WO2007145179A1 - Immobilized erythrocyte stably holding virus receptor on the surface thereof, method of producing the same and use of the same - Google Patents

Abstract

[PROBLEMS] It is known that a chick erythrocyte suspension immobilized with a 10% formalin solution still holds the ability to bind to a viral erythrocyte agglutinin. In this method, however, a part of a receptor binding to the erythrocyte agglutinin loses its function and thus the avidity to the virus is lowered. Moreover, the erythrocytes cannot be preserved over a long period of time while sustaining the function of the receptor. Thus, such an erythrocyte suspension should be prepared in situ. [MEANS FOR SOLVING PROBLEMS] Stably immobilized chick erythrocytes can be obtained by conducting the following steps in the presence of a monosaccharide or a disaccharide: (a) the step of optionally subjecting a suspension of chick, guinea pig or human blood O type erythrocytes to a bubbling treatment with carbon monoxide; and (b) the step of treating the dark red erythrocyte suspension thus obtained with a formalin solution. Moreover, a freeze-dried product of the immobilized erythrocytes containing a monosaccharide or a disaccharide can remain stable over a long period of time of 5 years or longer and sustain a high avidity to a virus. A filter such as a mask carrying these immobilized erythrocytes is highly useful as a mask for preventing influenza viruses.

Description

 Specification

 Immobilized erythrocytes stably holding a virus receptor on the surface, method for producing the same, and use thereof

 Technical field

 [0001] The present invention relates to an immobilized chicken, guinea pig or human o-type red blood cell which stably retains a receptor having a high and specific binding ability for a long time against influenza virus, a method for producing the same, and a method for producing the same The present invention relates to the application of fixed-rich red blood cells to filters.

 Background art

 [0002] Influenza viruses belong to the Orthomyxoviridae family, and natural hosts are humans, pigs, horses, birds, and the like, and sometimes non-human primates, Inu and Ushi are also hosts.

 This influenza virus has the ability to be classified into several subtypes depending on the antigenicity of A, B, and C (any type, and in the case of type A, HA and NA) depending on the antigenicity of RNP (liponucleoprotein) Type A and type B are important for causing influenza in humans.There are two types of sugars on the envelope of type A and type B viruses: hemagglutinin (HAin) and neuraminidase (NA). Proteins form spikes with trimers and tetramers, respectively, and viruses of type C have spikes consisting of hemagglutinin-esterase (HE) protein trimers.

 [0003] It is known that natural type A influenza has 15 HA (hemagglutination) serotypes (HI to H15) and 9 NA serotypes (N1 to N9). H, 1, 2, 3 and 5 (H5N1 of a new virus isolated from the tracheal lavage fluid of a 3-year-old boy diagnosed with Reye syndrome in Hong Kong in 1997) and N Only viruses with 1 or 2.

[0004] Recently, avian influenza has occurred on a large scale in various places, and humans have been infected and killed. So far, the power of birds has been limited to human infections, and there have been no reports of an outbreak of human-to-human pandemic, a so-called pandemic, but there have been repeated infections from birds to humans. In addition, the human influenza virus and trifluenza virus genes were intermingled in the human body to acquire genes that can infect humans to humans. Nfluenza virus ”is said to have a high probability of appearing.

 [0005] “New influenza viruses” that are explosively prevalent in humans have repeatedly occurred every 10 to 40 years, and three types have been confirmed in the 20th century. Viruses in the 1918 Spanish flu (H1N1), the 1957 Asian flu (H2N2), the 1968 Hong Kong flu (H3N2), and the 1977 Soviet cold (H1N1) is there.

 [0006] The H5N1 avian virus, which has recently become a problem, was first found in Asia, but the infection has spread along migratory routes. This bird infection flew to Russia in 2005, and then spread to Europe and the Middle East, and even to Africa. In the region of avian virus infection, humans have been infected and more than 100 people have died.

 [0007] According to a study group document by the Ministry of Health, Labor and Welfare in Japan, if a new influenza pandemic occurs, 3 billion people will be infected worldwide and 60 million people will die if they do not have the antibody There is. In the worst case, there are speculations that the death toll will reach 500 million.

 [0008] The form of influenza virus is outer diameter 80-: LOOnm, generally spherical but rich in polymorphism. Usually, when the patient is separated, it initially exhibits a filamentous shape and becomes spherical when passaged. Two types of protrusions, hemdalchun and neuraminidase, protrude independently from the envelope.

 [0009] Influenza virus type A consists of 8 segmental RNAs. 1, 2 and 3 are synthases, 4 is HAin (hemglutinin), 5 is nucleoprotein, 6 is NA (receptor degrading enzyme; neurominidase), 7 is M protein, and 8 is RNA encoding NS protein. .

 [0010] The main infection routes of influenza virus are droplet infection and air infection (splash nucleus infection), and its strong transmission is one of the features of this virus. Clogged, the source of infection is a virus in the respiratory tract that is scattered by coughing and sneezing, particularly in patients with influenza virus, especially acute patients.

[0011] Infection of susceptible cells with influenza virus is a receptor that has N-acetylneuraminic acid at the tip of the sugar chain, protruding in a spike shape on the surface of the virus particle, and hemagglutin present on the cell surface. The force begins where it joins. Invasion into cells following this binding is thought to have two mechanisms: uptake by vacuoles and fusion of the plasma membrane. By the way, the mechanism of the above-mentioned adsorption phenomenon when adsorbing virus particles on the erythrocyte surface and infecting sensitive cells in the hemagglutination (HA) reaction by influenza virus is the same.

 [0012] Influenza viruses agglutinate erythrocytes of chicks, guinea pigs, human type O, and a wide variety of other birds. This hemagglutination (HA) reaction is also based on the same principle as the mechanism of adsorption to susceptible cells in the above-described influenza virus infection. Hemagglutination (HA) reaction by various viruses including influenza virus is not only used for serological diagnosis of various viral diseases (hemagglutination inhibition test: HI-test), but also for development of vaccines and basic research fields. This is an extremely powerful technique.

 [0013] The most suitable pH for hemagglutination by influenza virus is 7.0 to 7.8, and at pH 3.5 to 5.0 or less, the aggregation ability is lost. Therefore, the buffer used for influenza experiments is preferably in the range of ρΗ7.0 to 7.8 unless otherwise specified.

In experiments of the present invention, therefore, normal _P H7 two Dulbecco's phosphate buffered saline. (-) was a the use. In general, we used the so-called (1), which is free from Mg and Ca.

[0014] At 37 ° C, influenza virus adsorbed on erythrocytes is released from erythrocytes by destroying erythrocyte receptors by the action of an enzyme (neuraminidase; NA) present on the surface of the virus. This NA is a glycoprotein similar to hemagglutin and is thought to specifically cleave the a-ketoside bond of neuramin-lactose at the end of the mucoprotein receptor in the presence of Ca ^2+. Yes.

