JPH0746991B2 - Virus immobilization method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a method in which a liquid in which a virus is present is subjected to substantially vertical filtration through a porous membrane and then freeze-dried so that the virus, such as a protein, is contained in the pores of the membrane. The present invention relates to a method for immobilizing a virus that is immobilized without a coating agent. Immobilization in the present invention means
It is not something that can be achieved by coating a protein or the like on the membrane, but means that the substance is confined in the pores of the porous membrane and retained at one place so that it does not move. INDUSTRIAL APPLICABILITY The method of the present invention can be used for a virus test, for example, an immunological test for hepatitis virus or AIDS virus, and for immobilizing a target virus or virus group from a virus solution in which a plurality of viruses are mixed.

(Prior Art) A virus is an infectious substance that propagates only in cells and is potentially pathogenic, and has the following attributes.
It has either DNA or RNA as nucleic acid. Only the genetic material is replicated. It does not multiply in two divisions. Lack of energy production system. The host ribosome is used for protein synthesis.

At present, virus concentration methods include fractional centrifugation based on particle size, isoelectric point treatment using solubility differences, treatment with organic solvents, adsorption elution, particle shape and density. There are density gradient centrifugation method, oxygen treatment method, etc., and the method suitable for each virus is selected.
Among these, methods of concentrating viruses using a membrane include a method using adsorption and elution, a method by filtration, and a method by retained dissolution by an alginate membrane.

The method using adsorption and elution is a method in which the virus particles are adsorbed on the adsorbent-coated membrane by the surface charge of the virus particles under appropriate conditions, and then the membranes coated with the virus particles by changing the conditions such as salt concentration. This is a concentration method that makes it possible to elute more. For example, “Appl. Microbiol., Vol.23 (4) 76 (197
2)., Wallis, C. et al. ”, Enteric viruses suspended in water such as sewage are sterilized and deproteinized in advance, and MgCl ₂ or AlCl ₃ is added. The virus is adsorbed and filtered on the filter and then eluted. Also, in the virus culture medium, the adsorption inhibitory substances in the liquid were removed with protamine and the pH was adjusted to 5.0 and filtered, and the virus was adsorbed to the filter. A double concentrate has been obtained. However, in general, the virus particles are adsorbed,
When elution is performed with an appropriate eluent, the infectivity of the virus is likely to decrease. Moreover, it is necessary to determine the conditions for each virus, and the operation is very complicated. In addition, the amount of adsorption is limited and the concentration rate is low.

Next, a method by filtration, which is concentration by ultrafiltration. `` Canad.J.Chem., Vol.33,1452 (1955), Go
As described in “Rdon, JL & Mason, SG”, a sample such as a culture solution is caused to flow in parallel to a filter by a circulation pump, and components other than the virus are filtered by aspiration to concentrate the virus. This is because the protein is adsorbed on the filter, so that it is easily clogged and the virus concentration rate is slow. Further, it is not possible to select a membrane having a pore size suitable for viruses having various shapes and sizes.

Next, regarding the method using an alginate membrane, “Muscle virus research, general review (National Institute of Preventive Health, alumni association) Maruzen (197
3), p47 ”, this film was added to a solution of sodium alginate to produce La (NO ₃ ) ₃ · 6H ₂ O, AlCl ₃ · 6H ₂ O.
Gelled by adding three layers (capillary zone, inte
rmediatelayer, primary gel membrane). The virus is retained in this filter, thawed and recovered, and the virus is separated by animals or cultured cells. In this filter virus is retained in the middle layer. However, since it is gel-like, it is brittle, very difficult to handle, and the amount of virus that can be reserved is limited. Moreover, it is impossible to store the virus retained after filtration in a dry state. In addition, the filter must be thawed and the virus must be recovered, and the infectivity of the virus is likely to decrease. Thus, it can be said that the method using an alginate film is not practical.

(Problems to be Solved by the Invention) When a virus is concentrated, it has been difficult for the conventional methods to simultaneously satisfy the two requirements of maintaining the infectivity of the virus and enhancing the efficiency of concentration. Moreover, in many cases, the virus is suspended in a liquid and may be scattered in the air at all times, but the safety to the human body has not been considered when handling harmful viruses.

