RU2694836C1 - Method of producing rabies virus antigen for serological diagnosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to veterinary science, and can be used in preparing the rabies virus antigen for serological diagnosis. That is ensured by preparing a suspension of brain white mice experimentally infected with rabies virus. Brain tissue is homogenised in 0.1 M phosphate-buffer solution at pH 7.2–7.4. Obtained cerebral suspension is held in test tubes for at least 5 minutes until complete precipitation of silicon carbide balls. Supernatant is taken and 0.1 M phosphate-buffer solution is added to the test tubes, then shaken 3 times, held for at least 5 minutes and centrifuged for 50 minutes at 5,000 rpm. Supernatant is concentrated by ultracentrifugation at 37,000 rpm for 4 hours. Supernatant is then removed and the resulting resuspended residue is layered on a step gradient of 10–60 % sucrose. Ultracentrifugation for 3 hours at 30,000 rpm at temperature of +40 °C viral material is then fractionated by gel filtration. Immunoblot results are used to select active fractions, re-precipitate them with alcohol and obtain an antigen of rabies virus.

EFFECT: invention increases specificity and sensitivity of the obtained antigen and increases output of the active antigen.

1 cl, 1 tbl, 6 dwg

Description

The invention relates to veterinary Virology, immunology, in particular to a method of obtaining antigen of rabies virus from the production strain of rabies virus "Sheep" GNCI for the diagnosis of rabies.

The spread of rabies among animals is one of the most important international criteria for assessing the biological and ecological safety of the human environment. Globally, from 55 to 70 thousand people die of rabies each year, half of which occur in children, and up to 6.5 million people are exposed to post-exposure anti-rabies treatments (Smith A. et al. 2005; 3 (1): 29-37, "Ecological theory to enhance infectious disease control and public health policy" (2005). In Russia, 250-450 thousand cases of rabies are recorded annually. (Onishchenko GG, 2012 Resolution of the Chief State Sanitary Doctor of the Russian Federation of February 1, 2012 N 13 Moscow “On Strengthening Measures Aimed at the Prevention of Rabies in the Russian Federation”). Rabies is among the most dangerous viral diseases, according to WHO, among the top five infections common to humans and animals, causing the greatest social and economic damage. Taking into account the exclusivity of rabies with its inherent characteristics of many classic infectious diseases - world nosoareal, natural foci, extreme contagiousness, vector transmission by carnivores, susceptibility of most animal species, social and economic significance, this disease is included in the list of especially dangerous infectious diseases of the OIE (World Organization for Animal Health, 2015), and in Russia - in the List of infectious, including especially dangerous, animal diseases, according to which restrictive measures can be established (quarantine) (Order of the Ministry of Agriculture of the Russian Federation of December 19, 2011, No. 476).

Russia occupies a dominant position both in the number of unfavorable points (outbreaks) and in the incidence of animals. Over the past 20 years, Russia has recorded the highest mortality rate of rabies among developed countries. Despite ongoing activities, natural foci of rabies intensified in Russia over the last period, the incidence of wild carnivores increased, domestic animals are intensively involved in the epizootic process, creating a threat to the population, which predetermines the modern features of the epizootic and species composition of diseased animals. Today, the problem of the fight against rabies is one of the most pressing due to the prevalence of the disease and the absence of specific treatment [5].

The most important means of controlling the spread of rabies along with specific prevention is accurate and reliable laboratory diagnosis [7, 8]. Modern means of laboratory diagnostics have a specific set of "in vitro" tests, such as the indirect hemagglutination test (RNGA), the neutralization reaction (PH), the compliment binding reaction (RSC), the indirect immunofluorescence reaction (RNIF), the diffusion backward precipitation test (RDP), however, in screening studies, they have several disadvantages due to their length and complexity [1, 9]. To solve such problems, enzyme-linked immunosorbent assay (ELISA) methods and their various variants are developed. They are distinguished by sufficient sensitivity and specificity, which are determined on the basis of which test systems were designed to quantify antibodies to rabies virus in blood serum, but not all of them are sensitive enough. specificity determined by the biological properties of the antigens used in them [2, 3, 4].