 [0015] One of the reasons that influenza viruses are viral diseases that are still uncontrollable is that hemdalchun (HA) and neuraminidase (NA), which are spiked on the surface of these viruses, are easily antigenic. This is to cause mutation.

 [0016] There are two types of antigenic variations of the influenza virus. In other words, continuous mutation and discontinuous mutation.

A discontinuous mutation means that a virus with a new antigenic subtype, which is completely different from the previous one, appears suddenly. Discontinuous mutations occur almost every 10 years This is also accompanied by a pandemic of influenza!

 Sequential mutation refers to slight antigenic changes of HA and NA that occur every year within the same subtype.

 Whereas the nature of discontinuous mutation is the recombination (reassembly) of genes in nature, the nature of continuous mutation can be thought of as mutation and selection.

 [0017] Since the influenza virus gene is easily mutated in this way, it is virtually impossible to create a preventive vaccine that protects against the new influenza virus. Also, it takes at least six months even if influenza begins to become epidemic and manufacture of force vaccines, during which time the infection spreads. The influenza drug Tamiflu also has a limited stockpile, and viruses that are resistant to Tamiflu also appear!

 [0018] Therefore, a method for preventing human infection by supporting a substance that adsorbs and captures influenza virus on a gas filter such as a mask, regardless of type or subtype, has been highlighted. Among them, there is known a method (Patent Document 1) in which an adsorbent adsorbed on the hemagglutinin site of influenza virus, for example, sulfatide powder is supported on a mask. However, since its ability to adsorb viruses is not so high and unstable, development of a strong and stable virus adsorbent is awaited.

 Patent Document 1: Japanese Patent Application Laid-Open No. 2002-48730

 Disclosure of the invention

 Problems to be solved by the invention

[0020]-It has been known for a long time that chicken avian erythrocytes specifically bind to various hemdarchuns of viruses and cause agglutination. For example, viruses such as rubella, Japanese encephalitis, influenza, mumps, Nursing disease, Sendai, etc. It has been used for serodiagnosis of infectious diseases. However, red blood cells in birds are poorly preserved1, so it is necessary to prepare fresh blood after each test.

[0021] The present inventor is a person who has been working for many years on the stability of erythrocytes for virus diagnosis targeting influenza viruses, etc. As a result of their research,- — PBS that has been immobilized after standing for several days at low temperature also has a very high ability to bind virus hemagglutinin after lyophilization. I found that I have it.

 [0022] After that, there was a point that this method should be further improved. In other words, it was found that in the formalin fixation operation in which the avian avian erythrocyte isolated from fresh blood is brought into contact with formalin solution, a part of the receptor that binds to hemdarchun loses its function and the binding ability of the virus decreases. This phenomenon also causes a difference in binding ability from lot to lot, resulting in fluctuations in the agglutination titer of the obtained fixed erythrocytes by the virus.

 Means for solving the problem

 [0023] The present inventor has sought to prepare immobilized erythrocytes that stably retain high-level and specific binding ability for a long time against influenza virus. When the red blood cells were fixed with formalin in the presence of the used ALSVER solution, no functional decline of the virus receptor occurred. Therefore, as we gradually investigated which component force in this Alsbar solution brought about this effect, the Alsbar solution contained about 2% by weight! /, And glucose was used to prevent hemolysis of this red blood cell. I found out.

 [0024] Furthermore, if glucose is added to the freeze-dried product of fixed erythrocytes obtained as described above, the erythrocytes will remain in the influenza virus state after being stored at room temperature for several years or longer. It was found that the binding ability was kept high. Next, it was confirmed whether other saccharides had the same effect instead of glucose, and it was confirmed that other monosaccharides and disaccharides such as sucrose have almost the same effect.

 [0025] This highly stable immobilized erythrocyte having a high virus-capturing ability can be obtained by treating erythrocytes other than chickens, such as guinea pigs and human type O red blood cells, with the same treatment as chicken erythrocytes. It was found that fixed rabbit erythrocytes stably holding the receptor can be obtained.

Furthermore, as a result of various investigations, the fixed erythrocytes cannot be used for the prevention of influenza virus infection. It was found to be very useful in preventing viral diseases that are infected with droplet nuclei. Of course, this fixed erythrocyte is infected with various viral infections such as rubella, Japanese encephalitis, dengue fever, influenza, mumps, Newcastle, Sendai, etc. It can also be used advantageously for serodiagnosis of the disease. The present invention has been completed based on these findings.

That is, the present invention

(1)

 -Stabilize the virus receptor on the surface obtained by carrying out the step (b) of treating an aqueous suspension of chicken red blood cells, guinea pig red blood cells or human type O red blood cells with formalin solution in the presence of monosaccharides or disaccharides. Retained fixed red blood cells,

(2)

 Before the step of treating the aqueous suspension with a formalin solution, the step (a) of publishing with monoacid-carbon gas in the presence of a monosaccharide or disaccharide is performed. 3)

 The immobilized erythrocyte according to (1) or (2), wherein the concentration of monosaccharide or disaccharide in step (b) is 0.1 to 15% by weight,

(Four)

 The immobilized red blood cell according to any one of (1) to (3), wherein the monosaccharide is glucose and the disaccharide is sucrose,

(Five)

 The immobilized red blood cell according to any one of (1) to (4), wherein the concentration of the formalin solution in step (b) is 3 to 20 v / v%,

(6)

(8) the immobilized red blood cells according to any one of (1) to (5),

 The immobilized erythrocyte according to (6), which contains 0.1 to 10% by weight of monosaccharide or disaccharide in the lyophilized product,

(8)

-Immobilization with stable retention of the viral receptor on the surface of step (b) in which an aqueous suspension of chicken red blood cells, guinea pig red blood cells or human type O red blood cells is treated with formalin solution in the presence of monosaccharides or disaccharides Manufacturing method of erythrocytes, (9)

 Before the step of treating the aqueous suspension with the formalin solution, perform the step (a) of publishing with a monoacid-carbon gas in the presence of a monosaccharide or disaccharide. Manufacturing method,

(Ten)

 The method for producing immobilized erythrocytes according to (8) or (9), wherein the concentration of monosaccharide or disaccharide in step (b) is 0.1 to 15% by weight,

(11)

 The method for producing immobilized erythrocytes according to any one of (8) to (10), wherein the monosaccharide is glucose and the disaccharide is sucrose,

(12)

 (8) to (11), wherein the concentration of the formalin solution in step (b) is 3 to 20 v / v%;

(13)

 An influenza virus trapping filter comprising the air-permeable filter carrying the immobilized erythrocyte according to any one of (1) to (7);

 (14)

 The influenza virus capturing filter according to (13), wherein the breathable filter is a mask filter,

It is.

