JP5721336B2 - Virus adsorption material - Google Patents

Description

  The present invention relates to a virus adsorption material.

  Preventing inhalation of pathogenic viruses such as influenza virus into the body during breathing is extremely important in preventing infection and preventing spread of infection.

  However, it is difficult to protect against the inhalation of pathogenic viruses. For example, the most familiar normal mask does not provide sufficient anti-suction effect even if it is worn correctly. There is a problem that the effect of preventing the virus from being sucked is hardly obtained due to the sucking from. Moreover, if a mask with a high virus collection rate such as an N95 standard mask is correctly mounted, it is possible to prevent inhalation of the virus to a considerable extent (about 95%), but there is a problem that it is stuffy.

  It is known that hemagglutinin present on the surface of influenza viruses of type A, B and C is adsorbed to erythrocytes, and based on this finding, an adsorbent adsorbed on the hemagglutinin site of influenza virus is supported on a sheet. There has been proposed an influenza virus trapping filter or an influenza virus trapping filter in which an adsorbent adsorbed on a hemagglutinin site is adsorbed on a porous material and the porous material is supported on a sheet (Patent Document 1).

  On the other hand, studies on the prevention and prevention of influenza viruses have been made on influenza viruses. For example, glycoproteins having a molecular weight of 100,000 or more and molecular weights of 30,000 obtained from cell membranes of chicken erythrocytes capable of binding to influenza viruses. Contains a base sequence complementary to the base sequence of the target region containing the anti-influenza virus agent (Patent Document 2) consisting of 5,000 or less glycoproteins and the translation start codon AUG of the influenza virus PB2 gene or PA gene An anti-influenza virus agent (Patent Document 3) characterized by containing an oligonucleotide and a liposome stable in blood is known.

  Further, the present inventor found that when red blood cells were immobilized with formalin in the presence of the ALSVER solution used as a blood coagulant, the function of the virus receptor did not deteriorate at all. In addition, lyophilized products of immobilized red blood cells maintain high influenza virus binding ability for several years or more at room temperature by adding glucose, and almost the same effect can be obtained with disaccharides containing glucose. In other words, immobilized erythrocytes with high virus capture ability can be obtained by treating guinea pigs other than chickens and human O-type erythrocytes with the same stable treatment as chicken erythrocytes. Influenza virus capture filter using headline, formalin-immobilized red blood cells Proposes (Patent Document 4).

JP 2002-58730 A Japanese Patent Laid-Open No. 10-316583 Japanese Patent Laid-Open No. 10-313872 JP 2008-19247 A

  However, Patent Document 1 uses a lysoganglioside / poly-L-glutamic acid conjugate, chicken erythrocyte extract glycoprotein, sulfatide, etc. as a substance adsorbed on the hemagglutinin site of influenza virus, and has a binding ability to influenza virus. It is not stable over the long term.

  Moreover, although patent document 2 captures influenza virus, there exists a problem that processing operation for refine | purifying glycoprotein without reducing a binding ability is complicated, and patent document 3 suppresses the proliferation of influenza virus. There is a problem that it cannot be used for capturing influenza viruses.

  Furthermore, in the case of Patent Document 4, although the influenza virus capture filter captures influenza virus, there is a problem that formalin-immobilized erythrocytes cannot stably maintain the capture ability over a long period of time in an external environment.

  In addition, the formalin-fixed erythrocytes are removed from the filter while adsorbing the virus, enter the body, released from the receptor by the receptor-destroying enzyme of the virus itself, adsorbed to the receptor on the surface of the virus-sensitive cell, and infected. There was also concern that it would be established.

  The present invention can be easily produced, can stably maintain virus adsorption ability for a long period of time, and adsorbs virus particles on the entire surface of erythrocytes and prevents the virus particles adsorbed on the receptor from dropping off with the receptor. It is intended to provide materials.

