RU2748475C1 - Method for spectrometric determination of concentration of rabies virus ribonucleoprotein by estimating the number of viral rna molecules in raw materials for rabies vaccines - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: method is described for spectrometric determination of concentration of the rabies virus ribonucleoprotein by estimating the number of viral RNA molecules isolated from complete particles of rabies virus after their strain-specific binding (NRNA RV), based on the linear function of the dependence of NRNA RVon the content of rabies virus ribonucleoprotein in the viral suspension. The number of RNA molecules isolated from complete particles of rabies virus is determined by the spectrometric method when assessing the extinction at a wavelength of 260 nm. Based on the established optical density (OD260), the number of molecules of the isolated rabies virus RNA is calculated and the concentration of rabies virus ribonucleoprotein (CRNP RV) is determined using the developed linear function CRNP RV=9×10-10×NRNA RV+0.022.EFFECT: invention expands the arsenal of spectrometric methods for determining the concentration of rabies virus ribonucleoprotein in raw materials for anti-rabies vaccines.4 cl, 4 dwg, 3 tbl, 7 ex

Description

The invention relates to the field of biotechnology and the production of rabies vaccines, namely to a method for determining the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccines using a spectrometric method for measuring the number of RNA molecules isolated from full particles of the rabies virus, and a developed linear model of the dependence of the number of viral RNA molecules on rabies virus ribonucleoprotein concentration.

Rabies occupies the first place among viral diseases of humans and animals, is one of the most dangerous zoonoses, causing damage to the central nervous system, encephalomyelitis, paralysis with inevitable death [1]. The rabies causative agent belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus, species Rabies lyssavirus [2]. Rabies virus virions are bullet-shaped, ≈180 nm long, ≈75 nm in diameter. On the outer surface of complete viral particles, there are projections in the form of spikes 10 nm long, which are attached to a bilayer lipid membrane [1]. The rabies virus genome is represented by an unsegmented single-stranded negative helical RNA about 12000 nt long, which encodes 5 basic proteins: nucleoprotein (N-protein), phosphoprotein (P-protein), matrix protein (M-protein), glycoprotein (G-protein ), RNA-dependent RNA polymerase (L-protein). Outside, the RNA is coated with a nucleoprotein, resulting in the formation of a nucleocapsid. Ribonucleoprotein (RNP) consists of genomic RNA, N-protein, P-protein and L-protein. The nucleocapsid has a spiral type of symmetry. A pseudogene (ψ-fragment) is located between G- and L-cistrons [2].

The disease leads to significant economic costs associated with the death of animals, the elimination of the consequences of outbreaks of the disease, the implementation of preventive and quarantine measures, the regulation of the number of wild carnivores, the capture of stray cats and dogs and the implementation of laboratory tests for diagnosis [3]. The system of measures for the control and prevention of the disease provides for the immunization of susceptible animals, as well as control of the level of tension of post-vaccination immunity [4].

In the process of industrial production of rabies vaccines, special attention is paid to the concentration of ribonucleoprotein, which significantly affects the immunogenic activity of vaccine preparations [1]. In this regard, each batch of raw materials is examined to determine the concentration of ribonucleoprotein using the colorimetric method of Merion Bradford and / or the direct sandwich variant of the enzyme-linked immunosorbent assay (ELISA). Bradford's method is based on the reaction of Coomassie Brilliant Blue G-250 with arginine and hydrophobic amino acid residues. The bound form is blue in color with an absorption maximum at 595 nm. The absorbance of the solution at this wavelength is proportional to the amount of protein in the solution. However, this method has a number of limitations: low analytical sensitivity of the assay (not less than 2 μg / ml), low specificity of the method [5]. The direct sandwich ELISA method has proven itself well, but it also has a number of limitations. Namely:

1) the possibilities of increasing the sensitivity of the method are limited by the background of the analyzed compound (i.e., its presence not only in the analyzed sample, but also in the used reagents and solvents), the substrate specificity of the enzyme and the affinity of antibodies;

2) the limitations of ELISA include the presence of cofactors, inhibitors and stimulators of enzyme activity in the tested samples;

3) ELISA does not allow separating native proteins and their biological inactive fragments that have retained antigenic determinants;

4) a possible change in the catalytic activity of the enzyme when it is conjugated to the antigen;

5) the applicability of ELISA only to well-studied systems, where purified antigens and highly specific antibodies are used [1].