 As a raw material of the present invention, the chicken, guinea pig or human type O red blood cells can be obtained from each fresh blood.

Birds in the pheasant family, for example, eggs for leghorns, minor power, meat for chamo, brama, eggs for plymouth rock, appreciation for long-tailed chickens, Higashi Tianjiang, and other chobos. Any of those varieties are listed. Guinea pigs are guinea pigs (Cavia) in the guinea pig (Cavia) that are raised and sold for animal experiments. Human type O red blood cells are of no race. Since these blood are fixed with formalin, they are operated aseptically in the process until formalin treatment. There is no need.

 [0028] As monosaccharides, force glucose such as glucose, mannose, fructose, sorbose and other pentoses such as ribose and xylose is particularly effective. Examples of disaccharides include sucrose, trehalose, maltose, and ratatoose. Sucrose is effective.

 [0029]-Fresh chicken, guinea pig or human type O blood as it is or mixed with an aqueous solution containing monosaccharides or disaccharides (hereinafter sometimes referred to as saccharides) and centrifuged to precipitate erythrocytes. Precipitated red blood cells (Packed cells) are suspended in an aqueous solution containing-or physiological saline, preferably monosaccharides or disaccharides.

 The concentration of monosaccharide or disaccharide in the aqueous suspension is usually 0.1 to 15% by weight, preferably 0.5 to LO weight%, more preferably 1 to 7 weight%. As the monosaccharide-containing solution, a commercially available ALSVER solution containing 2.05 w% of glucose is conveniently used.

 [0030] Prior to immobilization with formalin, trierythrocytes suspended in an aqueous solution containing monosaccharides or disaccharides were bubbled by blowing monoxide-carbon gas in the presence of monosaccharides or disaccharides (step a). By doing so, the reddish brown power may be changed to bright red. The pH of the liquid at the time of publishing is 6.0 to 7.5, preferably 7.1 to 7.4, and one of the suitable liquids is a pulse bar liquid (pH 6.1).

 For publishing, for example, 20 to 200 mL of red blood cell suspension is put into a 1 L glass bottle, and carbon monoxide gas is blown from a cylinder for 40 minutes for about 1 to 40 minutes. This should be done until it changes to crimson or bright red methemoglobin. The liquid temperature during publishing is 10-25 ° C. By publishing with carbon monoxide gas in the erythrocyte suspension in the presence of monosaccharides such as glucose and disaccharides such as sucrose, hemolysis during operation, and denaturation, destruction, and binding power of erythrocyte virus receptors Can be prevented. The concentration of the monosaccharide or disaccharide is usually 0.1 to 15 wZv%, preferably 0.5 to: LOwZv%, particularly preferably 1 to 7 wZv%. The bright red erythrocytes can be advantageously used in antibody titer measurement tests against virus infection after formalin fixation.

Red blood cells treated with carbon monoxide gas are pulsed with Barsbar, PBS, and physiological saline. After removing the impurities by centrifugation, washing, etc., subject to fixation treatment with formalin.

 [0031] In the formalin-fixing treatment, erythrocytes are fixed in contact with formalin solution in the presence of monosaccharides or disaccharides (step b). Formalin is a 37wZv% formaldehyde aqueous solution, and the concentration of formalin in the fixed cocoon treatment is usually 3 to 20vZv% (ie, 1. lwZv% to 7.4wZv% aqueous formaldehyde solution), preferably 5 to 15vZv%. is there. The concentration of monosaccharide or disaccharide in this step (b) is usually 0.1-15 wZv%, preferably 0.5-: LOwZv%, particularly preferably 1-7 wZv%. This immobilization treatment is carried out by allowing the liquid to stand at a room temperature of 10 to 25 ° C. for 1 to 15 days, preferably 3 to 10 days with occasional stirring. Red blood cells fixed with formalin solution are centrifuged and washed using, for example, distilled water, physiological saline, PBS, or the like.

 [0032] Formalin-fixed マ リ ン-匕 avian erythrocytes are washed to remove formalin, and then freeze-dried from an aqueous suspension preferably containing a monosaccharide or disaccharide to obtain a stable lyophilized product. That is, suspended fixed red blood cells are suspended in water containing 0.1 to 10 wZv%, preferably 0.5 to 5 wZv%, more preferably 1 to 3 wZv% monosaccharide or disaccharide to about 10 v%. For example, it is rapidly frozen in dry ice and acetone and then freeze-dried. When monosaccharides or disaccharides are allowed to coexist in the lyophilized product, lyophilized erythrocytes are more stabilized, and the virus capturing ability is maintained at a high level for a long period of 5 years or longer.

 [0033] The ratio of monosaccharide or disaccharide in the erythrocyte freeze-dried product is usually from 0.1 to 2.0 in a state resuspended in distilled water or other aqueous solution immediately before entering the freeze-drying process and after drying. The blending ratio is 15% by weight, preferably 0.2 to 10% by weight, more preferably 0.5 to 5% by weight.

[0034] In the present invention, the filter used for virus capture is a woven or non-woven fabric made of synthetic fiber or natural fiber, and the material thereof is polyester, polyamide, polyacryl, polypropylene. , Rayon, cotton, wood pulp and the like. In addition, as a method for producing a nonwoven fabric, there are a resin bond, a thermal bond, a needle punch, a span bond, a melt blown, a spun lace, a wet nonwoven fabric, and the like, and it is preferable to use a nonwoven fabric suitable for the application. In particular, melt blown non-woven fabrics can reduce the fiber diameter and reduce the pore size, making it an ideal filter for masks, air purifiers and air conditioners. It is preferable.

[0035] The virus trapping filter of the present invention is a filter in which fixed red blood cells are supported on a filter. The amount of the immobilized avian erythrocyte supported on the filter is 10 × 10 ⁶ to 10 × 10 ^{1 (>} , preferably 10 × 10 ⁷ to 10 × 10 ⁹ per lcm ^{2 of} filter. From 10 × 10 ⁶ If it is less, influenza virus will not be captured sufficiently, but if it is more than 10 X 10 ^1G, there will be a significant change in the capture effect and it may be expensive.

More specifically, for example, there are about 2.6 billion erythrocytes in a 2 mL suspension of 0.2% immobilized avian erythrocytes, and if this is held in 5 x 5 cm ² gauze, 1 cm will ^{be 2} per Ri about 10 X 10 ⁸ red blood cells is carried, it has the ability to adsorb indeed about 500 billion pieces of influenza Enzawirusu infectious particles, Ru.