That is, the present invention includes a formalin-immobilized red blood cell having a virus-adsorbing ability and a fiber lattice material, and an aqueous suspension of the formalin-immobilized red blood cell that does not contain a binder is equivalent to a fiber lattice material. A material for adsorbing a virus, which is further coated with a fiber lattice material, which is attached to the fiber lattice material that is dried at 4 ° C. to 37 ° C. and carries the formalin-immobilized red blood cells,
The virus is either influenza virus or Newcastle disease virus, the formalin-immobilized erythrocytes are chicken erythrocytes immobilized with formalin, and the fiber lattice material is made of silk or cotton fibers, virus adsorption timber which is characterized in cloth or puffy der Rukoto.

In the present invention, a formalin-immobilized erythrocyte aqueous suspension obtained by suspending formalin-immobilized erythrocytes having virus-adsorbing ability in water not containing a binder is uniformly attached to a fiber lattice material at 4 ° C. to 37 ° C. A method for producing a virus-adsorbing material, comprising drying and supporting the formalin-immobilized red blood cells on a fiber lattice material, and further covering with a fiber lattice material .

  According to the present invention, since formalin-immobilized erythrocytes that efficiently adsorb viruses are stably maintained for a long period of time, the effect of capturing viruses can be maintained over a long period of time, and the immobilized erythrocytes can be used as a fiber lattice material. Since it is enclosed, there is no possibility that the adsorbed virus will be taken into the body together with the immobilized red blood cells. Moreover, since all of the commercially available fiber products have a fiber lattice, it is easy to adsorb viruses by a simple method in which the immobilized red blood cells having the ability to adsorb viruses are sprayed on this and then covered with a fiber lattice material. There is also an advantage that it can be used as a textile product for medical and daily use such as gauze, sheets, curtains, pillow covers, pajamas, lab coats, examination clothes, masks, etc., which are excellent in performance.

  Further, according to the present invention, since the fiber lattice material is silk or cotton fiber, without producing a fiber product for virus adsorption, the fiber product used in the most wide range of daily life holds formalin-immobilized red blood cells, By covering this with a fiber lattice material, it has the advantage that it can be set as various virus adsorption materials.

  Further, according to the present invention, a formalin-immobilized chicken erythrocyte having strong influenza virus or Newcastle disease virus adsorption ability is encapsulated in a silk or cotton gauze or cloth, whereby a gauze product excellent in influenza virus adsorption ability, for example, a mask, is obtained. And the mask has the advantage that it can efficiently capture influenza without inhibiting respiration.

The present invention comprises a formalin-immobilized erythrocyte having a virus-adsorbing ability and a fiber lattice material, and an aqueous suspension of the formalin-immobilized erythrocyte and not containing a binder is evenly divided into a fiber lattice material. A material for adsorbing a virus, wherein the fiber lattice material attached and dried at 4 ° C. to 37 ° C. and supporting the formalin-immobilized red blood cells is further coated with a fiber lattice material,
The virus is either influenza virus or Newcastle disease virus, the formalin-immobilized erythrocytes are chicken erythrocytes immobilized with formalin, and the fiber lattice material is made of silk or cotton fibers, a virus adsorption timber, wherein the fabric or floss silk der Rukoto.

In the present invention , a formalin-immobilized erythrocyte aqueous suspension in which formalin-immobilized erythrocytes having virus adsorption ability are suspended in water not containing a binder is evenly adhered to the fiber lattice material, and the temperature is 4 ° C. to 37 ° C. The method of producing a virus-adsorbing material is characterized in that the formalin-immobilized erythrocytes are supported on a fiber lattice material after drying, and further covered with a fiber lattice material .

In the present invention, the viruses to be adsorbed target, human influenza virus to mammals or birds as a host, Men u Castlebridge virus (NDV) and the like.

In the present invention, erythrocytes fixed with formalin include chicken erythrocytes.