It is an object of the present invention to provide a method for immobilizing a virus that can be concentrated while maintaining the infectivity of the virus regardless of the type of virus, and that is easy to handle and highly safe.

(Means for Solving Problems) In the method of the present invention, a liquid in which a virus is present is subjected to substantially vertical filtration with a porous membrane made of regenerated cellulose having a copper-ammonia method, and then freeze-dried to obtain a membrane pore. It is characterized by immobilizing the virus inside.

There are various methods for producing regenerated cellulose, such as the viscose method, the saponification method of cellulose ester, and the copper ammonia method. However, there are differences in physical and scientific properties due to differences in production conditions. It is not uniformly discussed as "regenerated cellulose". In the copper-ammonia method, the generation of fine pores and the formation of a unique molecular chain aggregate structure due to the removal of copper are recognized by the indispensable acid treatment, and therefore the copper-ammonia method regenerated cellulose has a unique property.

Its characteristic feature is that it is hydrophilic and has low protein adsorption. The present inventors have examined the correlation regarding the adsorptivity between the protein and the polymer material, and as a result, in general, the hydrophilic material has a lower protein adsorptivity, and the copper-ammonia method regeneration used in the method of the present invention It has been found that the porous hollow fiber made of cellulose is the smallest.

Viscosity average molecular weight of regenerated cellulose with copper ammonia method is 7 ×
It is preferably 10 ⁴ or more, and the smaller the dissolved component in the 0.1N NaOH aqueous solution, the more preferable. 40 ° C, 48 hours, 0.
This hollow fiber is most suitable for concentrating viruses when it is immersed in 1N NaOH aqueous solution and the dissolved content is 10 ppm or less.

To make a film made of cellulose as described above, use a high-purity cellulose stock solution to make regenerated cellulose by the cuprammonium method, or use 0.1N NaOH after making the film.
It may be washed with an aqueous solution for 72 hours or more. The use of a high-purity cellulose raw material is more preferable because the dissolved content is significantly reduced. Here, "high-purity cellulose raw material"
Means that the α-cellulose content is 95 wt% or more and the degree of polymerization is
Over 500 cotton linters and wood pulp. For these raw materials, it is preferable to always use purified water in order to prevent bleaching, decomposition and oxidation during the washing process, and avoid mixing of impurities.

When immobilizing a virus on a porous membrane made of regenerated cellulose by the cuprammonium method, assuming that the virus diameter is V (nm), the average water flow velocity pore diameter is D (nm), and the membrane thickness is T (μm), It is preferable to satisfy the condition of.

Here, the virus diameter V refers to the diameter of a spherical virus, and the diameter of the minor axis when approximated to an ellipsoid for a non-spherical virus. When the value of the formula (1) is 9.5 or more, the virus blocking rate of the membrane is high, and the virus cannot penetrate into the membrane pores.

If the value of the formula (1) is less than 1, the loss of virus is large, and the substance that constitutes the porous membrane (called the medium of virus, water in the undiluted solution, the membrane in the case of virus trapped in the membrane The regenerated cellulose used as the medium) cannot increase the virus density per 1 g higher than the virus density per 1 g of virus stock solution (= 1 ml).

As the separation mechanism of virus particles by the membrane, there are a "sieving mechanism" that sifts according to the difference between the pore size of the membrane and the particle diameter of the virus particles to be separated, and "adsorption mechanism" that adsorbs the virus particles on the membrane surface. . Considering the results of the study by the present inventors that the adsorption property of protein is the smallest in the porous membrane made of the copper-ammonia method regenerated cellulose as compared with other polymer materials, the porous membrane made of the copper-ammonia method regenerated cellulose is considered. Viral separation by membranes is considered to be mostly a "sieving mechanism".

As the porous membrane, either a flat membrane or a hollow fiber can be used, but a hollow fiber is preferable.