In this regard, one of the most important tasks of rabies prophylaxis is the improvement and selection of technical solutions for the isolation of highly purified rabies virus antigen for further use in the creation of modern diagnostic tests and the production of specific antibodies. The solution of this problem is of great national economic importance and contributes to the expansion of laboratory diagnostics, detection of the rabies virus and determination of the immune response of vaccinated animals and people.

The improvement of virus cleaning methods is aimed at finding new technical approaches to increase the viral mass yield, preserve its serological activity, obtain highly purified discrete and monomeric structures of the pathogen without impurities of other associated biological structures tropic to the rabies virus, with constant monitoring of each purification step using methods analytical electrophoresis, immunoblotting and ELISA using hyperimmune blood sera of rabbits and sheep.

A method of obtaining antigen for the diagnosis of rabies, based on intracerebral infection of newborns of the foal with rabies virus, taking brain tissue, preparing a suspension, an additional twofold treatment of brain tissue with ultrasound at 15-20 kHz for 25-30 minutes and separation of the rabies virus in a sucrose gradient (innovative Patent KZ # 23848, А61К 39/205, published on 15. 04.2011 Bull. # 4).

This method has a significant drawback consisting in a large amount of ballast material (Fig. 1), the presence of which reduces the proportion of specific antigen in this material and leads to a decrease in the specificity of hyperimmune serum. Its further use in serological tests, such as ELISA, MFA reduces the effectiveness of their use. Obviously, this phenomenon is caused, on the one hand, by the duration of ultrasound treatment (25-30 min), which leads to heating of the dispersible material, as a result of which irreversible destructive changes in the biological material (cell suspension and virus) are possible, on the other hand - long-term cavitation effect leads to the complete destruction of cells and, as a consequence, increases the yield of the virus.

The presence of a huge amount of destroyed material has supposedly the same buoyant density with the virus, and therefore the presence of proteins of the brain tissue is observed in the zones of localization of the viral mass after centrifugation in a sucrose density gradient.

The closest to the proposed invention is a method of obtaining rabies antigen for serological diagnosis, including obtaining suspensions from the brain of white mice experimentally infected with the rabies virus - the sheep strain of GNCI, disintegration on the FastPrep 24 ™ device at a vibration speed of 6 ml / s for 60 in the presence of homogeneous particles of silicon carbide, precipitation of brain tissue by low-speed centrifugation, concentration of the supernatant by ultracentrifugation at 25,000 rpm, and separation of the rabies virus in steps the sucrose gradient (15-50%) (see Art. Efimov MA and others. "Detection, purification and evaluation of the serological activity of the antigens of the rabies virus" / "Problems of especially dangerous infections." Scientific and practical., Saratov, Issue 4, 2017, pp. 27-31).

However, the disadvantage of this method is the low yield of the rabies virus antigen and low sensitivity and specificity.

The technical result, the achievement of which the invention is directed, is to increase the yield of monomeric antigen, increasing its sensitivity and specificity.

To achieve the above technical result in the proposed method, which includes obtaining suspensions from the brain of white mice experimentally infected with the rabies virus - sheep strain, GNCA, disintegration on a FastPrep 24 ™ device at a vibration speed of 6 ml / s in the presence of uniform particles of silicon carbide, centrifuging the resulting supernatant to remove foreign particles, ultracentrifuge for precipitating viral particles and separating the rabies virus in a stepwise sucrose gradient, before brain disintegration This tissue is additionally homogenized in a glass homogenizer for at least 3-5 minutes in 0.1 M phosphate buffer solution, pH 7.2-7.4, and disintegration is carried out three times for 30 seconds with an interval between treatments of 5 minutes, then obtained the brain suspension is kept in Blue Lysing Matrix tubes for at least 5 minutes until the silicon carbide balls are completely settled, the supernatant is taken in a separate container, and 0.1 M phosphate buffer solution is added to the tubes, shaken 3 times on Vortex V1 for 30 s and incubated for at least 5 minutes, the resulting solution is taken and all n The resulting supernatants are combined, centrifuged for 50 minutes at 5000 rpm, the supernatant taken into a sterile container, concentrated by ultracentrifugation at 37,000 rpm for 4 hours at + 4 ° C, the supernatant is removed, and 0.1 M phosphate buffer solution, pH 7.2-7.4, in a volume equal to 1/10 of the original eluate, shaken 3 times for 30 s with an interval of 3 minutes between shaking, then the resuspended precipitate is layered on a step gradient of 10-60 % sucrose and ultracentrifuge for 3 aces at 30,000 rev / min at + 4 ° C, followed by viral material is fractionated by gel filtration and the results of immunoblotting active fractions were collected, recrystallised their alcohol to give rabies virus antigen.