In this regard, it is advisable to search for a method for determining the concentration of rabies virus ribonucleoprotein in vaccinated raw materials for a vaccine based on an alternative method.

The objective of the present invention was to develop a highly sensitive, specific, rapid method for determining the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine in order to eliminate the above disadvantages.

This problem was solved thanks to the creation of a new method for determining the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine using the spectrometric method of RNA analysis. Using this method, a model was proposed for determining the number of RNA molecules isolated from full particles of the rabies virus, of the type:

N _{RNA bb} = 4.373410 ⁹ × K × (OD ₂₆₀ sample _-OD 260K- _-OD 320 sample),

where: K is the dilution factor of the eluate,

OD ₂₆₀ is the extinction value of the eluate of the rabies virus RNA at a wavelength of 260 nm,

About _260K ^_ extinction value of the negative control at a wavelength of 260 nm,

_Sample OD 320 is the extinction value of the rabies virus RNA eluate at a wavelength of 320 nm.

Using the developed method between the values of the concentration of the ribonucleoprotein of the rabies virus (C _{RNP Wb} ) and the number of RNA molecules extracted from the total viral particles (N _PHKIB ), a relationship was revealed, reflected in the form of a linear function C _{RNP WB} = 9 × 10 ^-10 × N _RNPB +0.022 with high accuracy of approximation R ² = 0.9998. The proposed model makes it possible to estimate the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine.

The essence of the invention is reflected in the graphics.

FIG. 1 - Dependence of rabies virus ribonucleoprotein concentration on the number of RNA molecules extracted from full particles after immune uptake (the number of molecules is expressed in exponential format);

FIG. 2 - Spectrograms of dilutions of the standard 30x eluate of RNA virus of rabies (from top to bottom graphs are shown for some dilutions of eluates corresponding to the concentrations of total viral particles: 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, 0.1 μg / ml);

FIG. 3 - The absorption spectrum of the eluates of the RNA virus of the rabies virus strain PB-97 to assess the purity and determine the concentration of ribonucleoprotein in the original suspensions;

FIG. 4 - Absorption spectrum of RNA eluates of the rabies virus of the ARRIAH strain to assess the purity and determine the concentration of ribonucleoprotein in the initial suspensions.

The essence of the invention lies in a new approach to determine the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine using the spectrometric method and the developed power-law model.

The inventive method is based on carrying out the reaction of strain-specific binding of the rabies virus, isolating RNA from the formed immune complexes, spectrometric analysis of the number of viral RNA molecules and calculating the concentration of total viral particles using the proposed model. Based on the analysis results, the purity of the obtained rabies virus RNA extract is assessed using spectral analysis, the number of viral RNA molecules is determined using a calculation formula based on the extinction values. Based on a sample of known values of the concentration of rabies virus ribonucleoprotein and the amounts of viral RNA molecules established by the spectrometric method, a linear model has been developed that makes it possible to determine the content of ribonucleoprotein in raw materials for rabies vaccine based on the results of spectrometric analysis.