In the porous hollow fiber made of regenerated cellulose by the cuprammonium method, when the pore diameter in the plane perpendicular to the film thickness direction from the inner wall surface to the outer wall surface is expressed by the in-plane average pore diameter, the in-membrane average at the inlet and outlet of the through-membrane hole It is preferable to use a structure in which at least one set in the thickness direction of the hollow fiber has a structure in which a minimum portion, a portion larger than the minimum portion, and another minimum portion are arranged in this order between the pore diameters. The minimum mentioned here refers to the minimum in a mathematical sense. That is, in the present invention, a membrane in which the in-plane average pore diameter of the membrane surface is smaller than the in-plane average pore diameter of the immediately inner surface thereof is not said to be a "minimal membrane surface portion".

With this structure, compared with the conventional porous hollow fiber, it is possible to increase the virus blocking rate of the porous hollow fiber made of regenerated cellulose by the copper ammonia method, trap the virus in the pores, and increase the filtration rate. You can speed it up. On the other hand, in the case of a porous hollow fiber in which at least one set of minimum in-plane average pore diameter does not exist, the permeation rate must be slowed in order to achieve a blocking rate of 90% or more.
Also, the virus cannot be trapped in the hollow fiber pores and concentrated.

In the porous hollow fiber made of regenerated cellulose by the cuprammonium method, the minimum in-plane porosity is preferably 10% or more. Below 10%, the ultrafiltration rate drops sharply.
It is preferably 30% or more. The effect of the in-plane porosity on the ultrafiltration rate is as follows:
The ultrafiltration rate increases in proportion to about the second power at 10 to 30% and to the first power at more than 30%. On the other hand, the minimum in-plane porosity is 80%
If it exceeds, the mechanical properties of the porous hollow fiber may be remarkably deteriorated, and a defect such as a pinhole may be generated, or the cellulose molecules constituting the hollow fiber may be dropped and dispersed in the filtrate or the liquid to be filtered. .

The thinner the thickness of the hollow fiber is, the more preferable it is because the filtration rate is generally increased. However, the film thickness is 10
When it is less than μm, pinholes frequently occur in the hollow fiber,
Viral particles leak out into the filtrate. Also the film thickness is 100
If it is more than μm, the filtration rate will be significantly reduced.
If the minimum in-plane porosity is large, the number of trapped viruses may be increased by designing a thicker film thickness.

The porous hollow fiber made of the cuprammonium method regenerated cellulose used in the method of the present invention has a layered structure in the film thickness direction from the inner wall surface to the outer wall surface of the hollow fiber, and has protein permeability,
It has a minimal part that further controls virus blocking and virus trapping properties. The thickness of the extremely small portion in the film thickness direction corresponds to the diameter of the cellulose dense phase particles because the porous hollow fiber is produced by the micro phase separation method. Therefore, its thickness is 2 μm or less.

Examples of the method for producing a porous hollow fiber made of regenerated cellulose of the copper-ammonia method used in the method of the present invention include:
Cellulose linter (α-cellulose content 96% or more,
A solution obtained by dissolving an average molecular weight of 2.6 × 10 ⁵ ) in a copper ammonia solution prepared by a known method at a concentration of 8 wt% is used as a spinning stock solution. This spinning dope is obtained by using an acetone / ammonia / water mixed solution as a coagulant and a hollowing agent to cause microphase separation, and then coagulating and regenerating. Here, the micro phase separation means a state in which a concentrated layer or diluted layer of a polymer is dispersed in a solution as particles having a diameter of 0.02 to several μm and is stabilized. The occurrence of microphase separation can be confirmed by direct visual observation of the devitrification phenomenon of the yarn during spinning, or by electron microscopic observation of the yarn after spinning, in the presence of particles having a diameter of 1 μm or less and 0.02 μm or more.

A known method can be used for freeze-drying. The lower the freezing temperature and the shorter the freezing time, the better for maintaining the infectivity of the virus. For example, a method is preferred in which the hollow fibers trapped with viruses are instantaneously frozen in liquid nitrogen and immediately dried in vacuum. In order to freeze immediately after filtration,
It is desirable to freeze the module in its form, without disassembling the module and removing the hollow fibers. Disassembling the module before freeze-drying is not preferable because it not only increases the degree of contamination of hollow fibers with bacteria and makes it difficult to use it under aseptic conditions thereafter, but also reduces virus infectivity.

Prior to the description of the examples according to the method of the present invention, the definitions of the main technical terms (physical property values) used in the present specification and the measuring methods thereof will be shown below.