 [0036] The method for supporting the fixed erythrocytes on the filter is arbitrary. It can be supported using an appropriate binder. Examples of binders that can be used include water-soluble and emulsion-type binders. Examples of water-soluble types include PVA, CMC, polyacrylic acid, and their sodium salts. Examples of emulsion types include acrylic, SBR, and NBR. And EVA. As a specific method for carrying the binder, for example, an immobilized red blood cell is dispersed in water by adding a small amount of a surfactant to PBS or physiological saline, and then an appropriate amount of an aqueous binder is used. And a method of dropping, coating, spraying or impregnating the dispersion liquid on the sheet can be exemplified. The amount of the binder is preferably about 2 to 20% by weight with respect to the immobilized red blood cells. As the surfactant, for example, PEG or sorbitan monooleate can be used in an amount of about 0.1 to 10 w%.

 [0037] The filter carrying the fixed erythrocytes may be cut and sewn as a sanitary mask, or may be sandwiched between the fixed erythrocyte-carrying gauze and used as a sandwich structure mask. For further perfection, the fixed erythrocyte-carrying gauze can be used as a nasal pillow so that almost all of the inspiration passes through the nasal pillow.

 Furthermore, it is possible to prevent infection and transmission of influenza to domestic birds by using this fixed red blood cell-carrying filter for air purifier filters in poultry houses, livestock houses and pet shops.

[0038] Masks and filters after use are positive cleaning agents (reverse sarcophagus) such as commercially available males. By contacting with a 100 to 500-fold diluted solution of van solution (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.), the infectivity of the virus can be easily lost.

 The invention's effect

 [0039] According to the method of the present invention, when the erythrocytes are fixed with formalin, the virus receptor is not destroyed or altered, so that extremely stable fixed erythrocytes can be obtained in a high yield and with no lot difference. Can be prepared. The lyophilized fixed soy erythrocytes together with monosaccharides or disaccharides obtained in this way have almost the same virus binding ability as fresh erythrocytes even after storage at room temperature for 5 years or more. For example, one fresh-bird avian erythrocyte has the ability to bind to more than 5000 influenza virus-infected particles! /, But the fixed-bird erythrocyte lyophilized product obtained in the present invention is more than 5 years old. There is almost no change in its ability after storage.

 The fixed 匕 red blood cells obtained by the method of the present invention are used as suspensions at any time, for example, antibody titer measurement tests for viral infections such as influenza A, B and C, Newcastle disease, mumps, Japanese encephalitis, dengue fever (H Test). If this immobilized red blood cell is supported on a mask or an air filter, it will strongly adsorb and remove the virus, and will exhibit an extremely high effect in preventing influenza virus infection. BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described in more detail with reference to examples and comparative examples.

 Example 1

[0041] Preparation of immobilized avian erythrocytes

 (1) Blood collection

 Collect 15 ml of blood in total, each with 5 mL of wing venous force for each of three white Leghorn and Nagoya Cochin species, 12 months after hatching, using a syringe containing the same amount of blood-bar liquid (Note 1) as the amount of blood collected in advance. Pool blood was used.

 (Note 1) Composition of ALSVER solution

 Glucose 2. 05% by weight

 Salt 0.42% by weight

Sodium Kuen acid 0.80 wt _^0/0 Chenic acid 0.55% by weight

 Distilled water 96. 18% by weight

 [0042] (2) —Coloring by blowing carbon dioxide gas

 60 mL of an equal volume mixture of blood and pulse bar solution obtained in (1) above was repeatedly washed with PBS three times (1500 rpm, 5 minutes), and 3 mL of precipitated erythrocytes were accommodated in each of the three centrifuge tubes. Next, solutions of the following three types of compositions were poured so that each became 10 v% of precipitated red blood cells.

 (a) -PBS

 (b) —PBS + 5w% glucose

 (c) PBS + 5w% sucrose

 Put three kinds of fresh erythrocyte suspensions (30 mL each) into 1 L glass bottles with clear stoppers, and blow carbon monoxide gas from a cylinder at a rate of 60 bubbles for 1 minute for about 5 minutes. It was confirmed that the red hemoglobin was changed to crimson methemoglobin. Thus, each suspension was centrifuged at 1500 rpm for 5 minutes, separated into precipitated erythrocytes and supernatant, and the supernatant was transferred to a transparent test tube. In order to see the degree of hemolysis, the color tone of each of the obtained supernatants was visually judged according to the following criteria. This test was repeated three times and the results are summarized in Table 1.

 Judgment criteria:

 1: colorless and transparent (no hemolysis)

 Shi: A faint red color

 +: Light red

 ++: Medium red

 +++: Crimson

[0043] [Table 1] Composition test of fresh erythrocyte suspension in CO force 'Fring' o.

 1 PBS 1 PBS + 5w% course — PBS + 5w close

 1 Sat

 2 Sat

 As is clear from Table 1, the supernatant of the erythrocyte suspension suspended only in PBS showed a faint red color, indicating that slight hemolysis occurred. In contrast, the supernatants of erythrocyte suspensions suspended in PBS containing glucose or sucrose were both colorless and transparent, indicating that hemolysis did not occur.

[0044] (3) Immobilization of red blood cells with formalin

 The red blood cells obtained in (2) above were combined, suspended in the following three types of solutions so that the volume was 10v%, and placed at 4 ° C for 7 days. Meanwhile, the sunk red blood cells were suspended by shaking the container once a day.

 (a) Contains 10% formalin — PBS (no sugar)

(b) glucose 5 weight _^0/0, PBS containing formalin 10v%

(c) sucrose 5 weight _^0/0, including formalin 10v% - PBS

 After fixing with formalin for 7 days, centrifugal washing with distilled water (1500 rpm) is repeated 6 times, and the precipitated erythrocytes are fixed again in the original solutions (a), (b), and (c) to 10 v% each.匕 Red blood cells were suspended and dispensed to 2 mL in a 20 mL lyophilized vial. This suspension was rapidly frozen with dry ice'acetone, and then freeze-dried to obtain a freeze-dried product of fixed erythrocytes, which was stored at room temperature.

[0045] (4) Measurement method of hemagglutination titer (small test tube dilution method)

 (a) -Measurement of viral hemagglutination titer using fresh avian erythrocyte

3. Blood of white leghorn males (12 months after hatching) obtained by collecting blood with a syringe containing 1% of the expected amount of 8% sodium quenate was washed with PBS three times (1500 rpm). Precipitated red blood cells—PBS is added to form a 10v% red blood cell suspension and stored in a 4 ° C ice chamber. It was.

 Dilute the sample continuously with -PBS (pH 7.2) twice, add an equal amount of the above red blood cells to 0.5 mL, shake and mix well, and let stand at room temperature for 1 hour. The reciprocal of the highest dilution before the addition of erythrocytes of the material showing clear aggregation was taken as the hemagglutination number (HA value).

