A method for concentrating virus

 

(57) Abstract:

The invention relates to the application of Virology, specifically to the processes of separation, purification, modification of viruses and viral drugs, i.e., processes related to the concentration of viruses. When the concentration of the virus through its binding affinity sorbent containing antibodies to the virus immobilized on a polymer basis, leaching and subsequent desorption, as the polymer base bioaffinity adsorbent using a polyvinyl alcohol cryogel with a pore size of 0.04 to 2.0 μm. In addition, affinity sorbent may, according to this invention, contain, along with antibodies immobilized enzymes, modifying the virus. The proposed method has advantages in comparison with analogues and prototype; and improved the efficiency of the method; b) increased versatility. The invention can be used both in research and in the production of vaccines and diagnostic, as well as in medical practice. 1 C.p. f-crystals.

The invention relates to the application of Virology, specifically to the processes of separation, purification, identification, modification of viruses and viral drugs, i.e., processes associated with conc the ve vaccines and diagnostics, as well as in medical practice.

Known methods of concentration of viral particles in modern Virology are multistage operation long high-speed centrifugation in density gradient of sucrose or glycerol, as well as zone electrophoresis and others [1], also used for concentration of viruses using bioaffinity adsorbents containing immobilized antibodies to the virus [2]. The latter approach is characterized by greater efficiency and technological facilities.

Thus, the known method of bioaffinity purification of measles virus, which use a carrier containing the immunoglobulins in the blood serum of monkeys, covalently grafted to the surface modified glass beads [3]. The method includes passing virusanderica fluid through the chromatographic column Packed with bioaffinity adsorbent with antibodies, covalently-grafted to glass media, with subsequent washing of the column of 0.14 M NaCl and elution of the virus in 0.1 M glycine buffer at pH 2.3.

The disadvantages of this method is low biospecific the capacity of the used media (works only the surface of the glass granules), NASPE.

The known method of bioaffinity purification of viruses of different sizes, where to clean the virus uses silica gel, controlled pore sizes of 0.05 to 1.0 μm) with grafted affine ligands [4]. The method consists of similar transactions chromatographic purification of the target product, as in the previous case, only to wash using 0.1 M Na-phosphate buffer (pH 7.0), and elution is used 0.05 M Na-citrate buffer (pH 3.0).

The disadvantages of this method are the difficulty of obtaining immunosorbent assay associated with the multistage scheme modification inorganic media, as well as the characteristic of such materials fragility. In addition, in the working fluid even with a slightly alkaline reaction (pH 7.5 to 8.5) silica matrix hydrolytically unstable, which leads to the dissolution of the active surface and the progressive reduction of biospecific capacity.

Closest to the proposed technical substance is selected as a prototype method of purification of the virus marsupial disease of chickens by its sorption preconcentration by passing virusanderica suspensions through a column of an immunosorbent assay, representing an insoluble carrier - granularity immunoglobulins [5] . Such a bioaffinity adsorbent are used for cleaning is not very large viruses whose dimensions exceed its 0,08 ám.

However, the concentration of the virus ineffective when working with large (0.1-0.5 micron) viruses. In addition, granular agarose gels, in particular sepharose, are of high value and therefore can not find large-scale application. Brazian used to activate the media is a very toxic compound, it is dangerous for personnel.

The task of the invention is to develop an effective, versatile and technologically simple method for concentration of various viruses, including the largest virus with a particle size of 0.5 μm.

The solution to this problem is achieved by the fact that when the concentration of the virus through its binding affinity sorbent containing antibodies to the virus immobilized on a polymer basis, leaching and subsequent desorption, as the polymer base bioaffinity adsorbent using a polyvinyl alcohol cryogel with a pore size of 0.04 to 2.0 μm. In addition, affinity sorbent may, according to this invention, contain, along with anticellulite (PVA) as a polymer base bioaffinity adsorbent enables technological simplification of the method for concentrating virus improving the efficiency of the method in General, its universality, including in relation to the largest virus with a particle size of 0.5 μm.

Very important properties of PVA cryogels is their biocompatibility, non-toxicity, as well as its availability and relative cheapness. The PVA cryogels formed by the freezing of concentrated aqueous solutions of PVA, storage in a frozen state for a certain period of time and subsequent thawing [6]. Thus formed macroporous cryogel is stable at temperatures up to 60-65oC and has a melting temperature of 80-100oC. He has expressed macroporosity [6], i.e., corresponds to one of the main requirements for holders suitable for use with viruses. According to the present invention as a polymer base bioaffinity adsorbent provides for the use of PVA cryogel having a pore size of 0.05 to 2.0 μm. These pores allow free penetration of even the largest viruses inside the pellet carrier, i.e. the binding of viruses involved affine ligands of the total volume of media.