[Water Flow Rate Average Pore Diameter] A porous hollow fiber module made of regenerated cellulose prepared by the copper-ammonia method was prepared, and the outflow amount of water from the hollow fiber was measured in the module state, and the water flow rate average pore diameter was calculated from the equation (2). It was

V: Outflow rate (ml / min) T: Film thickness (μm) ΔP: Pressure difference (mmHg) A: Membrane area (m ² ) Prρ: Porosity (−) μ: Water viscosity (cP) Porosity The ratio Prρ was calculated by the equation (3) from the apparent density ρaw at the time of water swelling and the polymer density ρp. In the case of cellulose, ρp =
1.561 was used.

Prρ (%) = (1-ρaw / ρp) × 100 (3) [Average molecular weight] Intrinsic viscosity number [η] (ml / g) measured in copper ammonia solution (20 ° C) The average molecular weight (viscosity average molecular weight) Mv is calculated by substituting.

Mv = [η] × 3.2 × 10 ³ (4) [Minimum in-plane porosity] After embedding porous hollow fiber made of regenerated cellulose with copper ammonia method with acrylic resin, ultra microtome (LKB
Using a glass knife attached to a company (Sweden Ultratome III8800 type), samples with a thickness of about 1 μm are sequentially cut out from the outer wall surface along the film thickness direction. The sample sections are deembedded with chloroform, and then electron micrographs of each section are taken. The number of pores having a radius of (r) to (r + dr) per 1 cm ^{2 of the} target slice is indicated as N (r) dr. The third and fourth order average pore radii (3 and 4, respectively) are defined by the following equations.

Average pore size is It is calculated from the equations (5) and (6). The average pore size is calculated from the electron micrograph of each section, and the plane showing the minimum in-plane pore size is determined from the drawing of the average in-plane pore size from the inner wall surface. The porosity of the determined surface is defined as the minimum in-plane porosity. The minimum in-plane porosity is calculated by the equation (7).

[Virus inhibition coefficient] Number of viruses per unit volume of aqueous solution to be filtered
No, number of viruses per unit volume of filtrate permeated through membrane N
Then, it is defined by the following equation (8).

φ = −log (N / No) (8) (Effect of the invention) According to the present invention, a virus is immobilized from a liquid in which the virus is present in the human body safely without lowering the infectivity of the virus. Can be concentrated. Then, an immunological test for infectious viruses such as serum hepatitis virus and AIDS virus can be performed with the virus fixed in the pores of the hollow fiber. In addition, it can be used for separating or immobilizing a target virus or virus group from a virus solution in which a plurality of viruses are mixed.

(Example) Hereinafter, an example of a method for immobilizing a virus according to the method of the present invention will be described.

(Example 1) Cellulose linter (α-cellulose content of 96% or more,
The average molecular weight of 2.6 × 10 ⁵ ) was decomposed in a copper ammonia solution adjusted by a known method at a concentration of 8 wt%, and filtered and defoamed,
This was the spinning dope. The spinning dope is 2.5 ml / min from the outer spinning outlet (outer diameter 2 mmφ) of the annular spinning port, while as a hollowing agent,
Acetone 45wt% / Ammonia 0.575wt% / Water 54.425wt%
45wt% of acetone / 0.5mm of ammonia at 1.7ml / min from the central spinning outlet (outer diameter 0.6mmφ)
It was directly discharged into a mixed solution (coagulant) of 75 wt% / water 54.425 wt% and wound at a speed of 12 m / min. The transparent blue fibrous material immediately after discharge gradually became white, microphase separation occurred, and subsequently solidification occurred, and the shape as a fiber was maintained. Then, it was regenerated with a 2 wt% sulfuric acid aqueous solution and then washed with water. The hollow fiber after washing with water was replaced with acetone to replace the water content inside the hollow fiber, and then vacuum-dried in a 10% stretched state (25 ° C. × 1.5 hours). The thus obtained copper-ammonia method regenerated cellulose porous hollow fiber had an inner diameter of 300.2 μm, a film thickness of 28.5 μm, a water flow average pore diameter of 10.8 nm, and a minimal in-plane porosity of 28%. (1)
The value calculated from the formula was 9.4.