The red blood cells of chickens, Leghorn, Minorca, Tokyo silkie, Shamo, Nagoya Cochin, southern chicken, Hinai chicken, Tosa Giraud, Plymouth Rock, bantams, such as the crowning in the morning, for egg collection, for the meat, such as for appreciation Regardless of the application of the chicken, it can be used in the present invention.

  As a method for immobilizing these erythrocytes, a known method can be used. For example, erythrocytes can be efficiently immobilized by the method described in Patent Document 4.

  Specifically, for example, erythrocytes can be immobilized by contacting with a formalin solution in the presence of monosaccharides or disaccharides.

  The concentration of formalin in the immobilization treatment may be usually 3 to 20 v / v% (that is, 1.11 w / v% to 7.4 w / v% aqueous formaldehyde solution), and preferably 5 to 15 v / v%. Moreover, the density | concentration of a monosaccharide or a disaccharide is 0.1-15 w / v% normally, Preferably it is 0.5-10 w / v%, Most preferably, it is 1-7 w / v%. This immobilization treatment is carried out by allowing the solution to stand with stirring at room temperature of 10 to 25 ° C. for 1 to 15 days, preferably 3 to 10 days. Red blood cells fixed with a formalin solution may be centrifuged and washed using, for example, distilled water, physiological saline, -PBS, or the like.

  In addition, prior to immobilization with formalin, erythrocytes can be changed from hemoglobin (red) to methemoglobin (bright red) by bubbling carbon monoxide gas in the presence of monosaccharide or disaccharide. Good. The pH of this bubbling solution is 6.0 to 7.5, preferably 7.1 to 7.4, and suitable solutions to be used include Arsbar solution (pH 6.1), -PBS, Hanks. Liquid etc.

  In bubbling, 20 to 200 mL of red blood cell suspension is put in a suitable container, and carbon monoxide gas is blown from a cylinder at a rate of 40 to 100 bubbles for 1 to about 3 to 10 minutes so that red hemoglobin becomes deep red or bright red. This can be done by changing the color to methemoglobin. The liquid temperature during bubbling is suitably 10-25 ° C. By performing bubbling with carbon monoxide gas in the presence of monosaccharides such as glucose and disaccharides such as sucrose, it is possible to prevent hemolysis during operation, denaturation and destruction of erythrocyte virus receptors, and reduction in binding power. The concentration of the monosaccharide or disaccharide is usually 0.1 to 15 w / v%, preferably 0.5 to 10 w / v%, particularly preferably 1 to 7 w / v%. This bright red erythrocyte can be advantageously used in an antibody titer measurement test against viral infection after formalin fixation. Red blood cells treated with carbon monoxide gas can be subjected to immobilization treatment with formalin after removing impurities by centrifuging and washing with Arsbar solution, -PBS, physiological saline and the like.

  The fiber lattice material of the present invention may be any material, material, and manufacturing method as long as the fibrous material is processed into a relatively thin and wide plate shape and has a lattice made of fibers. In addition to the lattice in which the vertical and horizontal fibers are orthogonal to each other, the lattice may be one in which the fibers intersect at various angles, or the lattice may be superimposed on each other.

  Examples of fibers constituting the fiber lattice include cotton, hemp, silk, linen, moya, wool, cashmere, glass wool, and other natural fibers derived from animals, minerals, cupra, rayon, polynosic, lyocell, and acetate. Recycled fibers such as fleece, fibers produced from synthetic fibers such as nylon, vinylon, polyester, acrylic and polyurethane, plant fibers such as mulberry, honey and husk.

Any of these fibers can be suitably used. However, when silk is used as raw silk, the immobilized red blood cells and fibers are more strongly fixed by the glue components such as sericin contained in the raw silk and the irregularities and protrusions of the fibers. More preferable. In this case, in addition to the encapsulation by the fiber lattice of the present invention, the immobilized red blood cells are adhered to and retained by the raw silk itself due to the adhesive force of the raw silk. Supplementary power will be further strengthened.
Furthermore, natural fibers other than silk also have irregularities and protrusions in the fiber before purification as fibers, and contain proteins, wax substances, resinous substances, fats, etc., but denature the immobilized red blood cells. If the adhesion between the fiber and the immobilized erythrocyte is strengthened, unpurified natural fiber can be preferably used.

