RU2329505C1 - Method of immunosorbent production for virus-specific antibody binding - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to virology, namely, to methods of immunosorbent production. Method of immunosorbent production virus-specific antibody binding is offered. Method includes inorganic sorbent-carrier incubation with virus-containing liquid. Ultradisperse oxygen-containing graphite is used for inorganic sorbent-carrier as foamed particles of the stratified graphite, containing oxygen in amount 12-14 mass % per 73-76 mass % of carbon. Specific surface of graphite is 1500-2000 m³/g, particle size is 25-50 mcm. Graphite is pre-boiled in distilled water. Produced suspension of sorbent-carrier at graphite content not less than 2 mass % is incubated with virus-containing liquid at temperature 5-35°C. Virus-containing liquid contains, e.g. influenza virus.

EFFECT: produced immunosorbent for virus-specific antibody binding has high sorptive power, enables to bind completely virus-specific antibodies of immune serum.

2 cl, 5 tbl

Description

The invention relates to medicine and biology, in particular to virology - to methods for producing immunosorbents, and may find application in the diagnosis of viral infections.

Immunosorbents are the main bioactive components of test systems for immunodiagnostics. Immunosorbents for detecting antibodies to viruses are obtained by immobilizing viruses or antigens on a polymer (RU 2138286, А61К 38/36, С07К 14/00, G01N 33/53, 09/27/1999; SU 1517545, G01N 33/53, publ. 15.12.1993 ) or inorganic carrier (RU 2140084, G01N 33/551, G01N 33/552, G01N 33/533, B01J 20/02, B01J 20/10, publ. 20.10.1999; RU 2192013, G01N 33/543, G01N 33 / 531, publ. 10/27/2002).

For example, a method for producing an immunosorbent for diagnosing viral hepatitis C is known, including incubating the polymer carrier with a virus-specific protein for 20-24 hours at 4 ° C, washing the excess protein and drying the resulting immunosorbent. As a virus-specific protein, a protein produced by a recombinant E. coli strain is used as a solution in acetate or phosphate buffer. To reduce the non-specific sorption of the immunosorbent, it is treated after drying with a 10% solution of clarified cattle serum or 5-7% solution of skim milk (RU 2095815, G01N 33/543, publ. 10.11.1997).

The disadvantages of this known method of producing an immunosorbent are its limitedness to only one virus-specific protein, as well as the difficulty of its use for other carriers, inorganic in particular.

Closest to the proposed method for producing an immunosorbent is a method for producing an immunosorbent capable of binding antibodies to influenza viruses, which consists in incubating barium sulfate powder (BaSO ₄ ) (as an inorganic sorbent carrier) with a liquid containing influenza virus at 4 ° C for 1 hour based on 1 g of BaSO ₄ per initial virus concentration equal to 1 million hemagglutinating units (CU) or 1000 CU per 1 mg of BaSO ₄ - use a concentrated viral preparation containing at least 2000 CU in 0.2 ml. The resulting suspension is centrifuged at 2000 rpm, the precipitate is washed from the excess virus with physiological saline with a pH of 7.2. Immunosorbent can be stored in lyophilized form for up to 18 months. or in physiological saline in the form of a 50% suspension at 4 ° C for up to 2 months. (Zakstelskaya L.Ya., Shenderovich SF. Use of an immunosorbent to remove anti-influenza antibodies from diagnostic sera. Laboratory, 1979, No. 12, pp. 748-749 - prototype).

The disadvantage of the prototype method is the low sorption ability of the sorbent carrier used in it, which leads to low efficiency of the immunosorbent in the process of binding of influenza antibodies. In addition, the method requires strict adherence to temperature conditions, which complicates the process.

The objective of the invention is to develop such a method of producing an immunosorbent for binding of virus-specific antibodies, which will significantly increase the sorption ability of the sorbent carrier and will provide high efficiency of the immunosorbent in binding of viral antibodies and, in addition, will simplify the process.

The solution of this problem is achieved by the proposed method for producing an immunosorbent, including incubation of an inorganic sorbent carrier with a virus-containing liquid, in which, according to the invention, ultrafine oxygen-containing graphite in the form of expanded particles of layered graphite containing oxygen in an amount of 12-14 wt is used as an inorganic sorbent carrier % by 73-76% by weight of carbon, which is preliminarily treated with water at 95-100 ° C, then centrifuged and the resulting precipitate is suspended in one buffer solution, the resulting suspension of the sorbent carrier with a graphite content of at least 2 wt.% incubated with a virus-containing liquid at a temperature of 5-35 ° C.

In the method, you can use ultrafine oxygen-containing graphite with a specific surface of 1500-2000 m ³ / g and with a particle size of 25-50 microns.