Coliphage φ × 174 (IFO20009, virus diameter 25nm)
Was infected with E. coli (IFO13898) to prepare a phage stock solution. The phage concentration was 1.9 × 10 ⁹ (PFU / ml) as determined from the number of plaques formed by the agar overlay method. The phage stock solution was fed into the hollow portion of the hollow fiber through the inlet of the glass mini-module consisting of 100 copper-ammonia method regenerated cellulose hollow fibers, with the hollow portion outlet closed,
11 ml of the phage stock solution was filtered at a pressure of 200 mmHg to obtain 5 ml of the filtrate. The phage concentration in 1 ml of the filtrate was quantified by the plaque method, but no phage was confirmed. Therefore, φ was 9.2 or more. Next, open the module outlet, and while supplying physiological saline to the module with a peristaltic pump at a flow rate of 10 ml per minute, adjust the pressure applied to the membrane to about 200 mmHg and thoroughly wash the hollow part and the inside of the hole. did. No phage was confirmed by the plaque formation method in the filtrate immediately before the end of washing and in the washing liquid that passed through the hollow portion. After the module was washed, the module was frozen in liquid nitrogen as it was, placed in a chamber of a freeze dryer (FD-1) manufactured by Tokyo Rikaki Co., Ltd., and the hollow fiber was freeze dried for 24 hours.

About 100 mg of freeze-dried hollow fibers (40 hollow fibers) to measure the amount of phage trapped in the hollow fiber pores
Was dissolved in 10 ml of 0.5% cellulase solution (pH 5.0, 0.1N acetic acid buffer solution dissolved in Yakult's Onozuka R-10) at 37 ° C to decompose the cellulose, and the pH was adjusted to 7.0.
After centrifugation at rpm for 10 minutes, the phage concentration in the supernatant was measured by the plaque method. Since the phages immobilized on the hollow fibers were infected with Escherichia coli and formed clear plaques, it was confirmed that the phages were immobilized in the pores while maintaining their infectivity. The phage concentration in the supernatant was 3.8 × 10 ⁷ (PFU / ml). Thus hollow fibers
There are 3.8 × 10 ⁹ PFU of phage in 1g, and 1g of medium
The virus density inside was higher than the stock solution.

(Comparative Example 1) Cellulose concentration 10%, ammonia concentration 7%, copper concentration 3.6
%, And a viscosity of 2000 poises of a copper ammonia regenerated cellulose stock solution was added to the outer side of the double annular spinneret (outer diameter 5 m
m) at 20 ml / min, and at the same time, perchlorethylene was extruded at 5 ml / min from a central tube having a diameter of 1 mm. Next, the extruded linear spinning dope was dropped directly into the air by 300 mm, then introduced into an 11 wt% NaOH aqueous solution, and passed through an 8 m coagulation bath at a winding speed of 100 m / min. The yarn drawn from the coagulation bath was washed with water, regenerated with 3% sulfuric acid, and then washed again with water.
Then, it was dried at 130 ° C., and then the organic solvent in the hollow portion was extruded. The hollow fiber thus obtained has an inner diameter of 265
μm, film thickness 25 μm, average pore size of water flow velocity was 8 nm, and in-plane porosity was 10% or less. The value calculated from the equation (1) was 9.7.

The same phage stock solution as in Example 1 was filtered to obtain 5 ml of filtrate. No phage was confirmed in the filtrate by the plaque method. When the hollow fibers were decomposed by cellulase treatment in the same manner as in Example and the concentration of phage in the centrifugal supernatant was quantified by the plaque method, no phage was confirmed. Therefore, the phage was hardly trapped in the hollow fiber pores.

(Example 2) A porous hollow fiber was produced in the same manner as in Example 1 under the following conditions. Cellulose concentration: 7%, hollow agent composition; acetone / ammonia / water = 40 / 0.56 / 59.44, coagulant composition; same as hollow agent composition, hollow agent discharge rate; 2.2 ml / min, spinning dope discharge rate;
2.2 ml / min., Spinning speed 10 m / min. The hollow fiber obtained had an inner diameter of 238.4 μm, a film thickness of 29.0 μm, an average pore diameter of 48.6 μm,
The minimum in-plane porosity was 30.2%. The value calculated from the equation (1) was 1.2.