Based on the analysis of scientific data, the proposed method for assessing the concentration of rabies virus ribonucleoprotein in raw materials for a vaccine has not yet been used. To determine the concentration of the ribonucleoprotein of the rabies virus, the method of M. Bradford and the direct sandwich variant of ELISA are used [1]. In comparison with M. Bradford's method, the developed method is characterized by higher analytical sensitivity and specificity. In comparison with the direct sandwich ELISA version, the proposed method is more economical, does not involve the use of enzymes and changes in their catalytic activity, and allows the study of samples in 2-3 hours. Proceeding from this, the proposed method can be used to determine the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine. The key element of the proposed method is to determine the dependence of the number of RNA molecules of the rabies virus on the extinction value at a wavelength of 260 nm, as well as to establish a linear relationship between the content of rabies virus ribonucleoprotein in the raw material for the vaccine and the number of RNA molecules extracted from the complete viral particles.

Comparative analysis with the prototype allows us to conclude that the novelty and inventive level of the claimed invention lies in the use of a spectrometric method for assessing the number of RNA molecules isolated from full particles of the rabies virus, and the developed linear model for determining the concentration of rabies ribonucleoprotein in non-inactivated raw materials for the vaccine.

The authors did not find information on the development of the proposed method for determining the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccine.

The essence of the invention is illustrated in the graphic material - a graph of the dependence of the concentration of ribonucleoprotein of the rabies virus on the number of RNA molecules extracted from full particles after strain-specific binding (Fig. 1).

Immunological 24-well plates are sensitized, the surface of which is free of DNA and RNA, DNase and RNase, with purified strain-specific polyclonal antibodies against rabies virus in a volume of 3.0 ml of suspension with a concentration of immunoglobulins G 10 μg / ml at a temperature of 4 ± 2 ° C for 18 hours. The bottom of the wells of the plates is washed three times with 1/15 M phosphate buffered saline (PBS), the open binding sites are blocked with a 0.5% suspension of bovine serum albumin (BSA) at 37 ± 1 ° C for 1 hour and the wells are washed with 1 / 15 M FSBR 5 times. These operations for the preparation of the tablet are carried out in advance, before the start of the study.

For the analysis of rabies virus samples with an unknown concentration of ribonucleoprotein, a positive control is used (a suspension of the rabies virus strain PB-97 reproduced in a suspension culture of kidney cells of a newborn Syrian hamster (BHK-21)); negative control (a suspension of BHK-21 cells not infected with the rabies virus with a concentration of 2.8⋅10 ⁶ -3.0⋅10 ⁶ cells / ml). Into wells with sensitized polyclonal strain-specific antibodies against rabies virus add 1.0 ml of control samples and samples, incubate at 37 ± 1 ° C for 1 hour. As a result of the interaction of antibodies and antigens of the rabies virus, immune complexes are formed on the surface of the wells. Immune complexes are washed from ballast components using 1/15 M PBS and they are resuspended in 0.5 ml of 1/15 M PBS.

RNA bound by the immune complex is isolated from the ribonucleoprotein of the rabies virus by lysis of polypeptide and nucleoprotein structures with subsequent fractionation of RNA from DNA, denatured proteins and lipoproteins. Lysis is carried out using 10 ml of a solution containing 50% carbolic acid (pH 6.5) and 50% 3M guanidine isothiocyanate (GTZ) solution, which is mixed with 0.5 ml of the immune complex suspension. The mixture is incubated at a temperature of 22-25 ° C for 15 minutes for complete dissociation of the polypeptide and nucleoprotein complexes. The resulting lysate is purified from denatured proteins and conglomerates by centrifugation at 9000 g for 5 minutes. The supernatant is transferred into a centrifuge tube, 2.5 ml of freon-20 is added and the contents are incubated for 5 minutes with periodic stirring. After incubation, the mixture is centrifuged at 9000g for 10 minutes at a temperature of 4 ± 2 ° C. As a result of sedimentation, the contents of the test tube are divided into three phases: 1) the lower phase of a straw color, containing a complex of carbolic acid, GTZ and freon-20 with bound proteins and lipoproteins; 2) the middle phase of white color, which includes peptide components and cellular DNA; 3) the upper phase, which is a transparent extract of viral RNA [6, 7]. The upper phase is completely taken, without affecting the rest of the fractions, and ballast components are removed using 5 ml of 100% propanol-2. The mixture is incubated for 10 minutes at a temperature of 22-25 ° C, after which the contents of the tube are centrifuged at 9000g for 6 minutes at a temperature of 23-25 ° C. The supernatant is removed, leaving a precipitate of rabies virus RNA, to which 3.0 ml of 80% ethanol is added. The contents are mixed and centrifuged at 10,000 g for 6 minutes at a temperature of 23-25 ° C. The supernatant is removed, the RNA pellet is dried in a stream of air at room temperature for 3 minutes. To the dried sediment, add 100 μl of TE buffer (10 mM tris (oxymethyl) aminomethane, 1 mM EDTA, pH 7.3), free of RNase and Mg, warm the contents of the tube at 58-60 ° C for 2-3 minutes to complete dissolution of the RNA of the rabies virus. The TE buffer with the indicated acidity value protects the rabies virus RNA from degradation [8]. As a result, 100 μl of 30-fold (30x) extracts of rabies virus RNA are obtained relative to the initial volume of the viral suspension (3000 μl) introduced for incubation into the plate at the stage of strain-specific binding.