The virus-containing fluid may contain an enveloped virus.

An enveloped virus in a virus-containing fluid may be an influenza virus.

When choosing a carrier sorbent, the main criterion was a high sorption ability with respect to viruses.

One of the most known sorbents for viral binding in virology is a suspension of formalized chicken erythrocytes (Rovnova ZI Issues of Virology, 1959, No. 4, p. 465). However, this sorbent did not find wide practical application, since the sorption process requires strict adherence to a special temperature regime, in violation of which the complex of red blood cells with viruses is destroyed, and the virions enter the solution.

Until recently, it was believed that sorbents based on activated carbon were not able to remove viruses from water (RU 2237022, C02F 1/28, C02F 1/50, publ. 09/27/2004, US priority of 05/20/1999).

However, a number of studies indicate the possibility of using various modifications of activated carbon for the adsorption of viruses.

Clay (dark KJ, Sarr AB et al. Vet. Microbiol. 1998, v.63 (2-4), p.137-146) used clay or charcoal as sorbents for rotaviruses and coronaviruses of cows that cause gastroenteritis in mammals and birds. In (Zadorozhnaia V.I. Mikrobiol. J., 1996, v. 58 (3), p. 83-7), a carbon sorbent was proposed for removal of enteroviruses (type 2 polioviruses) from solutions. The patent (RU 2237022, C02F 1/28, C02F 1/50, publ. 09/27/2004, US priority 05/20/1999) proposes a carbon sorbent, which is an activated carbon particle having a size of from 0.1 to 5000 microns, capable of remove nano-sized pathogens, such as viruses, from water (using the bacteriophage MS-2 as an example). The sorbent for use is placed on a filter of a special design, while preliminary extrusion of activated carbon particles in the form of hollow tubes is provided.

It should be noted that, depending on the composition and method of producing activated carbon, a wide range of their sorption properties is observed, in particular, pore size distribution and specific surface area.

Currently, various forms of modified graphite are known in the form of layered graphite compounds that are used in various fields of economy (SU 1614350, СВВ 31/04, publ. 02.20.1995; SU 1813711, СВВ 31/04, publ. 07.05.1993; RU 2089495, СВВ 31/04, publ. 09/10/1997; RU 2090498, С01В 31/04, publ. 09/20/1997).

An ultrafine oxygen-containing graphite (oxygen content of 12-14 wt.% Per 73-76 wt.% Carbon) with a specific surface area of 1500-2000 m ³ / g, containing expanded particles of layered graphite 25-50 microns in size, the preparation of which was described, was selected for research. in patent RU 2198137, СВВ 31/04, publ. 02/10/2003. The choice of this ultrafine graphite was due to the fact that it is a relatively cheap material and has a high sorption capacity for aromatic and aliphatic chemical compounds. For the adsorption of biological objects from aqueous media, this graphite sorbent is not convenient due to the extremely low specific gravity (0.25 g / cm ³ ).

As a result of the experiments, the proposed method for producing an immunosorbent was developed, including preliminary processing of ultrafine graphite with water at 95-100 ° С, which allowed significantly increasing the efficiency of the interaction of the sorbent with objects adsorbed from liquid media: during hydrothermal treatment (at 95-100 ° С) In the initial ultrafine graphite, the gas component is replaced by a liquid medium. At the same time, the sorbent carrier is sterilized.

To obtain the immunosorbent, enveloped viruses were used, which include, for example, orthomyxoviruses, paramyxoviruses, arboviruses, etc. Reference and epidemic strains of influenza viruses A (H1N1, H3N2) and B (belonging to the family of orthomyxoviruses) isolated during 1982 were taken -2004, with a different structure of surface proteins and, accordingly, physico-chemical properties. Influenza viruses were cultured in various systems (chicken embryos (CE), tissue culture MDCK). The work also used 10-50% concentrated sucrose allantoic influenza viruses (grown on TBE) purified in a gradient of sucrose concentration.

Determination of the concentration of viruses in solution was carried out using the reaction of hemagglutinating activity (RGA). Determination of the concentration of antibodies in serum - using the inhibition of hemagglutinating activity (rtga). Both reactions were carried out using a 0.75% suspension of human blood red blood cells with a 0 (I) blood group. Protein concentration in solutions was determined according to the Lowry method (Lowry O.K. et al. Protein measurement with folin reagent. J. Biol. Chem., 1951, V.193, p.265-267).

The proposed method for producing immunosorbent is as follows.