  Further, as described above, the fiber lattice material may be a material obtained by processing a large number of fibrous substances into a relatively thin and wide plate shape, regardless of the material, the material, and the manufacturing method. It may be a kind of so-called cotton.

  For example, the production method can be classified into woven fabric, knitted fabric, lace, felt, non-woven fabric, etc., and any of these can be used preferably.

  Specific examples of these include gauze, towels, towel towels, cotton, Japanese paper, and western paper.

  In addition, the fiber lattice material of the present invention may be any fiber lattice that has a size that retains immobilized red blood cells and does not allow the passage of the immobilized red blood cells and has a size that does not significantly impair air permeability. Even if it is a thing, it can be used conveniently. Further, the size of the fiber lattice does not have to be uniform, and fiber lattices of various sizes may exist in one fiber lattice material.

  For example, the size of the fiber lattice may be 200 to 5000 nm, preferably 450 to 1000 nm.

  However, these are merely examples. For example, even if the fiber lattice is larger than the above, it is possible to make the range of the lattice by overlapping a plurality of fiber lattices, or to fix the immobilized red blood cells. Any fiber lattice that can be encapsulated can be suitably used in the present invention.

Furthermore, the number of immobilized red blood cells enclosed in the fiber lattice may be 1 × 10 ⁵ to 1 × 10 ⁸ per square centimeter, preferably 1 × 10 ⁶ to 1 × 10 ⁷ , particularly preferably 2 × ¹⁰ 6 ~1 × ^{10 7,} and most preferably may be encapsulated 2 × ^{10 6} ~5 × ¹⁰ 6 cells become so.

  Encapsulation can be easily performed by holding the immobilized red blood cells on a carrier and covering the outside of the carrier with a fiber lattice material.

  As the carrier for immobilized erythrocytes, any carrier can be suitably used as long as it does not impair the ability of the erythrocytes to adsorb viruses and is air-permeable to the extent that it can adsorb airborne viruses. It is also possible to use a fiber lattice material itself, a material constituting the fiber lattice material having an appropriate shape such as a plate shape or a spherical shape, or a porous material such as sponge, sponge, or porous ceramic.

Specifically, for example, the suspension of immobilized erythrocytes is uniformly adhered to the carrier by means such as spraying or spraying, or the carrier is immersed in the suspension of immobilized erythrocytes and then dried. The spread surface of the immobilized red blood cells may be folded inward, or the carrier may be covered with a fiber lattice material having the same or different lattice, and then sewn or molded into a desired shape.
The medium for suspending the immobilized erythrocytes may be any medium that does not impair the ability to adsorb viruses and does not contain a binder . For example, simple media such as distilled water, isotonic saline, physiological saline, Arsbaal solution, glucose, etc. A saccharide solution, Dulbecco's phosphate buffer and the like are preferable. Moreover, drying can be implemented at 4 degreeC-37 degreeC.

  Examples of the molded article of the virus adsorbent of the present invention include clothing such as outer clothing, inner clothing, and inner clothing, medical products such as surgical clothing, bedding such as bed covers, pillow covers, sheets, and mats, masks, and towels. It is done.

  Among these, a mask is particularly preferable, and a mask in which a carrier encapsulating immobilized red blood cells is coated with gauze or gauze in which immobilized red blood cells are encapsulated, or a normal mask in which the immobilized red blood cells are encapsulated (what is cotton in this case) It is easy to use what is covered with the original meaning of silk fibers that are tangled together, and is effective in defense against infection.

  Further, by using such a mask and the outer garment together with a hood, even when the risk of infection is very high, the virus can be completely captured and effective in preventing infection.

