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WO2007145179A1 - Érythrocytes immobilisés dont la surface porte un récepteur de virus de façon stable, leur procédé de fabrication et leur utilisation - Google Patents

Érythrocytes immobilisés dont la surface porte un récepteur de virus de façon stable, leur procédé de fabrication et leur utilisation Download PDF

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WO2007145179A1
WO2007145179A1 PCT/JP2007/061754 JP2007061754W WO2007145179A1 WO 2007145179 A1 WO2007145179 A1 WO 2007145179A1 JP 2007061754 W JP2007061754 W JP 2007061754W WO 2007145179 A1 WO2007145179 A1 WO 2007145179A1
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red blood
erythrocytes
immobilized
blood cells
erythrocyte
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Homu Ito
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae

Definitions

  • 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.
  • 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.
  • HE hemagglutinin-esterase
  • 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.
  • 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.
  • 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.
  • 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. .
  • influenza virus 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.
  • 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.
  • HA hemagglutination
  • 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.
  • 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 ⁇ 7.0 to 7.8 unless otherwise specified.
  • 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.
  • NA neuroaminidase
  • 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.
  • 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.
  • HA hemdalchun
  • NA neuraminidase
  • 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.
  • discontinuous mutation is the recombination (reassembly) of genes in nature
  • continuous mutation can be thought of as mutation and selection.
  • 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!
  • Patent Document 1 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.
  • Patent Document 1 a method in which an adsorbent adsorbed on the hemagglutinin site of influenza virus, for example, sulfatide powder is supported on a mask.
  • Patent Document 1 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
  • 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.
  • - 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the step (a) of publishing with monoacid-carbon gas in the presence of a monosaccharide or disaccharide is performed.
  • step (b) 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,
  • the immobilized red blood cell according to any one of (1) to (3), wherein the monosaccharide is glucose and the disaccharide is sucrose,
  • 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%,
  • the immobilized erythrocyte according to (6) which contains 0.1 to 10% by weight of monosaccharide or disaccharide in the lyophilized product,
  • 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)
  • step (a) of publishing with a monoacid-carbon gas in the presence of a monosaccharide or disaccharide 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.
  • step (b) 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,
  • step (11) wherein the concentration of the formalin solution in step (b) is 3 to 20 v / v%;
  • An influenza virus trapping filter comprising the air-permeable filter carrying the immobilized erythrocyte according to any one of (1) to (7);
  • the chicken, guinea pig or human type O red blood cells can be obtained from each fresh blood.
  • 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.
  • glucose such as glucose, mannose, fructose, sorbose and other pentoses such as ribose and xylose
  • disaccharides include sucrose, trehalose, maltose, and ratatoose. Sucrose is effective.
  • 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%.
  • a commercially available ALSVER solution containing 2.05 w% of glucose is conveniently used.
  • step a 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).
  • red blood cell suspension 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.
  • 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.
  • erythrocytes are fixed in contact with formalin solution in the presence of monosaccharides or disaccharides (step b).
  • Formalin is a 37wZv% formaldehyde aqueous solution
  • 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.
  • Formalin-fixed ⁇ ⁇ ⁇ - ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method for supporting the fixed erythrocytes on the filter is arbitrary. It can be supported using an appropriate binder.
  • 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.
  • 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.
  • the amount of the binder is preferably about 2 to 20% by weight with respect to the immobilized red blood cells.
  • the surfactant for example, PEG or sorbitan monooleate can be used in an amount of about 0.1 to 10 w%.
  • 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.
  • the fixed erythrocyte-carrying gauze can be used as a nasal pillow so that almost all of the inspiration passes through the nasal pillow.
  • Masks and filters after use are positive cleaning agents (reverse sarcophagus) such as commercially available males.
  • positive cleaning agents reverse sarcophagus
  • van solution trade name, manufactured by Nippon Pharmaceutical Co., Ltd.
  • 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.
  • 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.
  • one fresh-bird avian erythrocyte has the ability to bind to more than 5000 influenza virus-infected particles! /
  • 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 ⁇ 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.
  • 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.
  • 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.
  • HA value hemagglutination number
  • 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.
  • 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 .
  • 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.
  • 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.
  • CAM chicken embryo chorioallantoic membrane
  • 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.
  • a tissue culture system was established.
  • 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
  • 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)
  • a porcine kidney cell PS-Y15 cell 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.
  • 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.
  • MEM Eagle Minimal Essential Medium
  • 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 .
  • 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.
  • RDE dry receptor degrading enzyme
  • Example 1 Contains 5 x 10 lc> ZmL of formalin fixed ⁇ - ⁇ 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.
  • 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
  • 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.
  • the virus solution of one aggregation unit contains about 10 7 ⁇ 2 worth of virus particles, so 120 ⁇ 10 7 ⁇ 2 to 128 ⁇ 10 7 ⁇ 2 The virus particles are adsorbed on the receptor.
  • Immobilization Red erythrocyte red blood cell agglutination titer (HA titer)
  • 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.
  • [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.
  • Receptor destruction Red blood cells: room temperature after preparation obtained in the same manner as in Example 1. Storage 5 mL of formalin-fixed ⁇ - ⁇ 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.
  • 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.
  • PBS phosphate-free saline solution of Dulbecco's pH 7.2 without Ca
  • 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.
  • 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).
  • 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.
  • healthy horse serum that had been inactivated by heating at 56 ° C for 30 minutes was added at a rate of 0.2%.
  • Example 1 The same influenza virus strain as that in Example 1 was used.
  • RDE powder was dissolved in 20 mL of physiological saline.
  • 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
  • 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.
  • 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.
  • 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.
  • H reaches 120 units or more and 128 units. Ain is adsorbed by the receptor.
  • the virus liquid of one aggregation unit contains about IX 10 7 ⁇ 2 registered virus particles, 12010 7 ⁇ 2 to 128 ⁇ 7 ⁇ 2 virus particles are on the surface of these immobilized red blood cells. It is adsorbed by the upper receptor.
  • 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
  • the red blood cell group of the receptor (1) almost completely lacked the ability to adsorb the infected virus particles.
  • 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
  • the virus 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.
  • erythrocyte suspension 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.