 (b) Measurement of hemagglutination value of erythrocyte freeze-dried product

 The hemagglutination value was measured using the three erythrocyte freeze-dried products obtained above. First, three types of freeze-dried products were each suspended in PBS so as to be 10 v%. Then 0.25 v% —diluted with PBS and inactivated healthy horse serum was added at a rate of 0.2% to prevent non-specific aggregation.

(5) Tissue culture of influenza virus and measurement method of infectious titer

 (a) Influenza virus strain used

 As the influenza virus strain used, type A PR8 strain obtained from the former National Institute of Preventive Health was used. This PR8 strain is a reference species of influenza virus type A, and was AZPR8Z34 (H1N1) type isolated from patients in 1934 by T. Francis Jr. during the outbreak in Puerto Rico.

 Seed virus of type A PR8 strain was inoculated on the 10th day of hatching in the chick embryo chorioallantoic fluid and in suspension culture using a slice of chicken chick embryo chorioallantoic membrane (Maitland method, Yamaguchi Medical Division 9th, 5th) No., September 1960, pages 1490-1510), and subcultured and maintained, and dispensed into small test tubes—used at cryogenic temperatures below 40 ° C .

(b) Influenza virus tissue culture

The culture solution is Hanks solution, which is supplemented with 3% of healthy horse serum that has been inactivated by heating at 56 ° C for 30 minutes, and penicillin and streptomycin are added to each 1 mL. The one added to 00 units and 100 g was used. As a culture tissue for influenza virus, a minced chicken embryo chorioallantoic membrane (CAM) on the 11th day of hatching was used. CAM was taken out, washed 3 times with Hanks solution, and then chopped to about 1 mm ^{2 with} a scissors. Hank's solution was added to the tissue pieces and allowed to stand for 5 minutes, and the supernatant was discarded. This operation is repeated 3 times to remove mixed blood cells and tissue ultrafine fragments, and then put into a Spitz glass, lOOOrp The precipitate was obtained by centrifugation at m for 1 minute. A suspension of an equal amount of Hank's solution in this sediment is dropped into a culture bottle (round vaccine bottle with an internal volume of 20 mL) dropwise to form a cultured tissue, and 2 mL of the culture solution is dispensed into this. Thus, a tissue culture system was established.

[0047] (c) Infectious titer measurement method of influenza virus liquid

 Influenza virus infectivity was measured by inoculating 0.2 mL of the influenza virus solution diluted in decimal using a tissue culture solution into the above tissue culture system and plugging the culture bottle with a rubber stopper. After incubating this at 37 ° C for 3 days, the culture broth was examined for the presence or absence of hemagglutination (HA) to determine the infectious titer. In both culture bottles, the reciprocal of the terminal dilution concentration of HA plus was taken as the tissue culture infectious titer (TCID) (Yamaguchi Medical No. 9, No. 5, September 1960, 1490-1510

 100

 page).

 [0048] (6) Sendai virus (HVJ) hemagglutination and black number measurement method

 (a) Sendai virus (HVJ)

 As the Sendai virus, MN strain belonging to Paramyxovirus type I was used. Sendai virus virus was serially subcultured by inoculation of embryonated chicken egg chorioallantoic cavity, and was used after being distributed by the Institute of Medical Science, University of Tokyo. The origin of the MN strain was first isolated in 1954 by Dr. Hideo Fukumi of the National Institute of Preventive Health. (Jap. J. Med. Sci. Virol. Vol. 9: 169-177 (1954)

 [0049] (b) Black formation test

 As a cell for monolayer culture necessary for Sendai virus black formation method, a porcine kidney cell PS-Y15 cell (Journal of Clinical Microbiology, Vol. 3, No. 2, P91 (1976)) was used. As a culture medium for cell growth, Eagle Minimal Essential Medium (MEM) dissolved in Earle's salt solution with 5% fetal bovine serum and 10% Tryptose Phosphate Broth was used.

 [0050] (c) Black number measurement method

A 5 mL growth medium containing 2 x 10 ⁵ individually dispersed PS cells by trypsin digestion is placed in a 60 mL black bottle and cultured at 37 ° C for 2 days to form a cell monolayer. Formed. Next, the culture solution was discarded, and 0.2 mL of the virus solution diluted decimally with MEM without sodium bicarbonate and serum was inoculated on the cell monolayer in the culture bottle. This virus inoculation bottle 3 Leave at 7 ° C for 1 hour to adsorb the virus, cover the cell layer with 5 mL of growth medium containing 0.8% agarose incubated at 40 ° C, and incubate at 37 ° C for 7 days . Next, the monolayer culture layer of the virus-infected cells was stained in vivo, and 0.8% agarose and 0.007% -EUTRAL RED were preliminarily warmed to 40 ° C to count the black number with the naked eye. Sodium bicarbonate-free MEM was layered and solidified on the solid culture layer in the black bottle. These plastic bottles were placed at room temperature in the dark for 2 days before black counts (PFU) were counted. PFU was expressed as the arithmetic average of two bottles showing 10-50 plaque formation per black bottle.

 Example 2

 [0051] Measurement of adsorptive capacity of influenza virus type A PR8 by using formalin-immobilized chicken avian erythrocyte receptor

 (1) Preparation of receptor-disrupted formalin-fixed 匕-ヮ avian erythrocytes

A commercially available dry receptor degrading enzyme (RDE) was used to destroy the receptor on the erythrocyte surface. First, according to the instruction manual, RDE powder was dissolved in 20 mL of physiological saline. Next, with respect to 2 mL of the solution obtained by suspending formalin-fixed 匕-ヮ avian erythrocytes obtained in Example 1 at room temperature for 5 years in physiological saline at 10 v% (equivalent to 6.5 X 10 ^1C ZmL) Then, the mixture containing 6 mL of the RDE solution was allowed to stand at 37 ° C. for 6 hours to allow RDE to act. Residual RDE was removed by centrifugally washing the RDE-treated erythrocytes with PBS three times.

 [0052] (2) Measurement of chicken erythrocyte aggregation titer

Contains 5 x 10 ^lc> ZmL of formalin fixed 匕-ヮ avian erythrocytes or (1) RDE-treated erythrocytes obtained in Example 1 stored at room temperature for 5 years-a tissue of 128 HA units 5 mL of the culture-derived type A PR8 strain influenza virus solution was mixed and sampled at intervals of 5, 10, and 15 minutes. The avian avian hemagglutination value of these sampling samples was measured by the small test tube dilution method described in Example 1 using a centrifugal supernatant at 3000 rpm10 minutes.