To determine the amount of viral RNA molecules and to assess the purity of the eluates, spectrometric analysis is performed, assessing the absorption of monochromatic ultraviolet light by the sample under study. Measurements of the spectral absorption capacity of the samples are carried out in quartz cuvettes (the optical path (1) is 10 mm, the minimum analyte volume is 50 μl) at wavelengths in the range 205-325 nm and a temperature of 23-25 ° C. The content in the preparation of phospholipid residues, polysaccharides and GTZ, carbolic acid, polypeptides and large conglomerates is estimated by determining the optical density (OD) at wavelengths 205, 235, 270, 280 and 320 nm, respectively [9]. An RNA extract is considered free of polypeptide impurities if the extinction coefficient R ₁ (OD ₂₆₀ / OD ₂₈₀ ) is in the range of 1.8-2.2 and optimally is about 2.0. Lower R ₁ values indicate the presence of DNA, protein constituents and residues of carbolic acid in the eluate. Higher values of the coefficient R ₁ indicate degradation of RNA and the presence of free ribonucleotides. The RNA extract of the rabies virus is considered uncontaminated with polysaccharides if the extinction coefficient R ₂ (OD ₂₆₀ / OD ₂₃₅ ) corresponds to a value of 2.0 [10]. When replacing 1% of RNA with polysaccharide components, R ₂ decreases by 0.002. R ₂ values greater than 2,000 may indicate degradation of RNA. The absence of large particles in the obtained extract is confirmed if OD _{320 is} close to zero [10, 11]. If OD _205-259 and / or OD _261-325 exceed OD ₂₆₀ , and pronounced double (and more) peaks are observed on the spectrogram, then the eluate contains ballast components and the reliability of determining the number of RNA molecules and, as a consequence, the concentration of the ribonucleoprotein of the rabies virus decreases significantly. With a low degree of purification of the rabies virus RNA extract, it is necessary to repeat the extraction step and spectral analysis of the newly obtained eluate.

From the RNA extract isolated from the ribonucleoprotein of the rabies virus, a series of standard dilutions is obtained, corresponding to the concentrations of ribonucleoprotein from 0.1 to 3.5 μg / ml in increments of 0.2 μg / ml. The extinction of the obtained standard samples is measured at a wavelength of 260 nm (OD ₂₆₀ ) and a relationship is established between the number of RNA molecules of the rabies virus (N _{RNA wb} ) and OD ₂₆₀ to obtain a mathematical model for calculating N _{RNA wb} . The determination of the dependence of the concentration of ribonucleoprotein in the raw material for rabies vaccine on the number of RNA molecules extracted from the rabies virus is carried out in the process of constructing a positive linear model. Determine the degree of reliability of the approximation (R ² ) and calculate the value of the concentration of the ribonucleoprotein of the rabies virus in the raw material for the rabies vaccine.