  Furthermore, since the immobilized red blood cells used in the present invention are fixed with formalin, the virus adsorbing ability is not lost even after the adsorbed virus is inactivated by formalin fumigation or short-time ultraviolet irradiation. The virus-adsorbing material of the present invention is characterized by being reusable after the inactivation treatment.

  In the present invention, the measurement of viral hemagglutination titer (HA titer) was carried out as follows.

  The method of measuring viral hemagglutination titer (HA titer) using fresh chicken erythrocytes is a male male of Nagoya Cochin species obtained by collecting blood with a syringe containing 3.8% sodium citrate 1/5 of the scheduled blood collection amount ( About 12 months after hatching, and washed with PBS three times with PBS, -PBS was added to the resulting precipitated erythrocytes to form a 10% erythrocyte suspension, and stored in a 4 ° C ice chamber. Used within 3 days. In the HA test, it was further diluted with -PBS so as to be 0.25% by volume.

  For dilution of the specimen, 0.2% by volume of horse serum inactivated by heating at 56 ° C. for 30 minutes was added-diluted continuously twice with PBS, and 0.25% by volume of the above-described 0.25% by volume was suspended in red blood cells. Add an equal amount of suspension, shake and mix well, let stand at room temperature for 1 hour, determine the presence or absence of erythrocyte aggregation, and calculate the reciprocal of the maximum dilution factor before adding erythrocytes of the material that showed obvious aggregation. The hemagglutination titer (HA titer) was used.

  In addition, measurement of viral hemagglutination titer using virus erythrocytes using freeze-dried erythrocytes is carried out by first suspending freeze-dried erythrocytes in -PBS so as to be 10% by volume, and storing in this state in a 4 ° C. ice room for one year. At the same time, the HA test and the HI test (red blood cell aggregation inhibition test for measuring the antibody titer in blood) were performed in the same manner as in the case of using fresh red blood cells.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the examples.

Example 1
(1) Manufacture of formalin-fixed erythrocytes Using a syringe pre-filled with an equal volume of Alsbar's solution (） 1), about 13 months after hatching, from each of the three sub-wing veins of white leghorn and Nagoya cochin Each 5 mL, a total of 30 mL of blood was collected and used as pooled blood.
(Note 1) Composition of Alsbar solution 2.05% by weight of glucose, 0.42% by weight of sodium chloride, 0.80% by weight of sodium citrate, 0.55% by weight of citric acid, 96.18% by weight of distilled water

  60 mL of an equal volume mixture of blood and Alsbar solution obtained above was centrifuged three times with Dulbecco's -PBS (1500 rpm, 5 minutes), and washed repeatedly to accommodate about 10 mL of precipitated red blood cells.

Subsequently, -PBS + 2% by weight glucose was poured so as to be 10% by volume of precipitated red blood cells.
The obtained fresh erythrocyte suspension is put into a 1 L transparent glass bottle with a stopper, and carbon monoxide gas is blown from a cylinder at a rate of 60 bubbles per minute for about 5 minutes to convert red hemoglobin into crimson methemoglobin. I confirmed that it changed.

  Glucose 2 volume% so that the obtained crimson methemoglobin erythrocyte may be 10 volume%. The suspension was added to Dulbecco's PBS containing 10% by volume of commercially available formalin and left standing at 4 ° C. for 7 days. Meanwhile, once a day, the container was shaken by hand to float the settled red blood cells. After fixing with formalin for 7 days, centrifugal washing with distilled water containing 2% by weight of glucose (1500 rpm, 5 minutes) is repeated three times, and the precipitated immobilized red blood cells are again returned to the original solution (distilled water with 2% glucose by weight). The mixture was suspended in 10% by volume and stirred as it was with a magnetic stirrer, and dispensed aseptically to 2 mL into a 20 mL capacity lyophilization vial. This suspension was quickly frozen with dry ice acetone, then lyophilized as it was, filled with nitrogen gas, covered with a rubber stopper and an aluminum cap, wrapped and stored at room temperature as a freeze-dried product of fixed erythrocytes. .