 Example 3

[0053] Adsorption of influenza virus type A strain PR8 haemagglutinin or influenza virus particles by formalin-immobilized and avian avian erythrocyte receptor adsorbed to and absorbed on gauze Noh

Place 3 pieces of 5 x 5 cm ² gauze in a petri dish and pipette 2 ml (2 drops) of 2 x 10 ^1G ZmL immobilization solution of 2 x 10 ^1G ZmL PBS suspension (approx. 40 drops) over the entire surface. did. Next, 2 mL of a PBS suspension containing 128 units of influenza virus type A strain PR8 hemagglutinin (HAin) was dropped evenly with the same pipette. 23 gauze above. C. After standing for 30 minutes, 12 mL of 1 PBS was added to the petri dish, and the gauze was shaken vigorously for several seconds while using tweezers. The liquid in the petri dish together with the gauze was transferred to a 50 mL centrifuge tube, centrifuged at 3000 rpm for 20 minutes, and the erythrocyte aggregation titer of the supernatant was measured by the HA test using a small test tube method.

Similar tests were performed with 2 x 10 ⁸ ZmL-avian avian erythrocyte PBS suspension, 2 x 10 ^{1 G} ZmL receptor disruption-amber aerythrocyte PBS suspension and 2 x 10 ⁸ ZmL receptor disruption. -The hemagglutination value was measured using a suspension of rabbit red blood cells. The results are shown in Table 2.

[0054] [Table 2]

[0055] Results: In the formalin-fixed avian avian erythrocyte in Table 2, the amount of residual HA units was measured from the 8-fold dilution stage of the initial HA value (128 units). That is, in the receptor (one) -immobilized chicken erythrocyte group, 128 units remained as they were in terms of the original HA value, and the ability to adsorb to HAin or virus particles was almost completely absent.

On the other hand, in the receptor (+)-fixed erythrocyte group, more than 120 units and 128 units of HAin are adsorbed by the receptor. In addition, the virus solution of one aggregation unit contains about 10 ⁷ · ² worth of virus particles, so 120 Χ 10 ⁷ · ^{2 to} 128 Χ 10 ⁷ · ² The virus particles are adsorbed on the receptor.

 Example 4

[0056] 1 X 10 ⁵ TCID instead of 2 mL of the influenza virus A type PR8 strain hemagglutinin (HAin) 128 unit solution in Example 1 except that the residual virus-containing test sample was measured from a 10-fold dilution. Influenza virus type A PR8 strain infectious particle-containing solution 2m

 X X 100

 L was used to measure TCID infectivity. The results are shown in Table 3.

 100

 [0057] [Table 3]

[0058] Results: 2 X 10 ⁸ and 2 X 10 ^1G formalin-fixed-avian avian erythrocyte receptors each adsorbed 1 X 10 ⁵ TCID of type A PR8 strain influenza virus infected particles. to this

 100

 On the other hand, the red blood cells of receptor (-) almost completely lacked the ability to adsorb infectious virus particles.

 Example 5

 [0059] Influenza virus type A PR8 strain haemagglutinin (HAin) 128 unit in Example 3 Instead of PBS 2 mL, suspension containing noramyxo type I virus (Sendai virus) hemagglutinin (HAin) 128 units A similar test was performed using 2 mL of the suspension, and the results shown in Table 4 were obtained.

 [0060] [Table 4] Residual center virus in the supernatant

 Immobilization ;: Red erythrocyte red blood cell agglutination titer (HA titer)

 Form and number (number / mL)

 Converted from the 8-fold dilution stage to the original HA value

 0 <8

 Recef'ta (!)

 0 times 8

2 X 1 0 ^{1 0} 1 6 1 28

 Recef. -(-)

2 Χ 1 0 ^s 1 6 1 28 [0061] Results: The results were completely consistent with the results of the adsorption experiment of fixed erythrocytes [Receptors I (+) and (I)] against the influenza virus (HAin) of Example 3. That is, receptor (−) erythrocytes hardly adsorbed Sendai virus HAin.

 Example 6

 [0062] Contains 128 units of influenza virus HAin in Example 3 except that the residual virus-containing test sample was measured from a 10-fold diluted solution-Instead of 2 mL of PBS-Sendai derived from chick embryo-sensitized chorioallantoic fluid 200 PFUZ0. 2 mL of virus (PFU per plaque bottle) and receptor (+) and (1) suspension of formalin-fixed red blood cells in equal amounts (2 mL + 2 mL) were mixed with PFU. It was. The results are shown in Table 5. In this example, however, the infectivity titer was measured by the decimal method after diluting the virus solution before centrifugation by 10-fold dilution.

 [0063] [Table 5]

[0064] Results: 2 X 10 ⁸ and 2 X 10 ^1G formalin-fixed avian erythrocytes (receptor (+)) each adsorbed almost 200 PFU of Sendai virus-infected particles. On the other hand, it was found that the red blood cell of the receptor (one) corresponding to the above red blood cells almost completely lacked the ability to adsorb Sendai virus.

 Example 7

 [0065] Prepare each of the following red blood cells, add influenza virus hemagglutinin or infectious particles to the suspension, mix, leave for 5, 10, 15 minutes, centrifuge, and centrifuge supernatant of each red blood cell. The hemagglutination value in the medium was measured by a small test tube method.

 (a) Fresh-bird avian erythrocyte:

(b) Immobilized erythrocyte lyophilizate: obtained in Example 1

(c) Receptor destruction (RDE treatment) Red blood cells: room temperature after preparation obtained in the same manner as in Example 1. Storage 5 mL of formalin-fixed 匕-ヮ avian erythrocytes suspended in physiological saline at 10v% (6.5 X 1 (equivalent to ^ ZmL)) 6 mL of a solution dissolved in physiological saline was added, and the mixture was left at 37 ° C for 6 hours to destroy the receptor.In addition, RDE-treated erythrocytes were washed with PBS three times by centrifugation, Residual RDE was removed.

Mix 2 x 10 ^1G ZmL suspensions of the above three types of red blood cells and tissue culture-derived type A PR8 strain influenza virus solution (including 64HA units) in equal amounts (5 mL each) and bring to room temperature. Oh, and sampled every 5, 10 and 15 minutes. The chicken erythrocyte agglutination value of these sample samples was measured by a small test tube method with respect to a centrifugal supernatant at 3000 rpm for 10 minutes. The results are listed in Table 6.

[0066] [Table 6]

Results: Formalin-fixed avian erythrocytes treated with receptor-disrupting enzyme lacked the ability to adsorb influenza virus hemagglutinin. On the other hand, the fresh and formalin-fixed erythrocytes of Example 1 have an erythroagglutinin (HAin) of 32 HA units (64 units in terms of the original HA value), which is further converted to this figure ( It means that 64 X 10 ⁷ · VmL ³ ) infected virus particles were adsorbed.