Dry ultrafine oxygen-containing graphite with a specific surface of 1500-2000 m ³ / g, containing expanded particles of layered graphite with a size of 25-50 microns, is mixed with distilled water at a mass ratio of graphite: water of 1: 1000-5000, the mixture is heated to boiling and boiled for 10-15 minutes, then stirred for 20 minutes and centrifuged for at least 10 minutes at 10,000 rpm. The resulting precipitate was suspended in an aqueous buffer, for example, in Tris-HCl buffer (pH 7.2). The graphite content in the suspension is at least 2 wt.%. The resulting suspension of graphite is incubated with a virus-containing liquid on a shaker at a temperature of 5-35 ° C for 15-30 minutes and centrifuged at 2000-3000 rpm for 3-5 minutes. The supernatant is examined for the presence of the virus using RGA. The precipitate, an immunosorbent, is washed with physiological saline with a pH of 7.2 (RF) until the virus is absent in the supernatant (RGA control) and is used in experiments on the binding of virus-specific antibodies from immune serums. The immunosorbent is stored in physiological saline at 4 ° C.

A study of the sorption of different strains of influenza A and B viruses on hydrothermally modified ultrafine graphite (carrier sorbent) showed that all viruses successfully interacted with the carrier sorbent under different conditions, regardless of the antigenic properties of surface proteins. Influenza viruses A and B were sorbed from various virus-containing fluids: the allantoic fluid CE of the MDCK culture fluid, FR, and buffer solutions. In the experiments, the weighed suspension of the carrier sorbent ranged from 50 to 200 mg with a constant volume of added virus-containing liquid V _o = 200 μl.

The invention is illustrated by the following examples presented in tables.

Table 1 shows the data on the immobilization of various viruses depending on the concentration of the virus (titer in GE) in the virus-containing fluid. The range of initial titers of the virus in viral preparations is from 16 to 32768 GE in 200 μl. The initial titers of viruses in terms of 1 mg suspension of the sorbent carrier were in the range from 0.3 to 650 GE (in terms of dry graphite - 15-32500 GE for 1 mg). The final titers (after sorption) in the solution amounted to 2-256 GE (in terms of 1 mg suspension of the carrier sorbent 0.04-5.0 GE, respectively), that is, a drop in the hemagglutinating titer of influenza viruses after sorption was 8-128 times. With an increase in the concentration of the virus in the initial solution, sorption processes proceed more efficiently.

Table 1 Adsorption of influenza A and B viruses (50 mg of a 2% suspension of the carrier sorbent in 200 μl of the viral preparation) Virus Antigenic formula Cultivation system, purification The titer of the virus in the RGA, GE in 0.2 ml Before sorption After sorption A / Texas / 1/77 A / H3N2 CE 16 <2 A / Sichuan / 2/87 A / H3N2 CE purified 2048 16 A / H3N2 CE purified 32768 256 A / Texas / 1/77 A / H3N2 CE 16 <2 Epidemic strain A / H1N1 CE 32 four B / Shanghai / 361/02 AT CE 32 four Epidemic Strains A / H1N1 MDCK 64 2 A / H3N2 64 <2 AT 128 8

In addition to detecting a drop in the virus titer in a virus-containing liquid as a result of virus immobilization on a sorbent carrier, control experiments were carried out to determine (by the Lowry method) the sorption of proteins present in a virus-containing liquid (including viral ones). To assess the sorption ability of the sorbent carrier, proteins with different molecular weights and different molecular sizes were used: allantoic proteins, bovine serum albumin, and immune serum proteins.

It was shown that bovine serum albumin is completely sorbed from a 1% aqueous solution onto a sorbent carrier upon contact for 30 minutes at 22 ° C. Serum protein sorption occurred up to 53%. When studying the interaction of the sorbent carrier with the allantoic fluid containing and without influenza viruses, it was found that proteins from the allantoic fluid containing no virus were sorbed by approximately 64%. The percentage of protein sorption on a sorbent carrier from an allantoic virus-containing liquid ranged from 69 to 83%, that is, adsorbed viruses account for about 19% of the protein. It should be noted that protein sorption increased slightly if the allantoic virus was diluted with buffer.

The effect of temperature and the ratio of the mass of the suspension of the sorbent carrier to the volume of the solution containing viruses was also analyzed. The mass of the suspension of the sorbent carrier in the samples varied from 50 to 200 mg, the volume of the virus-containing liquid was constant and equal to 200 μl. As studies have shown (see table 2), the immobilization of viruses (for example, A / Sichuan / 2/87 A / H3N2) occurs equally intensively at temperatures from 5 to 35 ° C. A change in the weight of the suspension of the carrier sorbent from 50 to 200 mg does not affect the intensity of sorption of viruses.

table 2 Effect of sorption temperature and carrier sorbent concentration on the adsorption of a purified reference strain of influenza A virus (H3N2) Virus Antigenic formula RGA virus titer Temperature ° C A portion of the suspension of the sorbent carrier, mg Before sorption After sorption 64 fifty twenty 64 one hundred A / Sichuan / 2/87 A / H3N2 32000 twenty 128 twenty 150 64 200 twenty 128 150 35 128 150 5

To determine the optimal time of contact of the virus-containing liquid with the carrier sorbent, studies were carried out on the effect of the duration of its incubation with the carrier sorbent on the sorption process. The results of the experiments (for example, the virus A / Sichuan / 2/87 A / H3N2) showed (see table 3) that sorption occurs almost identically in the time range from 15 to 120 minutes.