Distinctive features of the proposed method are the additional homogenization of brain tissue before disintegration in a glass homogenizer for at least 3-5 minutes in 0.1 M phosphate buffer solution, pH 7.2-7.4, carrying out 3 times disintegration within 30 s with an interval between treatments 5 minutes, keeping the brain suspension in Blue Lysing Matrix B tubes for at least 5 minutes until complete precipitation of silicon carbide pellets, selection of the supernatant into a separate container, shaking 3 times a day on Vortex V1 for 30 seconds and holding for at least 5 minutes, sampling the resulting soup batching, combining all the resulting supernatants, centrifuging for 50 minutes at 5000 rpm, selecting the supernatant into a separate sterile container, concentrating by centrifuging at 37000 rpm for 4 hours at + 4 ° C, removing the supernatant and adding to sediment 0 , 1 M phosphate buffer solution, pH 7.2-7.4, in a volume of 1/10 of the initial eluate, 3 times shaking for 30 s with an interval of 3 minutes between shaking, layering of the resuspended sediment on a step gradient of 10-60 % sucrose, ul rocentrifuging for 3 hours at 30,000 rpm at + 4 ° C, fractionating viral material using gel filtration, selecting active fractions based on immunoblotting results, re-precipitating them with alcohol and obtaining a rabies antigen, which allows to obtain monomeric, highly purified and specific antigen of rabies virus and increase the yield of antigen by 2 times, increase the specificity and sensitivity by 8 times.

The invention is illustrated by drawings, which depict:

FIG. 1. - Results of analytical electrophoresis in 12.0% PAAG and immunoblot with rabbit hyperimmune serum sediment of viral material, divided by buoyant density, obtained after UFC at 37000 rpm.

FIG. 2. - Distribution of buoyant sediment density of viral material obtained after UCF at 37000 rpm in a stepwise sucrose gradient (10-60%).

FIG. 3. - Results of analytical electrophoresis in 12.0% PAAG and immunoblotting with rabbit hyperimmune serum sediment of viral material divided by floating density in a stepwise sucrose gradient (10-60%) obtained after UCF at 30,000 rpm.

FIG. 4. - The histogram of the fractionation of viral material, selected according to the results of immunoblotting after UCF in the sucrose density gradient obtained by gel filtration on an Enrich ™ SEC 70 column.

FIG. 5. - Results of immunoblot of viral material after gel filtration on an Enrich ™ SEC 70 column with hyperimmune rabbit serum.

FIG. 6. - Densitogram of immunoblot of the initial viral material before chromatography, fraction No. 5, and alcohol residue of fraction No. 5.

Antigen for the diagnosis of rabies is obtained as follows:

To obtain used a production strain of rabies virus "Sheep" GNKI (collection FSBI "FTSTB-VNIVI) organo-tissue origin with an infectious titer of 5.25 lgLD ₅₀ / 0.03 ml. Pre-rabies virus-strain “Sheep” GNKI ”refreshed through the brain 12-14 daily mice, conducting 3-4 blind passages. Then lambs of 2-3 months old were taken from seronegative sheep, under local anesthesia they were infected with a dose of 0.5–2.0 cm ^{3 with a} 20% suspension of the brain of white mice infected with the rabies virus (“Sheep” strain of GNKI) after 4 th passage. At 7-14 days after infection during the period of pronounced clinical symptoms - paralysis of lambs were killed by decapitation, the brain was sterilely removed. Brain tissue was ground in a porcelain mortar and a 20% brain suspension was prepared in 0.1 M phosphate-buffered saline (PBS). Next, the brain tissue was homogenized in a glass homogenizer for at least 3-5 minutes to obtain a homogeneous mass in a 0.1 M phosphate buffer solution, pH 7.2-7.4. Then the homogenate was subjected to disintegration on the device FastPrep 24 ™ Classic Instrument (MP Biomedicals) for the complete destruction of the cells of the brain tissue. For maximum virus extraction, the following treatment mode was used: the vibration rate was 6 ml / s, the treatment time was 30 s, and the number of treatments was 3 with an interval between treatments of 5 minutes. The disintegrated 20% brain suspension was kept in Blue Lysing Matrix tubes for at least 5 minutes until the silicon carbide balls were completely precipitated and after that the disintegrated material was sampled. The supernatant was carefully collected in a separate container, and 0.1 M of the FBI, pH 7.2-7.4 was again added to the Blue Lysing Matrix B tubes, shaken on Vortex V1 for 30 s, then kept for at least 5 minutes until the balls were completely deposited silicon carbide. The supernatant was carefully collected. This procedure was repeated 3 times. All the disintegrated supernatants obtained were pooled. A 20% brain suspension disrupted on a FastPrep 24 ™ instrument was precipitated by centrifugation at 5000 rpm for 50 minutes at a temperature of + 8–10 ° C in an Eppendorf 5804 R centrifuge to remove large foreign particles. The supernatant was collected in a separate container, and the precipitate was removed. The collected supernatant was concentrated by ultracentrifugation at 37000 rpm for 4 hours at + 4 ° C on a Beckman L-7 ultracentrifuge, SW-41 rotor, to precipitate virus particles. After concentration, the supernatant was removed, and 0.1 M phosphate buffer solution, pH 7.2-7.4, in a volume equal to 1/10 of the volume of the eluate was added to the sediment, then shaken 3 times on Vortex V1 with an interval of 3 minutes between agitations. . Next, the resuspended precipitate was layered on a stepwise density gradient of sucrose with a concentration of 10-60%. 1 ml of the concentrated virus suspension was layered on a 12 ml stepwise sucrose gradient (10–60% sucrose solution prepared in physiological solution, buffered with 0.01 M Na-phosphate buffer, pH 7.5) (FIG. 1), where 1 - source viral material (sediment), obtained after UCF at 37000 rpm; 2 - viral material selected to 10% sucrose; 3 - viral material selected from 10% to 20% sucrose; 4 - viral material selected from 20% to 30% sucrose; 5 - viral material selected from 30% to 40% sucrose; 6 - viral material selected from 40% to 50% sucrose; 7 - viral material selected from 50% to 60% sucrose; 8 - viral material selected from 60% sucrose to the bottom of the tube; 9 - visually missing sediment at the bottom of the tube; 10 — Broad Range molecular mass marker, “Bio-Rad”, was then centrifuged for 3 hours at 30,000 rpm at + 4 ° C in a Beckman L-7 ultracentrifuge, SW-41 rotor. After the end of centrifugation, the bottom of the tube was punctured and fractions were collected (5 fractions in total were obtained) of 2 ml each according to opalescence zones. The zone containing the virus was located on the border of a third of the gradient (Fig. 2), where 1, 2, 3, 4, 5 are the zones of visual opalescence of the distribution of floating density of sediment of the viral material.

Selected fractions were combined according to the results of immunoblotting. The most active fractions were located in the zone of 10–20% sucrose, this fraction was used for gel filtration on an Enrich ™ SEC 70 column (FIG. 3), where A is analytical electrophoresis, B is immunoblotting in FIG. 3A is shown:

1 - viral material, selected up to 10% sucrose; 2 - viral material selected from 10% to 20% sucrose; 3 - viral material selected from 20% to 30% sucrose; 4 - viral material selected from 30% to 35% sucrose; 5 - viral material selected from 35% to 40% sucrose; 6 - viral material selected from 40% to 45% sucrose; 7 - viral material selected from 45% to 50% sucrose; 8 - viral material selected from 50% to 60% sucrose; 9 - viral material selected from 60% sucrose to the bottom of the tube; 10 - Broad Range molecular mass marker, manufacturer Bio-Rad.