 Example 8

 [0067] Storage effect of sugars on susceptibility of freeze-dried chicken avian erythrocytes to influenza virus hemagglutinin

In the same manner as in Example 1, first, carbon monoxide publishing was performed in the presence of Alsbar solution, then formalin was fixed in the presence of 5% glucose-containing PBS, and then centrifugally washed with distilled water. Was suspended in (a) distilled water, (b) 2% glucose-distilled water, or (c) 2% sucrose-distilled water to a concentration of 10 wZv%. These suspensions were rapidly frozen with dry ice'acetone and lyophilized as they were. And freeze-dried Immediately thereafter, the adsorption sensitivity of the erythrocyte receptor to influenza virus hemagglutinin at the time of storage at room temperature for 1 year and at room temperature for 5 years was measured by a small test tube dilution method to calculate the residual rate. The results are shown in Table 7.

[0068] [Table 7]

 NT: not tested

[0069] Result: The strength of adding the sugar to the freeze-dried product The adsorption sensitivity to viral hemagglutinin decreased to 25% after 1 year, and to 12.5% after 5 years. The lyophilized product with 2% glucose or 2% sucrose retained 100% sensitivity after 5 years.

 Example 9

 [0070] Preparation of immobilized guinea pig erythrocytes

 (1) Blood collection

 2 mL of blood from 10 commercially available experimental guinea pigs (12-month-old Hartley ^ ~ · Hartley species) was collected by cardiac puncture into a syringe that had been pre-filled with an equal volume (2 mL) of Alsbar solution. And pooled.

 (2) Preparation of precipitated erythrocytes

 40 mL of an equal volume mixture of blood and pulsed bar solution obtained in (1) above was repeatedly washed with PBS three times (1500 rpm, 5 minutes), and 3 mL of precipitated erythrocytes were accommodated in each of the three centrifuge tubes.

[0071] (3) Immobilization of red blood cells with formalin

Guinea pig erythrocytes obtained in (2) above are washed 3 times (1500 rpm, 5 minutes) with PBS (Mg, phosphate-free saline solution of Dulbecco's pH 7.2 without Ca), and the concentration of erythrocytes is SlOv% Suspend in 1 PBS containing 2% glucose and 10v% formalin so that And placed at 4 ° C for 7 days. During that time, the settled red blood cells were suspended by shaking the container once a day.

[0072] (4) Vacuum freeze-drying of formalin-fixed erythrocytes

 The guinea pig erythrocytes obtained above are fixed in formalin for 7 days, and then washed with distilled water (1500 rpm, 5 minutes) 6 times, and the precipitated erythrocytes are suspended in distilled water containing 2% glucose by 10%. Then, while stirring with a magnetic stirrer, 2 mL was dispensed into a 20 mL lyophilization vial.

 Suspensions of these guinea pig-colored fixed baboon erythrocytes were rapidly frozen with dry ice / acetone, then transferred to a vacuum freeze dryer and freeze-dried as they were. The freeze-dried bottle was filled with nitrogen gas, then sealed with a rubber stopper, and further tightened with an aluminum cap to obtain a freeze-dried product of guinea pig-immobilized erythrocytes. This lyophilized product was stored at about 20 ° C for a long period of one year.

 [0073] (5) Resuspension of freeze-dried fixed red blood cells

 When using the freeze-dried fixed erythrocytes obtained above for 1 year, the suspension was first suspended in PBS so that the volume was 10 v% (PB S was added so that the total content of the vial for freeze-drying was 2 mL. did).

[0074] (6) Method for measuring hemagglutination titer (small test tube dilution method)

 3. Fresh blood obtained by collecting the venous force of adult wings with a syringe containing approximately 1Z10 of 8% sodium quenate in the amount of blood to be collected is washed 3 times with PBS (1500 rpm for 5 minutes) and washed. The precipitated erythrocytes were made into a 10 v% suspension with PBS, stored in an ice chamber at 4 ° C, and diluted to 0.25 v% as needed. In order to prevent nonspecific hemagglutination, healthy horse serum that had been inactivated by heating at 56 ° C for 30 minutes was added at a rate of 0.2%.

[0075] (7) Influenza virus strain used

 The same influenza virus strain as that in Example 1 was used.

 (8) Tissue culture of influenza virus and measurement method of infectious titer

 These were also carried out by the same method as in Example 1.

[9] (9) Measurement of adsorptive capacity for type A PR8 strain influenza virus using formalin-immobilized guinea pig erythrocytes

(9i) Preparation of receptor-disrupted formalin-immobilized guinea pig erythrocytes A commercially available dry receptor-degrading enzyme (RDE) was used to destroy the receptor on the surface of guinea pigs.

First, according to the instruction manual, RDE powder was dissolved in 20 mL of physiological saline. Next, a formalin-fixed guinea pig erythrocyte prepared as described above and stored at room temperature for 1 year is suspended in physiological saline at 10v% (equivalent to 6.5 x 10 ^{1 ()} ZmL) 2mL On the other hand, a mixture containing 6 mL of the above RDE solution was allowed to stand at 37 ° C. for 6 hours to allow RDE to act. These RDE-treated erythrocytes were washed with PBS three times (1500 rpm, 5 minutes) to remove the remaining free RDE.

 (9-ii) Measurement of guinea pig hemagglutination titer

Red blood cells from guinea pig fresh blood, formalin-fixed guinea pig erythrocytes obtained in Example 9 after storage for 1 year, and formalin-fixed guinea pig erythrocytes treated with receptor-disrupting enzyme after storage for 1 year, 2 × 10 ^{Three 1G} ZmL were mixed with 5mL each of the 128HA units of tissue culture-derived type A PR strain influenza virus hemagglutinin (HAin) and sampled at 10-minute and 30-minute intervals. The hemagglutination values of these samples were measured by the small test tube dilution method described in Example 1. The results are shown in Table 8.

 Example 10

 [0077] Three types of red blood cells were also prepared for human type O red blood cells by the same steps (1) to (9) as in the case of guinea pigs. The hemagglutination values of these samples were measured by the small test tube dilution method described in Example 1. The results are shown in Table 8.

 [0078] [Table 8]

[0079] Results: One-year-old formalin-fixed guinea pig erythrocytes treated with receptor-disrupting enzyme. And human o type erythrocytes lacked the ability to adsorb influenza virus hemagglutinin. On the other hand, fresh and formalin-fixed 1-year-old erythrocytes of Example 9 * 2 HA units (64 units in terms of the original HA value) are further converted to this value. The infected virus particles of the outline (64 X 10 ⁷ · VmL ³ ) were adsorbed.

 Example 11

 [0080] Adsorption ability of formalin-immobilized guinea pig adhering to gauze and human type O red blood cells against influenza virus type A PR8 hemagglutinin (HAin) or infectious influenza virus particles

Three 5 x 5 cm ² gauze layers are placed in a petri dish and spread over the surface of 2 x 10 ¹⁰ ZmL of ³ immobilized guinea pig or human O red blood cells—2 mL of PBS suspension (approximately 40 drops) Each was dropped with a pipette.