    PBS composition: salt 0.8% by weight, potassium chloride 0.02% by weight, dibasic sodium phosphate 0.115% by weight, primary potassium phosphate 0.02% by weight, calcium chloride 0.01% by weight, magnesium chloride -Hexahydrate 0.01% by weight dissolved in phosphate buffered saline.

    -PBS composition: the above PBS composition excluding calcium chloride and magnesium chloride.

(2) Production of virus adsorbing material using gauze Formalin obtained in (1) above on the surface of two sheets of gauze with a fiber lattice of 0.8 to 1.2 mm and a size of 5 cm × 5 cm Immobilized red blood cells were suspended in distilled water so as to be 8 × 10 ⁸ cells / mL, and 2 mL thereof was evenly dispersed. Subsequently, the gauze was blown and dried at 25 ° C. for 24 hours. The gauze carrying the formalin-immobilized erythrocytes is further overlapped with a gauze having a fiber lattice of 0.8 to 1.2 mm, and sewn so as to form a square having a side of 0.5 cm. The material was obtained.

Further, a virus adsorbing material of Comparative Example 1 was obtained in the same manner as in the above Example, except that a suspension suspended in a distilled aqueous solution containing 2% by volume of PVA so as to be 8 × 10 ⁸ pieces / mL was used.

  Furthermore, using the immobilized erythrocytes in which the influenza virus receptor produced in Reference Example 1 described later was destroyed, the same procedure as in the above Example was performed to obtain a virus-adsorbing material of Comparative Example 2.

  The obtained three types of gauze were put into individual dishes, and 5 mL of the supernatant (3000 rpm, 5 minutes) of tissue culture solution containing 128 units of influenza virus obtained in Reference Example 2 described later and 35 mL of -PBS were dispensed. Using tweezers, stir for several seconds, leave these dishes at 4 ° C for 1 hour, then transfer the contents of the dishes together with gauze to a centrifuge tube, and measure the HA value of the supernatant after centrifugation at 3000 rpm for 10 minutes. By doing so, the adsorption degree with respect to influenza virus HAin was investigated.

The experimental results are as shown in Table 1.

<Result>
In the adsorption material of the present invention in which formalin-immobilized chicken erythrocytes were directly encapsulated in gauze, all 128 units of influenza virus HAin were adsorbed. However, in Comparative Example 1 in which formalin-immobilized erythrocytes were attached to gauze with PVA, 25% of influenza virus HAin remained in the solution without being adsorbed. Furthermore, in Comparative Example 2 using immobilized erythrocytes in which the influenza virus receptor was destroyed, the influenza virus HAin was not adsorbed at all.

Example 2
A silk layer having a thickness of 0.8 to 1.2 mm was layered on a silk cloth having a size of 5 cm × 5 cm. Next, the formalin-immobilized red blood cells obtained in the above (1) were suspended in distilled water so as to be 8 × 10 ⁸ cells / mL, and 2 mL thereof was evenly spread on the cotton layer. Subsequently, the cotton layer and the silk cloth were blown and dried at 25 ° C. for 24 hours. On the obtained cotton layer carrying formalin-immobilized red blood cells, a cotton layer having the same thickness was layered, and the same silk cloth was further stacked to form a four-layer structure. Then, the virus adsorbing material of the present invention was obtained by sewing so as to form a square having a side of 0.4 cm.

  Using the obtained material for adsorbing virus and the centrifugal supernatant of the tissue culture solution containing 128 units of influenza virus obtained in Reference Example 2 described later, the degree of adsorption to influenza virus HAin was examined in the same manner as in Example 1. . As a result, with the adsorbing material of the present invention, all 128 units of influenza virus HAin were adsorbed.

Example 3
In Example 2, the material for adsorbing the virus of the present invention was obtained in the same manner as in Example 2 except that cotton was used instead of cotton and cotton was used instead of silk.