Example 1. Obtaining the eluate of the RNA virus of rabies strain PB-97 and assessment of the degree of purity of the resulting extract.

At the first stage of the study, strain-specific binding of the rabies virus strain PB-97 was carried out using an immunological plate sensitized with purified strain-specific polyclonal antibodies. A non-inactivated suspension of the rabies virus strain PB-97 reproduced in the suspension cell line BHK-21 with a ribonucleoprotein concentration of 3.5 μg / ml is used as a standard. A negative control sample is a suspension of the BHK-21 cell line with a cell concentration of 2.8 × 10 ⁶ -3.0 × 10 ⁶ cells / ml, not infected with the rabies virus.

In the wells of an immunological plate with sensitized antibodies against rabies virus strain PB-97, the standard and negative control are introduced in 2.0 ml in 3 wells and incubated at 37 ± 1 ° C for 1 hour. The study is carried out in 3 repetitions. As a result, immune complexes are formed. The suspension is removed from the wells of the plate, the surface of the wells is washed three times using 1/15 M PBS. The resulting immune complexes are resuspended in 0.5 ml of 1/15 M PBS.

Mix 0.5 ml of the resulting suspensions and 10.0 ml of a solution containing 50% phenol (pH 6.5) and 50% 3M GTZ solution. The mixture is incubated at 22-25 ° C for 15 minutes. The resulting lysates are subjected to centrifugation at 9000 g for 5 minutes. After sedimentation, the supernatant is transferred into centrifuge tubes, 2.5 ml of Freon-20 are added, and the contents are incubated for 5 minutes with periodic stirring. The suspension is centrifuged at 9000g for 10 minutes at a temperature of 4-5 ° C. As a result, the contents of the tube are divided into three phases: lower, middle and upper. The upper fraction is carefully taken into new test tubes without residue and 3.0 ml of 100% propanol-2 are added. The resulting mixtures are incubated for 10 minutes at a temperature of 23-25 ° C. The contents of the tubes are precipitated at 10000g for 6 minutes at a temperature of 23-25 ° C. The supernatant is removed, leaving the RNA virus strain PB-97. 3.0 ml of 80% ethanol is added to the RNA sediment, the contents of each tube are mixed and centrifuged at 10000 g for 6 minutes at a temperature of 23-25 ° C. The supernatants are removed, the RNA pellet is dried in a stream of air at room temperature for 3 minutes. RNA is dissolved in 100 μl TE buffer (10 mM tris (oxymethyl) aminomethane, 1 mM EDTA, pH 7.0-7.2), the contents are heated at 60 ± 2 ° C for 2-3 minutes, thereby obtaining a 20x eluate Rabies virus RNA strain PB-97 (n = 3).

Evaluation of the purity of the eluate of the RNA virus of the rabies virus strain PB-97 standard and its dilutions using spectral analysis. The RNA sample is scanned into a quartz cuvette (1 = 10 mm) at a temperature of 22-25 ° C and the extinction values are recorded in the range from 205 to 325 nm every 2 nm, carrying out a complete recording of the RNA absorption spectrum using the Specrtrum v. 5.0 (Fig. 2).

