test liquid.

[Preparation of virus concentration material]
(Preparation of cellulose membrane)
As a material for the cellulose membrane, Whatman (registered trademark) Qualitative Filter Paper No. 602h was prepared. This was cut into a circle with a diameter of 13 mm to obtain a cellulose membrane. This cellulose membrane contains 90% by mass or more of α-cellulose.
The thickness of this cellulose membrane was 160 μm, and the basis weight was 84 g/m 2 .
The retained particle size of this cellulose membrane was 2.5 μm or less, and the drainage time was 750 seconds.

[Antibody fixation treatment]
The VHH antibody was immobilized on the prepared cellulose membrane by the same procedure as in Test Example 1. As a result, the sample of Example 2, which is a cellulose membrane on which CoVHH-E9 was immobilized, was prepared.
Note that a cellulose membrane on which CoVHH-E9 was not immobilized was used as a sample of Comparative Example 2.

[Filtration]
A plurality of identical filter units (SWINNEX filter holder 35 mm, Merck) were prepared (see Figure 6). One sample of Example 1 or Comparative Example 1 was attached to the housing of each filter unit. Each sample was attached so that the filter surface (adsorption surface) was perpendicular to the flow direction of the liquid.
1 mL of the SARS-CoV-2 solution (virus solution) prepared in Test Example 1 was aspirated into a 2.5 mL syringe. The end of each syringe was attached to one end of the filter unit. The plunger was pressed to feed the liquid at a rate of 800 μL/min. Flow-through was collected from the other end of the filter unit.
This flow-through was subjected to Western blotting in the same manner as in Test Example 1, and the bands were imaged. An image of this band is shown in FIG. "Initial 1/2" and "Initial 1/4" indicate the results obtained by diluting the initial virus solution 1/2 and 1/4 times with PBS, respectively.

[result]
As shown in FIG. 11, the flow-through obtained by filtration using the sample of Example 2 is higher than the flow-through obtained by filtration using the sample of Comparative Example 2. It was found that the amount of CoV-2 N protein was clearly decreased. Comparing the initial band intensity and the band intensity of the sample of Example 2, it is thought that the sample of Example 2 adsorbs about 20% of SARS-CoV-2. These results showed that the virus concentration material of Example 2 had a high ability to adsorb SARS-CoV-2 and was able to concentrate SARS-CoV-2.

1 Virus concentration material 2 Cellulose membrane 2s Adsorption surface 3 Antibody 4 Virus concentration kit 5 Container 6 Virus test kit 7 Test reagent

Claims (16)

A cellulose membrane whose main component is α-cellulose,
an antibody against the target virus immobilized on the cellulose membrane;
Equipped with
The cellulose membrane has a pair of adsorption surfaces each configured to be able to adsorb the target virus by the antibody. Virus concentration material. The cellulose membrane includes fibers and is configured in a filter shape,
The virus concentration material according to claim 1, wherein the antibody is immobilized on the surface of the fiber. The virus concentration material according to claim 2, wherein the cellulose membrane has a retained particle diameter of 2.5 μm or more and 11 μm or less. The virus concentration material according to any one of claims 1 to 3, wherein the cellulose membrane contains 90% by mass or more of α-cellulose. The virus concentration material according to any one of claims 1 to 4, wherein the cellulose membrane has a basis weight of 80 g/m ² or more and 200 g/m ² or less. The virus concentration material according to any one of claims 1 to 5, wherein the antibody is one or more selected from heavy chain antibodies and fragment antibodies. The virus concentrate material according to claim 6, wherein the antibody is a VHH antibody. The antibody is
CDR1 consisting of the amino acid sequence shown by SEQ ID NO: 9 or an amino acid sequence in which one amino acid is substituted with another amino acid, and the amino acid sequence shown by SEQ ID NO: 10 or one amino acid sequence in which one amino acid is substituted with another amino acid. A structure comprising a CDR2 consisting of an amino acid sequence in which another amino acid is substituted, and a CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence in which one amino acid is substituted with another amino acid in the amino acid sequence. The virus concentrate material according to any one of claims 1 to 7, having one or more domains. The virus concentrate material according to any one of claims 1 to 8, wherein the target virus is an enveloped virus. The virus concentrate material according to claim 9, wherein the target virus is SARS-CoV-2. A virus concentration material having a cellulose membrane mainly composed of α-cellulose and an antibody against a target virus immobilized on the cellulose membrane;
a container capable of holding therein the virus concentration material and a test liquid for testing the target virus;
Equipped with
The cellulose membrane has a pair of adsorption surfaces each configured to be able to adsorb the target virus by the antibody. The virus concentration kit. A virus concentration material having a cellulose membrane mainly composed of α-cellulose and an antibody against a target virus immobilized on the cellulose membrane;
a container capable of holding therein the virus concentration material and a test liquid for testing the target virus;
a test reagent for detecting the target virus using the test target liquid attached to the cellulose membrane;
Equipped with
The cellulose membrane has a pair of adsorption surfaces each configured to be able to adsorb the target virus by the antibody. The virus test kit. A pair comprising a cellulose membrane containing α-cellulose as a main component, and an antibody against a target virus immobilized on the cellulose membrane, wherein the cellulose membrane is configured to be able to adsorb the target virus by the antibody, respectively. Prepare a virus concentration material having an adsorption surface of
A virus concentration method, comprising: concentrating the target virus in the test liquid by bringing the cellulose membrane into contact with a test liquid for testing the target virus. The virus concentration method according to claim 13, wherein the test target liquid includes saliva. A cellulose membrane containing α-cellulose as a main component, and an antibody against a target virus immobilized on the cellulose membrane, and the cellulose membrane is configured to be able to adsorb each of the target viruses by the antibody. Prepare a virus concentration material having a pair of adsorption surfaces,
By bringing the cellulose membrane into contact with a test liquid for testing the target virus, concentrating the target virus in the test liquid,
A virus testing method for detecting the concentrated target virus. Prepare a cellulose membrane containing α-cellulose as the main component,
A method for producing a virus concentration material, comprising immobilizing the antibody on the cellulose membrane by bringing the cellulose membrane into contact with an antibody of a target virus.

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