  Using this virus-adsorbing material, the Newcastle disease virus obtained in Reference Example 3 was examined for the degree of adsorption of Newcastle disease in the same manner as in Example 1, using a centrifugal supernatant of a culture solution containing 256 units.

  Further, a virus adsorbing material similar to that of Comparative Example 1 and 2 of Example 1 was prepared, and the Newcastle disease virus obtained in Reference Example 3 was used to obtain a Newcastle disease virus using a centrifugal supernatant of a culture solution containing 256 units. The degree of adsorption was examined.

The results are as shown in Table 2.

<Result>
In the adsorbing material of the present invention, all 256 units of Newcastle disease virus HAin were adsorbed. However, in Comparative Example 1 in which formalin-immobilized erythrocytes were adhered with PVA, 25% of Newcastle disease virus HAin remained in the solution without being adsorbed. Furthermore, Newcastle disease virus HAin was not adsorbed at all in Comparative Example 2 using immobilized erythrocytes in which the receptor of Newcastle disease virus was destroyed.

Reference example 1
<Preparation of chicken erythrocytes with receptor-disrupted formalin>
For the destruction of receptors on the surface of formalin-immobilized erythrocytes, freeze the dried filtrate of Vibrio cholerae, which is used to remove nonspecific hemagglutinin inhibitors in the serum that hinder the serological diagnosis of influenza virus. RDE commercial products were used. First, RDE powder was dissolved in 20 mL of physiological saline according to the instruction manual. Subsequently, PBS was added so that the volume of the erythrocyte suspension in the vial containing the formalin-immobilized freeze-dried chicken erythrocytes stored in room temperature for 1 year obtained in Example 1 (1) was 2.0 mL. (The number of immobilized red blood cells is equivalent to 6.5 × 10 ¹⁰ cells / 2.0 mL).

For disruption of the erythrocytes, 4 mL of the RDE lysate is suspended by diluting the immobilized erythrocytes in PBS to a concentration of 2 × 10 ⁸ cells / mL after resuspension from the freeze-dried solution at 37 ° C. The receptor was destroyed by allowing RDE to act for 12 hours.

  Subsequently, the receptor (−)-immobilized erythrocytes were repeatedly washed by centrifugation three times with PBS (3000 rpm, 5 minutes), and then the settled erythrocytes were centrifuged with a tissue culture solution containing 128 units of influenza virus (3000 rpm, 5 minutes) and left at 4 ° C. for 1 hour, and then the HA titer is measured on the centrifugal supernatant (3000 rpm for 5 minutes), and the (−) receptor erythrocyte adsorbs the influenza virus particles. Existence and degree were examined.

  On the other hand, in the case of receptor (+)-immobilized erythrocytes, HA was treated and operated in the same manner as in the case of receptor (-)-immobilized erythrocytes except that RDE-free PBS was used in the series of experimental steps. The value was measured. The comparison results for the HAin adsorption capacities of both of these, receptors (−) and (+) are shown in Table 3 below.

  In the influenza virus adsorption test using formalin-immobilized chicken erythrocytes, 128 units of the original HA value remain as they are in the receptor (−)-immobilized erythrocyte group, and the ability to adsorb virus particles is completely lacking. On the other hand, in the receptor (+)-immobilized erythrocyte, 128 units of influenza virus were completely adsorbed by the receptor.

Reference Example 2 <Influenza Virus Tissue Culture>
(A) Influenza virus strain used As the influenza virus strain used, the A-type PR8 strain distributed by the former National Institute of Preventive Health was used. This PR8 strain is a reference species of influenza virus and was T in 1934. Francis Jr. is A / PR8 / 34 / (H1N1) type isolated from patients during the outbreak in Puerto Rico.

  Seed virus of type A PR8 strain was inoculated with chicken embryo chorioallantoic fluid on day 10 of hatching and cultured with chicken chorioallantoic membranes (Maitland method, Yamaguchi Medicine Vol. 9, No. 5, pages 1490-1510) (September 1960)) and subcultured and maintained, and dispensed into small test tubes and stored frozen at an ultra-low temperature of -40 ° C or lower.