Table 3 The effect of the duration of contact of the virus-containing liquid with the sorbent carrier Virus Antigenic formula Cultivation system, purification The titer of the virus in the RGA, GE in 0.2 ml Sorption conditions Before sorption After sorption Time min HS A / Sichuan / 2/87 A / H3N2 CE, purified, concentrated 131072 1024 fifteen 22 1024 thirty 22 512 60 22 512 120 22

Of particular interest was to determine how stable the virus is associated with the carrier sorbent in the immunosorbent. To do this, we studied the desorption of the virus from the immunosorbent in two solutions: Tris-HCl buffer and FR. In the experiments, the immunosorbent was mixed with one of these solutions (200 μl volume) and kept for 72 hours at 35 ° C or placed on a shaker for 1 hour at 20 ° C. Then the immunosorbent was centrifuged for 5 min at 2000-3000 rpm. The supernatant was examined for the presence of virus in the RGA. It was found that the virus in the immunosorbent is firmly bound to the carrier sorbent, since the desorption process is weakly expressed: the titers of the virus in the supernatant after desorption did not exceed 8 GE (see table 4).

The study of the interaction of immunosorbent with antibodies.

The prepared immunosorbent with immobilized virus A / Sichuan / 2/87 A (H3N2) was mixed with a solution of immune serum containing homologous antibodies to this virus. The mixture was kept under various conditions: 1) for 30 min at 22 ° C or at 37 ° C and 2) 16 hours at 4 ° C. Then the mixture was centrifuged. The supernatant was examined for the presence of antibodies in rtga. Analysis of the serum solution before and after interaction with the immunosorbent showed that the serum titer decreased from 1/640 to <1/20, which indicates the binding of antibodies to the influenza virus from serum by immunosorbent under the studied conditions (table 5). Similar results were obtained for immunosorbents with influenza A (H1N1) and B viruses when interacting with homologous sera.

Table 4 Desorption of influenza viruses from immunosorbent Virus Antigenic formula Cultivation system, purification VGA titers Desorption Conditions Before sorption After sorption After desorption T, ° С Time, hour. Buffer A / Sichuan / 2/87 A / H3N2 CE, cleared. 8000 64 <2 twenty one Tris-HCl 256 8 twenty one FR 64 four 35 72 Tris-HCl 32 8 35 72 FR B / Shanghai / 362/02 AT CE 256 32 four twenty one Tris-HCl Epidemic. strains A / H3N2 MDCK 128 2 <2 35 72 Tris-HCl AT 256 64 <2 35 72 Tris-HCl Table 5 Antibody binding by immunosorbent containing A / H3N2 influenza virus from homologous immune serum Immune serum titers in rtga Conditions for the interaction of serum with immunosorbent Before interaction with immunosorbent After interaction with immunosorbent temperature, ° С Contact time 640 <20 37 30 minutes 640 <20 22 30 minutes 640 <20 four 16 hour

Thus, the developed method for producing an immunosorbent can significantly increase the sorption ability of the sorbent carrier and provides the resulting immunosorbent with the ability to fully bind virus-specific antibodies from immune serums. The use of various viruses will allow you to get a wide range of immunosorbents to determine the spectrum of antibodies in immune sera. The method is simple and highly reproducible.

Claims (2)

1. A method of producing an immunosorbent for binding antibodies specific for enveloped viruses, comprising incubating an inorganic sorbent carrier with a virus-containing liquid, characterized in that ultrafine oxygen-containing graphite in the form of expanded particles of layered graphite with a specific surface of 1500-2000 is used as an inorganic sorbent carrier m ³ / g with a particle size of 25-50 microns, containing oxygen in an amount of 12-14 wt.% on 73-76 wt.% carbon, which is preliminarily boiled for 10-15 minutes at the ratio of graphite: distilled water is 1: 1000-5000 by weight, then mixed, centrifuged and the resulting precipitate is suspended in an aqueous buffer solution, the resulting suspension of a sorbent carrier with a graphite content of at least 2 wt.% incubated with a virus-containing liquid at a temperature of 5-35 ° C. 2. The method according to claim 1, characterized in that the virus-containing liquid contains influenza virus.