Phage stock solution of φ × 174 (phage concentration 7.3 × 10 ⁷ PFU / ml was filtered in the same manner as in Example 1. The virus concentration in the filtrate was
It was 7.1 × 10 ⁶ PFU / ml. Therefore, φ was 1.01. After thoroughly washing with physiological saline as in Example 1,
Lyophilized. No phage was found in the filtrate at the end of washing.

Cellulase treatment was carried out in the same manner as in Example 1, and the amount of phage trapped in the hollow fiber pores was measured. As a result, the concentration of phage in the centrifugal supernatant was 7.9 × 10 ⁵ PFU / ml. Therefore, 7.9 × 10 ⁷ PFU of phage was present in 1 g of the hollow fiber. Therefore, the phage density in 1 g of the medium was higher than that of the stock solution.

(Example 3) A porous hollow fiber was produced in the same manner as in Example 1 under the following conditions. Cellulose concentration: 8%, hollow agent composition; acetone / ammonia / water = 45 / 0.575 / 54.425, coagulant composition; same as hollow agent composition, hollow agent discharge rate; 1.7 ml / min, spinning dope discharge rate; 2.5 ml / min., spinning speed 10m / min. The obtained hollow fiber had an inner diameter of 310.84 μm, a film thickness of 22.9 μm, and an average pore diameter of 20.9 μm.
m, and the minimum in-plane porosity was 27.5%. The value calculated from the equation (1) was 4.4.

In the same manner as in Example 1, the φ × 174 stock solution of phage (phage concentration 1.1 × 10 ¹⁰ PFU / ml) was filtered. The virus concentration in the filtrate was 7.9 × 10 ⁵ PFU / ml. Therefore, φ was 4.1. It was thoroughly washed with physiological saline as in Example 1, and then freeze-dried. At the end of washing, no phage was confirmed in the filtrate and the washing solution that passed through the hollow portion.

Cellulase treatment was carried out in the same manner as in Example 1, and the amount of phage trapped in the hollow fiber structure was measured. As a result, the concentration of phage in the centrifugation supernatant was 2.1 × 10 ⁸ PFU / ml. Therefore, 2.1 × 10 ¹⁰ PFU of phage was present in 1 g of the hollow fiber. Therefore, the phage density in 1 g of the medium was higher than that in the stock solution, and the phage were concentrated and trapped in the hollow fiber pores.

Example 4 The same porous hollow fiber as in Example 2 was used. T4 phage (IFO20004) was used as an E. coli phage, and E. coli (IFO13168) was used as a host bacterium. The virus diameter of T4 was 85 nm.
Therefore, the value calculated from the equation (1) was 1.3. The phage concentration of the phage stock solution was 9.5 × 10 ⁹ (PFU / ml). This stock solution was filtered as in Example 1. The virus concentration in the filtrate was 4.3 × 10 ⁶ (PFU / ml). Therefore, φ
Was 3.3. Subsequent to the separation, washing was carried out with a physiological saline solution in the same manner as in Example 1, followed by freeze-drying. At the end of washing, no phage was confirmed in the filtrate and the washing solution that passed through the hollow portion.

Cellulase treatment was carried out in the same manner as in Example 1, and the amount of phage trapped in the hollow fiber pores was measured. As a result, the concentration of phage in the centrifugation supernatant was 1.2 × 10 ⁸ PFU / ml. Therefore, 1.2 × 10 ¹⁰ PFU of phage was present in 1 g of the hollow fiber.

Using the same hollow fiber and phage stock solution as above, filtration and washing were performed under the same conditions. The hollow fiber was vacuum-dried at room temperature (25 ° C) for 24 hours without freeze-drying, or left indoors (air-dried) for 24 hours. No phage was found in the filtrate during washing and the washing liquid that passed through the hollow portion. When the amount of phage trapped in the hollow fiber pores was measured by cellulase treatment in the same manner as in Example, the phage concentration in the centrifugation supernatant was 3.7 × 10 ⁷ PFU / ml (vacuum dried), 2.3 ×. 10 ⁶ PF
It was U / ml (air dried). Therefore, infectious phages were present in only 3.7 × 10 ⁹ PFU (vacuum dried) and 2.3 × 10 ⁸ PFU (air dried) in 1 g of the hollow fiber, respectively.