FIG. 3B shows immunoblotting of electrophoresis fractions of viral material transferred after electrophoresis to a nitrocellulose membrane divided by buoyant density with hyperimmune rabbit serum obtained against the sheep sheep virus production strain of 1: 100.

The protein composition of the separated samples was studied by analytical electrophoresis in 12% PAAG in the presence of 0.1% sodium dodecyl sulfate according to Laemmli UK [11] and transferred to a nitrocellulose membrane (Suppoted nitrocellulose membrane 0.45 μm, Bio-Rad) no Towbin H [12], the serological activity was determined by immunoblotting using hyperimmune serum obtained by hyperimmunization of rabbits with the production of the sheep OVE virus strain of INFR (infectious titer 5.25 lg LD ₅₀ / 0.03 ml) according to the method [10].

At each stage of purification of the virus, control was performed using analytical electrophoresis and immunoblotting to detect the localization of polypeptides and their serological activity. The protein concentration was determined on a UV5 spectrophotometer (Mettler Toledo) at a wavelength of 280 nm according to the instrument's instructions. The electrophoretic profile of the rabies virus was studied by the method of Laemmli U. K. [11]. The method of immunoblotting according to Towbin N. [12] determined the serological activity of the obtained material with hyperimmune serum of sheep against the production strain of the sheep virus “Sheep” GNKI and with the serum of sheep vaccinated with rabies vaccine — Shchelkovo-51 strain (FKP Schelkovo Biokombinat).

As shown by the results of immunoblotting, serological activity was observed in almost all zones of visual opalescence, except for the zone of 60% sucrose. The greatest activity was localized in the 10–30% sucrose gradient boundary (Fig. 2, zones 1 and 2).

The combined viral material from the sucrose zone of 10-30% was separated on an NGC Discovery chromatograph (Bio-Rad) using gel filtration using an Enrich ™ SEC 70 column. Separation was monitored with a single-wavelength UV detector at a wavelength of 280 nm. Fractions were collected in 1 ml using the BioFrac fraction collector (Bio-Rad) (Fig. 4), where the abscissa shows the amount of elution buffer passing through the column, and the ordinate shows the optical density expressed in units of optical density (units .) at a wavelength of 280 nm.

The activity of the fractional material was analyzed by immunoblotting using rabbit hyperimmune sera. Rabbits were immunized 3-fold, first with Freund's complete adjuvant in the ratio of antigen and adjuvant 1: 1 with intracutaneous injection of 0.1 ml in 5 points along the back on each side, the injection site was pre-cut, after 8-10 weeks re-immunized in a mixture with Freund's incomplete adjuvant in a dose of 0.5 ml subcutaneously in the lower third of the neck on each side, then after 6 weeks the last procedure was performed. Selected rabbit sera were used in the immunoblotting reaction (Fig. 5), where 1 is the original viral material prior to chromatography (area of 10-20% sucrose); 2 - fraction number 1; 3 - fraction number 2; 4 - fraction number 3; 5 - fraction number 5; 6 - fraction No. 6; 7 - fraction No. 7; 8 - fraction No. 8; 9 - empty hole; 10 - Broad Range, “Bio-Rad” molecular mass marker.

The protein concentration was determined on a UV5 spectrophotometer (Mettler Toledo) at a wavelength of 280 nm. The protein composition of the fractional material was studied by analytical electrophoresis in 12% PAAG, and the serological activity was determined by immunoblotting and ELISA.

The greatest interest in the results of immunoblot was represented by fraction No. 5.

The results of immunoblotting were fixed on the GelDoc XR + device (Bio-Rad), and were processed using Image Lab Software 5.1. The results of processing are presented in the form of densitograms (Fig. 6), where the path length is shown along the abscissa axis and the optical density expressed in units of optical density (unit O.) at a wavelength of 550-600 nm is shown on the ordinate axis.