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Abstract

Une suspension d'érythrocytes de poulet immobilisée avec une solution de formaldéhyde à 10 % conserve encore l'aptitude à se lier à une agglutinine d'érythrocytes virale. Cependant, dans ce procédé, une partie d'un récepteur se liant à l'agglutinine d'érythrocytes perd sa fonction et, de ce fait, présente une avidité moindre pour le virus. De plus, les érythrocytes ne peuvent pas être conservés pendant une longue période de temps et maintenir la fonction du récepteur. Une telle suspension d'érythrocytes devrait donc être préparée in situ. La présente invention permet d'obtenir des érythrocytes de poulet immobilisés de façon stable en conduisant les étapes suivantes en présence d'un monosaccharide ou d'un disaccharide : (a) soumettre facultativement une suspension d'érythrocytes de sang de poulet, de cobaye ou d'humain de type O à un traitement de barbotage avec du monoxyde de carbone ; et (b) traiter de la suspension d'érythrocytes rouge foncé ainsi obtenue par une solution de formaldéhyde. De plus, un produit lyophilisé des érythrocytes immobilisés contenant un monosaccharide ou un disaccharide peut rester stable pendant 5 ans ou plus et maintenir une avidité élevée pour un virus. Un filtre, tel qu'un masque portant ces érythrocytes immobilisés, est hautement utile comme masque protégeant des virus de la grippe.
PCT/JP2007/061754 2006-06-12 2007-06-11 Érythrocytes immobilisés dont la surface porte un récepteur de virus de façon stable, leur procédé de fabrication et leur utilisation Ceased WO2007145179A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5423119A (en) * 1977-07-25 1979-02-21 Takeda Chem Ind Ltd Erythrocyte for haemagglutination test
JPS54104896A (en) * 1978-02-06 1979-08-17 Takeda Chemical Industries Ltd Agent for preetreating serum to be tested of virus agglutination
JPS577419A (en) * 1980-06-17 1982-01-14 Toshiba Kagaku Kogyo Kk Erythrocyte storing solution
JPS63179256A (ja) * 1987-01-20 1988-07-23 Yamanouchi Pharmaceut Co Ltd コレラ菌凝集反応用固定赤血球および該赤血球を使用するエルト−ル型コレラ菌の鑑別方法
JPH05503304A (ja) * 1990-11-07 1993-06-03 バクスター、インターナショナル、インコーポレイテッド 赤血球貯蔵溶液
JP2002058730A (ja) * 2000-08-16 2002-02-26 Kohjin Co Ltd インフルエンザウイルス捕捉フィルター

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5423119A (en) * 1977-07-25 1979-02-21 Takeda Chem Ind Ltd Erythrocyte for haemagglutination test
JPS54104896A (en) * 1978-02-06 1979-08-17 Takeda Chemical Industries Ltd Agent for preetreating serum to be tested of virus agglutination
JPS577419A (en) * 1980-06-17 1982-01-14 Toshiba Kagaku Kogyo Kk Erythrocyte storing solution
JPS63179256A (ja) * 1987-01-20 1988-07-23 Yamanouchi Pharmaceut Co Ltd コレラ菌凝集反応用固定赤血球および該赤血球を使用するエルト−ル型コレラ菌の鑑別方法
JPH05503304A (ja) * 1990-11-07 1993-06-03 バクスター、インターナショナル、インコーポレイテッド 赤血球貯蔵溶液
JP2002058730A (ja) * 2000-08-16 2002-02-26 Kohjin Co Ltd インフルエンザウイルス捕捉フィルター

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