 The gauze was allowed to stand at 23 ° C for 30 minutes, then 12 mL of PBS was added to the petri dish, and the gauze was vigorously shaken for several seconds while using tweezers. The liquid in the petri dish together with the gauze was transferred to a 50 mL centrifuge tube, centrifuged at 3000 rpm for 20 minutes, and the supernatant was measured for hemagglutination by the HA test using a small test tube method. The results are shown in Table 9.

 [0081] [Table 9]

結果：表 9におけるホルマリン固定化、モルモットならびにヒト O型赤血球によるイン フルェンザウィルス HAin吸着試験では、残存 HA単位量が当初の HA価 ( 128単位 )の 8倍希釈段階力も測定されている。即ち、レセプター（―）固定赤血球群では、もと の HA価に換算して 128単位がそのまま残亜温しており、 HAinまたはウィルス粒子 に対する吸着能をほぼ完全に欠如していた。

Results: In Table 9, in the formalin immobilization, guinea pigs and influenza virus HAin adsorption test with human type O erythrocytes, the residual HA unit amount was also measured as an 8-fold dilution step force of the initial HA value (128 units). That is, in the receptor (−)-fixed erythrocyte group, 128 units were converted to the original HA value, and the residual temperature was left as it was, and the ability to adsorb HAin or virus particles was almost completely absent.

On the other hand, in the receptor (+) fixed red blood cell group, H reaches 120 units or more and 128 units. Ain is adsorbed by the receptor. In addition, since the virus liquid of one aggregation unit contains about IX 10 ⁷ · ² registered virus particles, 12010 ⁷ · ^{2 to} 128 Χ ⁷ · ² virus particles are on the surface of these immobilized red blood cells. It is adsorbed by the upper receptor. Example 12

[0083] Except that the test sample containing residual virus was measured from a 10-fold dilution, instead of 2 mL of the influenza virus type 8 PR8 strain hemagglutinin (HAAin) 128 unit solution in Example 1, 1 X 10 ⁵ TCID Influenza virus type A PR8 strain infectious particle-containing solution 2m

 100

 L was used to measure TCID infectivity. The results are shown in Table 10.

 100

 [0084] [Table 10]

[0085] Results: 2 x 10 formalin-fixed guinea pigs and human type O red blood cells each

X 10 ⁵ TCID type A PR8 strain influenza virus-infected particles were adsorbed. Against this

 100

 However, the red blood cell group of the receptor (1) almost completely lacked the ability to adsorb the infected virus particles.

 Example 13

 [0086] Room temperature storage test for influenza virus HAin adsorption ability of formalin-fixed guinea pigs and human type O erythrocytes

 For various formalin-fixed guinea pigs and human erythrocyte erythrocyte freeze-dried products stored at room temperature (20 ° C) for 1 year, the hemagglutination titer of influenza virus particles was measured by the HA test using a small test tube method. The results are listed in Table 11.

[0087] [Table 11] Adsorption to HAin Immobilized guinea '; and immediately after lyophilization

 Noh survival rate! 《状態 Status of type 0 red blood cells> H position of HA value

 Storage at room temperature 1 year later a) Distilled water only 128 50 Guinea pig b) Distilled water + 2% quarkose 128 100 Red im sphere c) Distilled water + 2SU ^-ス 8 100

 Freeze-dried Control) Fresh erythrocytes 128 NT As above a) Distilled water only 128 50 Sat (25-50) Suspension in human () type b) Steamed water + 2¾ quarkose 128 100 Erythrocyte fluid composition C) Distilled water + 2¾ sk D-s 128 100

 Control) Fresh red blood cells 128 NT Chicken red blood cells b) Distilled water + 2W Lucose 128 100

 (Control) Control) Fresh red blood cells 128 NT

 NT: Not tested Industrial applicability

 Formalin-fixed rabbits, guinea pigs or human type 0 erythrocytes, especially those freeze-dried in the presence of monosaccharides or disaccharides in the presence of monosaccharides or disaccharides of the present invention, even after storage for more than a few years Since the virus has the ability to bind hemagglutinin to the same degree as fresh blood erythrocytes, it can be carried on a filter such as a mask to provide a virus infection prevention mask or filter. Prior to fixation with formalin aqueous solution, erythrocyte suspension is published with carbon monoxide gas in the presence of monosaccharide or disaccharide, and the red blood cells become bright red, and sometimes influenza, Newcastle disease, mumps It can be used for tests that determine the presence or absence of infection by viruses such as Japanese encephalitis and dengue fever.

Claims

The scope of the claims  [I] Treatment of an aqueous suspension of avian red blood cells, guinea pig red blood cells or human o-type red blood cells with a formalin solution in the presence of monosaccharides or disaccharides. Fixed red blood cells held in  [2] The fixed column according to claim 1, wherein a step (a) of publishing with a monoacid-carbon gas in the presence of a monosaccharide or disaccharide is performed before the step of treating the aqueous suspension with a formalin solution. Red blood cells.  [3] The immobilized erythrocyte according to claim 1 or 2, wherein the concentration of the monosaccharide or disaccharide in step (b) is 0.1 to 15% by weight.  [4] The immobilized red blood cell according to any one of claims 1 to 3, wherein the monosaccharide is glucose and the disaccharide is sucrose.  [5] The immobilized red blood cell according to any one of claims 1 to 4, wherein the concentration of the formalin solution in step (b) is 3 to 20 v / v%. [6] The fixed erythrocyte according to any one of claims 1 to 5, wherein the fixed erythrocyte is a freeze-dried product. 7. The immobilized red blood cell according to claim 6, wherein the lyophilized product contains 0.1 to 10% by weight of monosaccharide or disaccharide.  [8] Treating an aqueous suspension of avian red blood cells, guinea pig red blood cells or human type O red blood cells with a formalin solution in the presence of monosaccharides or disaccharides. Method for producing immobilized red blood cells. [9] The production of immobilized red blood cells according to claim 8, wherein the step (a) of bubbling with carbon monoxide gas in the presence of a monosaccharide or disaccharide is performed before the step of treating the aqueous suspension with a formalin solution. Law.  10. The method for producing immobilized erythrocytes according to claim 8 or 9, wherein the concentration of monosaccharide or disaccharide in step (b) is 0.1 to 15% by weight.  [II] The method for producing immobilized erythrocytes according to any one of claims 8 to 10, wherein the monosaccharide is glucose and the disaccharide is sucrose. [12] The method for producing immobilized erythrocytes according to any one of [8] to [8], wherein the concentration of the formalin solution in step (b) is 3 to 20 v / v%. An influenza virus capture filter comprising the air-permeable filter supporting the fixed erythrocytes according to any one of claims 1 to 7.  14. The influenza virus capture filter according to claim 13, wherein the breathable filter is a mask filter.