In addition, in contrast to the fragile macropores the mi viscoelastic physical bodies, not only susceptible to abrasive wear even under vigorous stirring. Thus, the PVA cryogels along with macroporosity and satisfy the requirement of operating stability. And, finally, the PVA cryogels can, if necessary, be subjected to sterilization, for example, washing with disinfectant solutions, irradiation, or (for the destruction of waste material reused.

The method is as follows: virusological fluid incubated with affinity sorbent containing specific antibodies immobilized on the activated matrix of PVA cryogel, wash off unbound and free of extraneous components adsorbed virus from the complex with a bioaffinity adsorbent. For virus modification using affinity sorbent containing coimmobilized specific antibody and enzyme (enzymes) that can cause a modification of the viral particles. The specific implementation of the present invention is illustrated by the following examples:

Example 1.

For concentration of influenza virus (type a, H1N1, the size of the viral particles 0.1 ám) using affinity sorbent, representing the PVA cryogel with a maximum pore size of 1.0 μm,installed from the blood serum of rabbits, immunized with the corresponding antigen (the capacity of the sorbent by the immobilized antibody of 0.7 - 1.2 mg/g). A portion (0.5 g) bioaffinity adsorbent mixed with 2.0 ml virusanderica liquid for 20 hours at +5oC, then washed unbound components 0.1 M PBS (control - zero values of optical density at 260 nm and 280 nm, and a negative reaction hemaglutination (DSA)), and then hold desorption virus 0,65 NaCl in 0.05 M Tris-HCl (pH 7,4) portions of 1.0 ml for three hours before full removal of the virus. The capacity of the affinity of the sorbent for desorbed antigen - 1400 GUY/g media. (The GUY is the product of the inverse of the title on the volume of the sample). The specificity obtained bioaffinity adsorbent (with immobilized antibodies to influenza A, H1N1) check with influenza A, H3N2. Antigenic activity is determined by the reaction of direct haemagglutination (DSA). The results show that at this immunosorbent assay nonspecific antigens (H3N2) is not adsorbed, i.e., there is only biospecific sorption (concentration) of the antigen.

Example 2.

For concentration of influenza virus (type a, H1N1, the size of the viral particles 0.1 ám) using affinity sorbent, represents what the covalently sewn specific antibodies, obtained from the serum of rabbits immunized with the corresponding antigen (the capacity of the sorbent by the immobilized antibody of 0.5 - 0.6 mg/g), and elution of concentrated virus is carried out in a column mode using 2,5 M MgCl2in 0.05 M Tris-HCl (pH 7,4). The amount of desorbed virus is 800 GUY/g of carrier.

Example 3.

For concentration of influenza virus (type a, H3N2, the size of the viral particles of 0.12 μm) using affinity sorbent, representing the PVA cryogel with a maximum pore size of 0.7 micron, activated with bromine cyan at a pH of 11.5, which covalently sewn specific antibodies obtained from the serum of rabbits immunized with the respective antigens (the capacity of the sorbent by the immobilized antibody 1.5 mg/g), and elution of concentrated virus is performed using a 0.65 M NaCl in 0.5 M Tris-HCl (pH 7,4). The amount of desorbed virus is 2000 AI/g of carrier.

Example 4.

For concentration of parainfluenza virus SE6(the size of the viral particles of 0.15 μm) using affinity sorbent, representing the PVA cryogel with a maximum pore diameter of 1.5 μm, activated with glutaraldehyde at a pH of 0.5, which is sootvetstvuyuschim antigen (the capacity of the sorbent by the immobilized antibody 0.5 to 0.7 mg/g), and elution of concentrated virus is carried out with the help of 0.65 NaCl in 0.05 M Tris-HCl (pH 7,4). The titer of the purified antigen (parainfluenza virus SE6) determined by indirect enzyme-linked immunosorbent assay (ELISA). The capacity of the affinity of the sorbent with respect to this virus is 1266 units antigenic activity per 1 g of the carrier.

Example 5.

For concentration of smallpox (the size of the viral particles of 0.4 x 0.2 µm) using affinity sorbent, representing the cryogel surfactant with a maximum pore diameter of up to 2 microns activated with glutaraldehyde at a pH of 1.2, which covalently sewn specific antibodies obtained from the serum of rabbits immunized with the corresponding antigen (the capacity of the sorbent by the immobilized antibody 0.8 mg/g), and elution of concentrated virus is carried out with the help of 0.75 NaCl in 0.05 Tris-HCl (pH 7.8). The titer of the purified antigen (smallpox virus) define indirect variant of ELISA. The capacity of the affinity of the sorbent with respect to this virus is 2048 units antigenic activity per 1 g of the carrier.