As a result of the study of a 30-fold extract of the RNA virus of the rabies virus strain PB-97, it was determined that the values of OD _205-259 and OD261-325 did not exceed OD ₂₆₀ (0.001-0.904 <0.905 and 0.903-0.002 <0.905), which is a sign of a high degree of the purity of the resulting RNA eluate (n = 3). From the data of the spectral analysis of the standard, one can see the absence of pronounced peaks in the diagram at wavelengths of 205, 235, 270, 280 and 320 nm, which indicates the almost complete absence of contamination of the drug with impurities of phospholipids, polysaccharides and GTC, carbolic acid, polypeptides and large conglomerates, respectively. The extinction coefficient R ₁ is 2.002 (OD ₂₆₀ / OD ₂₈₀ = 0.905 / 0.0.452) (close to the norm of 2.000), which confirms the absence of cellular DNA and the presence of only trace amounts of protein impurities and residues of carbolic acid. Nucleic acid degradation and the presence of free nucleotides in solution are not observed, since the R ₁ coefficient does not exceed 2,000. The RNA extract of the rabies virus of the presented standard is not contaminated with polysaccharides and residues of GTZ, since the extinction coefficient R ₂ (OD ₂₆₀ / OD ₂₃₅ = 0.905 / 0.453) corresponds to a value of 1.998. Considering that when 1% of RNA is replaced by polysaccharide components, the value of R ₂ decreases by 0.002, the presence of carbohydrate impurities in the resulting eluate is no more than 1%. The resulting standard dilutions also met the requirements (Fig. 2).

Example 2. Determination of the relationship between the number of RNA molecules of the rabies virus (N _{RNA wb} ) and OD ₂₆₀ to obtain a mathematical model for calculating N _{RNA WB} .

At the next stage of the study, the dependence of the ribonucleoprotein concentration of the cultural rabies virus on the number of viral RNA molecules is revealed. The maximum level of absorption of single-stranded ribonucleic acids is observed at wavelengths of 252-271 nm, which is explained by the high spectral absorption capacity of ribonucleoside-5'-triphosphates in this range, namely, for adenosine-5'-triphosphate (λ _ATP ) - 259 nm, uridine -5'-triphosphate (λ _UTP ) - 262 nm, guanosine-5'-triphosphate (λ _GTP ) - 252 nm, cytidine-5'-triphosphate (λ _CTP ) - 271 nm [12, 13]. Spectral studies prove that the absorption of monomers of whole ribonucleic acids merge, providing the maximum extinction in the specified wavelength range [12, 14, 15]. After performing a nucleotide analysis of the RNA of the strains Shchelkovo-51, RV-97, ARRIAH, SAD B19, SAD Berne, CVS-11 of the rabies virus, we determined the average percentages of ribonucleoside-5'-triphosphates (W _ATP , W _utp , W _GTP , W _ctp ) as part of a nucleic acid. The research results are presented in table 1.

The data obtained made it possible to calculate the average values of the wavelengths (λ _max ) with the maximum level of RNA absorption for each of the presented strains of the rabies causative agent, using the formula:

λ _max = λ _ATP × W _ATP + λ _UTP × W _UTP + λ _GTP × W _GTP + λ _CTP × W _CTP .

Thus, it was determined that for the RNA of the rabies virus strain Shchelkovo-51 λ _max is 261.64 nm (259 × 0.250 + 264 × 0.209 + 252 × 0.321 + 271 × 0.220), for the PB-97 strain - 261.51 nm (259 × 0.251 + 264 × 0.210 + 252 × 0.319 + 271 × 0.220), for the ARRIAH strain - 261.68 nm (259 × 0.251 + 264 × 0.212 + 252 × 0.319 + 271⋅0.218), for the SAD B19 strain - 261.63 nm (259 × 0.252 + 264 × 0.209 + 252 × 0.310 + 271 × 0.229), for the SAD Berne strain - 261.74 nm (259 × 0.250 + 264 × 0.214 + 252 × 0.314 + 271 × 0.222), for the CVS- strain 11 - 261.70 nm (259 × 0.253 + 264 × 0.215 + 252 × 0.321 + 271 × 0.211).

In the production of antirabies vaccines at the FGBI "ARRIAH", strains RV-97 and ARRIAH are used, for which the _λmax values were determined spectrometrically. The data obtained was 260 nm, which confirms the truth of the calculated values.