(B) Tissue culture of influenza virus The culture solution was added to Hanks' solution with 3% healthy horse serum that had been inactivated by heating at 56 ° C for 30 minutes, to which penicillin and streptomycin were added. What was added so that it might become 100 units and 100 micrograms per mL, respectively was used. As the cultured tissue of influenza virus, a chicken embryo chorioallantoic membrane (CAM) minced tissue on the 11th day of hatching was used. The CAM was taken out, washed 3 times with Hanks solution, and then cut into pieces of about 1 mm ^{2 with} a scissors. Hank's solution was added to the tissue piece and left to stand for 5 minutes, and the supernatant was discarded. This operation was repeated three times to remove mixed blood cells and tissue ultrafine pieces, and then placed in a Spitz glass and centrifuged at 1000 rpm for 1 minute to obtain a precipitate. A suspension obtained by adding an equal amount of Hank's solution to the sediment is added dropwise to a culture bottle (round vaccine bottle with an internal volume of 20 mL) to form a cultured tissue, and 2 mL of the culture solution is dispensed thereto. The tissue culture system was established.

(C) Infectious titer measuring method of influenza virus liquid Influenza virus infectious titer was measured using the suspension culture method (Maitland method) of chicken embryo chorioallantoic membrane (CAM) as described in (b) above. That is, 1.8 mL of culture solution was dispensed into a culture bottle, and each dilution of virus diluted stepwise in decimal using Hanks's solution in a tissue culture system in which a chopped chorioallantoic membrane was dropped drop by drop. Each 0.2 mL of the solution was inoculated into two culture bottles and cultured at 37 ° C. Three days after the start of the culture, the centrifugal supernatant of the culture broth was subjected to hemagglutination, the maximum dilution (log) showing HA positivity was determined, and the value that the supernatant in two culture bottles was HA positive was designated as TCLD ₁₀₀ . .

Reference Example 3 <Newcastle disease virus (NDV virus tissue culture)>
(A) Newcastle disease virus strain used As the Newcastle disease virus, the “Tokyo strain” distributed by the former National Institute of Preventive Health was used. It is a lentigenic type separated in Tokyo immediately after the Second World War.
(Foundation, Kyoto Pasteur Research Institute report (2); March 1988, page 41)

  Seed virus of the Tokyo strain is inoculated with chicken embryo chorioallantoic fluid on the 10th day of hatching and cultured with chicken chorioallantoic membranes (Maitland method, Yamaguchi Medicine Vol. 9, No. 5, pages 1490-1510 (Showa) In September, 35)), the cells were subcultured and maintained, and dispensed into small test tubes and stored frozen at an ultra-low temperature of −40 ° C. or lower.

  In addition, culture of Newcastle disease virus and measurement of infectious titer were performed by the same method as that for influenza.

Claims (2)

A formalin-immobilized red blood cell having a virus adsorbing ability and a fiber lattice material, and an aqueous suspension of the formalin-immobilized red blood cell that does not contain a binder are evenly adhered to the fiber lattice material. A virus-adsorbing material obtained by further drying a fiber lattice material carrying the formalin-immobilized erythrocytes dried at a temperature of 37 ° C. to 37 ° C. with a fiber lattice material,
The virus is either influenza virus or Newcastle disease virus, the formalin-immobilized erythrocytes are chicken erythrocytes immobilized with formalin, and the fiber lattice material is made of silk or cotton fibers, virus adsorption timber which is characterized in cloth or puffy der Rukoto. A formalin-immobilized erythrocyte aqueous suspension in which formalin-immobilized erythrocytes having virus-adsorbing ability are suspended in water not containing a binder is uniformly attached to a fiber lattice material, dried at 4 ° C. to 37 ° C. and dried. A method for producing a virus-adsorbing material, wherein formalin-immobilized red blood cells are supported on a fiber lattice material and then covered with the fiber lattice material.