(Comparative Example 2) A porous hollow fiber was produced under the above conditions in the same manner as in Example 1. Cellulose concentration: 7%, hollow agent composition; acetone / ammonia / water = 40 / 0.56 / 59.44, coagulant composition; same as hollow agent composition, hollow agent discharge rate; 3.5 ml / min, spinning dope discharge rate;
2.2 ml / min., Spinning speed 10 m / min. The resulting hollow fiber had a film thickness of 19.2 μm and an average pore diameter of 56.3 nm. The value calculated from the equation (1) was 0.5.

In the same manner as in Example 1, φ × 174 stock solution of phage (phage concentration 6.3 × 10 ⁹ (PFU / ml) was filtered. The virus concentration in the filtrate was 1.8 × 10 ⁹ (PFU / ml). is 0.
Was 5. It was thoroughly washed with physiological saline as in Example 1, and then freeze-dried. At the end of washing, no phage was confirmed in the filtrate and the washing solution that passed through the hollow portion.

Cellulase treatment was carried out in the same manner as in Example 1, and the amount of phage trapped in the hollow fiber pores was measured. As a result, the concentration of phage in the centrifugal supernatant was 3.3 × 10 ⁷ PFU / ml. Therefore, 3.3 × 10 ⁹ PFU of phage was present in 1 g of the hollow fiber. The phage density in 1 g of medium was lower than that of the stock solution.

(Example 5) Cellulose linter (α-cellulose content of 96% or more,
An average molecular weight of 2.6 × 10 ⁵ ) was dissolved at a concentration of 8 wt% in a copper ammonia solution prepared by a known method. This solution at 25 ℃
After uniformly casting on a glass plate placed in an acetone / ammonia vapor atmosphere of 1 to 10 minutes under the atmosphere, 2 wt of acetone 25 wt% / 2.8% ammonia water at 20 ° C.
Glass plate in a mixed solution of t% / 73 wt% water for 10 minutes to 60
Soak for 20 minutes, then soak in a 2wt% sulfuric acid aqueous solution at 20 ° C for 10 minutes, wash with water, and then rinse with a filter paper to remove water.
The membrane was immersed in acetone (100 wt%) at ℃ for 15 minutes, the water content in the membrane was replaced with acetone, and it was sandwiched between filter papers and air-dried to obtain regenerated cellulose porous flat membranes having different average pore diameters.

The obtained film had an average pore size of 63 nm and a film thickness of 133 μm.
The value calculated from the equation (1) was 4.0. This membrane was mounted on a Millipore stainless steel filter holder, and the same stock solution of T4 phage (2 × 10 ⁷ PFU / ml) as in Example 4 was applied at a pressure of 200.
Vertical filtration was performed with mmHg to obtain 5 ml of a filtrate. The virus concentration in the filtrate was 3.2 × 10 ² (PFU / ml) and φ was 4.8. Then, after thoroughly washing with physiological saline, it was freeze-dried as in Example 1. No phage was found in the filtrate during washing. Cellulase treatment was carried out in the same manner as in Example 1 to quantify the amount of phage trapped in the membrane.
Phage of × 10 ⁷ PFU were captured.

Claims (2)

[Claims] 1. A porosity comprising a copper-ammonia method regenerated cell roll having a minimum in-plane porosity Pr (%) of 10 ≦ Pr ≦ 80 and a film thickness T (μm) of 10 ≦ T ≦ 100 and satisfying the formula (1). A liquid containing a virus having a virus diameter of V (nm) is substantially vertically filtered using a hollow hollow fiber to trap the virus in the pores of the membrane, and then freeze-dried into the pores. A method for immobilizing a virus, which comprises immobilizing the virus. (However, D represents water flow velocity average pore diameter D (nm)) 2. A porous membrane is arranged in the order of a minimum portion, a portion larger than the minimum portion, and another minimum portion between the in-plane average pore diameters at the inlet and the outlet of the in-membrane through-hole. The method for immobilizing a virus according to claim 1, which is a porous hollow fiber made of regenerated cellulose having a copper-ammonia method having at least one set of the above structure in the film thickness direction.