A - source viral material before chromatography (area of 10-20% sucrose zone);

B - fraction No. 5;

C - fraction No. 5, precipitated by alcohol.

As can be seen from FIG. 6, the protein fraction with a molecular weight of 65–67 kDa, selected from the sucrose zone of 10–30%, is the most purified (Fig. 6, position C). High specific activity of this fraction was confirmed in ELISA and reached 1: 10240, which made it possible to increase the yield of antigen by 2 times, to increase its specificity and sensitivity by 8 times compared with the prototype [3].

The maximally purified fraction from the 10–30% zone of sucrose was additionally replanted with alcohol at a ratio of 1: 2, and thus a purified active fraction of the rabies virus was obtained, which was used as a rabies antigen for the serological diagnosis of rabies. A precipitate with a protein concentration of 20 mg / ml was dissolved in 0.1 M PBS, pH 7.2-7.4 in a volume equal to 1/10 of the original. This material was used in further work as a rabies virus antigen for ELISA. Part of the material was frozen at -70 ° C for storage and creation of a biobank of antigens. The precipitated antigen is a clear, slightly opalescent liquid. The serological activity of the purified antigen after gel filtration and re-precipitation with alcohol was determined by ELISA: the purified antigen was introduced into the strip wells of the VNII Medopolimer tablet in a volume of 120 μl and kept the plates for 24 hours at room temperature, then washed 3 times with distilled water and dried at room temperature. The ELISA reaction was carried out with sheep hyperimmune serum obtained for the sheep sheep rabbit strain GNCI, and Anti-sheep IgG labeled with horseradish peroxidase, manufactured by Sigma, was used as a conjugate. The results are shown in the table. As can be seen from the table, the activity was distributed depending on the zones of sucrose concentration, reaching a maximum value in the zone of 10-20% sucrose (protein concentration of 29.9 mg / ml, corresponding to a mass of 65-67 kDa). The use of the obtained antigen for immunization of sheep-producers of hyperimmune serum allowed us to obtain a serum with a higher activity in the ELISA, which reached a titer of 1: 12800.

Thus, the resulting antigen of rabies virus allows you to increase the yield of active antigen by 2 times to increase the specificity and sensitivity of 8 times compared with the prototype.

Bibliography

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Claims (1)

The method of obtaining rabies virus antigen for serological diagnostics, including obtaining suspensions from the brain of white mice experimentally infected with the rabies virus - sheep strain GNCI, disintegration on a Fast Prep-24 ™ device at a vibration rate of 6 ml / s in the presence of uniform particles of silicon carbide, centrifuging the resulting supernatant to remove foreign particles, ultracentrifuging to precipitate the virus particles, and separating the rabies virus in a stepwise sucrose gradient, characterized in that By disintegration, the brain tissue is additionally homogenized in a glass homogenizer for at least 3-5 minutes, in 0.1 M phosphate buffer solution, pH 7.2-7.4, and disintegration is carried out three times within 30 seconds with an interval between treatments of 5 minutes, then the obtained brain suspension is incubated in Blue Lysing Matrix tubes. At least 5 minutes until complete deposition of silicon carbide balls, the supernatant is taken in a separate container, and 0.1 M phosphate buffer solution, pH 7.2-7.4, is added to the tubes, shaken 3 times on a Vortex V1 for 30 s and incubated for at least 5 minutes; The supernatant is collected and all the supernatants obtained are combined, and centrifugation is carried out for 50 minutes at 5000 rpm, the supernatant is collected in a sterile container and concentrated by ultracentrifugation at 37000 rpm for 4 hours at + 4 ° C, the supernatant is removed, and 0.1 M phosphate buffer solution pH 7.2-7.4 is added to the sediment in a volume equal to 1/10 of the volume of the starting eluate, shaken three times for 30 s with intervals between shaking for 3 minutes, then the resuspended precipitate is layered on step gradient of 10-60% sucrose and ultracentrifuged for 3 hours at 30,000 rpm at + 4 ° C, then the viral material is fractionated using gel filtration and according to the results of immunoblotting, active fractions are selected, they are sedimented with alcohol and a rabies antigen is obtained.

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