Example 6.

For concentration and enzymatic modification of FMD virus (the size of the viral particles of 0.02 μm) of a portion (0.5 g) bioaffinity with the m aldehyde at a pH of 1.0, to which covalently sewn specific antibodies to FMD virus (obtained from the blood serum of calves immunized with the corresponding antigen) and the modifying enzyme papain (the capacity of the sorbent by the immobilized antibody - 0.4 mg/g, papain - 0.2 mg/g) is stirred with 2.0 ml virusanderica fluid for 12 hours at +5oC. Similarly proceed with the control of bioaffinity adsorbent, which immobilized papain contains blocked Tilney group of the active centre. Then hold desorption virus 0.5 M NaCl in 0.05 M Tris-HCl (pH 7,4) portions of 1.0 ml for three hours. Capacity control bioaffinity adsorbent desorbed by the virus is 780 PFU/g of carrier (PFU - plaque-forming units), the carrier with the active papain - 32 PFU/g carrier, i.e. in 24.3 times less, that is the result of immobilized protease in the FMD virus is concentrated in the phase of bioaffinity adsorbent due to the interaction with the immobilized antibodies (the fact of such concentration prove the results of the experiment with the control of bioaffinity adsorbent), which ultimately leads to a significant reduction virulent virus activity.

Example 7.

oC. Similarly proceed with the control of bioaffinity adsorbent, representing the drug, which is immobilized on the carrier only antibodies specific to mumps virus (capacity control bioaffinity adsorbent for an antibody - 0.7 mg/g of carrier). Then wash off unbound components 0.1 M PBS (control - zero values of optical density at 280 and 260 nm) and then hold desorption of the virus in 0.5 NaCl in 0.05 M Tris-HCl (pH 7,4) portions of 1.0 ml for three hours. Capacity control bioaffinity adsorbent desorbed by the virus is 650 the GUY/g carrier, for sorbent with the modifying enzymes virologic activity is not recorded, due to the action of other RNA elements in released from the virion RNA, the result is the absence of virulent virus activity.

The proposed method concentration of the virus has the following advantages in comparison with analogues and prototype:

- improved the efficiency of the method, since, in contrast to methods analogous to the claimed method is used affinity sorbent-based viscoelastic non-fragile and hydrolytically stable PVA cryogel, so it is possible to carry out the concentration of the virus not only in chromatographic (column) variant, but in the reactors with mixing of the sorbent, which significantly intensifies mass transfer processes, i.e., improves the manufacturability of the way;

- increased the versatility of the method, because there is the possibility of concentration, along with small, even the largest viruses (0.5 µm or more), while in the method-prototype matrix bioaffinity media capable of diffusion inside the granules are relatively small (up to 0.08 μm) viruses; in addition, the versatility of the proposed method is also due to the possibility of simultaneous concentration and enzymatic modification of the virus when bioaffinity adsorbent along with immobilized the ve PVA cryogel inert and vysokospetsifichnymi, can be reused without significant specific activity, the polymer used to prepare the gel base cheap and available.

Literature

1. H. Fraenkel-Konrat. Chemistry and biology of viruses.// M,1972, pp. 33-37.

2. J. Turkova, Affinity chromatography,//M,1980, page 5-14.

3. Purification of measles virus by affinity chromatography and by ultracentrifufation: a comparative study//Njayou M., Quash G.// J. Yirol. Meth. in 1991, v. 32, N 1, p/67-77.

4. Separation and purification of biopolimers by affinity chromatography on controlled-pore silica gel./M. Colpan//Ger. offen. DE 3627063, cl. B 01 D 15/08, 1988.

5. Separation and purification of antigen for diagnosis of infectious bursa disease in chickens. Nagai Sh., Otaki Y., Ueda, S., JP 63210775, cl. G 01 N 33/569, 1989.

6. The use of polyvinyl alcohol cryogels in biotechnology. A review of literature data. //Lozinski Century. And., Vakula A. C., Teeth A. L.// Biotechnology, No. 4, 1992, S. 5-14.

1. A method for concentrating virus, including its binding affinity sorbent containing antibodies to the virus immobilized on a polymer basis, leaching and subsequent desorption, wherein as the polymer base bioaffinity adsorbent using a polyvinyl alcohol cryogel with a pore size of 0.04 to 2.0 μm.

2. The method according to p. 1, characterized in that the binding of the virus to carry out the z virus.

 

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