The content of RNA molecules in a pure preparation is calculated using the Bouguer-Lambert-Beer law, according to which a weakening of a parallel monochromatic light beam is observed when passing through an absorbing medium. Based on this law, it is possible to find the ratio of the concentration of molecules that absorb radiation and the amount of absorbed light [16]. At a wavelength of 260 nm, the average extinction coefficient for single-stranded RNA is 0.024 (μg / ml) ^-1 cm ^-1 . Based on this, a solution with a nucleic acid content of 1.00 mg / ml, when transmitted by ultraviolet light with a wavelength of 260 nm, has an OD ₂₆₀ corresponding to 24,000 [15], and, as a consequence, an optical density of 1.000 corresponds to an eluate containing 41.67 μg RNA / ml.

The application of the Bouguer-Lambert-Beer law is valid for determining the number of molecules of viral RNA of the rabies virus in dilute eluates, since at high concentrations of the analyte (more than 1.00 mg / ml) the distance between the molecules of the UV-absorbing substance is significantly reduced. For this reason, the influence of each molecule on the distribution of surface charge between neighboring molecules increases, which can change the ability of RNA to absorb radiation at a given wavelength. In other words, to determine the amount of rabies virus RNA molecules in high concentration eluates, it is required to obtain its dilutions using TE buffer. Before measuring the absorbance of the extract dilution, it is necessary to subtract the background values of the specified buffer. To calculate the RNA content in the eluate, it is necessary to take into account the conversion factor for the nucleic acid of the rabies causative agent (F _RHKwb = 41.67), the dilution factor (K), and also subtract the background value of the test sample (OD ₃₂₀ ) and negative control (OD _{260 K-} ) from OD ₂₆₀ sample. When determining the number of RNA molecules of the rabies virus (N _RNA ), Avogadro's constant (6.02214076 × 10 ²³ mol ^-1 ), the average molecular weight of the ribonucleoside (Mw _{ribonucleoside} = 340.5 Da), the length of the RNA of the rabies virus (L = 12000 n. o.) [1, 2], and also convert the mass of nucleic acid from [μg] to [g] in accordance with the requirements of the International System of Units Le Systeme International d'Unites [15, 16] (coefficient 1/10 ⁶ ) and recalculation of the number of molecules from 30x eluate into 1x material (factor 1/10 ^1.48 ) (total conversion factor is 1/10 ^7.48 ).

During the cultivation of the rabies virus in sensitive cell lines, ribonucleoprotein particles are formed, each of which contains one RNA molecule. It is believed that in rabies virus suspensions, RNA is not only contained in ribonucleoprotein (97-99%), but also in a small amount (1-3%) in a free state [1]. In other words, when calculating the amount of RNA isolated from the ribonucleoprotein of the rabies virus, a factor of 0.97 should be taken into account.

The number of RNA molecules extracted from the ribonucleoprotein of the rabies virus (N _{RNA wb} ) is calculated using the calculation formula: N _{RNA wb} = n × Na [16], where n is the amount of the RNA substance of the rabies virus. In this case, n = m / M, where m is the mass of the substance, M is the molar mass of the substance. The mass of the substance is estimated by the spectrometric method taking into account the dilution factor, the conversion factor for single-stranded RNA, the conversion factors of the mass units and the optical density of the studied eluate of the rabies virus RNA, taking into account the subtraction of the OD _{320 of the} sample and OD _{260 K-} : m = ^{1/10 7.48} × F _PHKvb × K × (OD _{260 samples} - О260 _к- - OD _{320 samples} ). The molecular weight of the average RNA of the rabies virus (using the example of strains PB-97 and ARRIAH) is found taking into account the length of the RNA of the rabies virus (L = 12000 n.a.) and the average molecular weight of ribonucleoside (Mw _{ribonucleoside} = 340.5 Da): M = L _{RNA WB} × Mw _{ribonucleoside} .

It follows that the amount of rabies virus RNA substance can be determined by the formula: n = (F _PHqb × K × (OD _{260 sample} - OD _260k - - OD _{320 sample} ) / (10 ^7.48 × L _{RNA WB} × Mw _{ribonucleoside} ). , the formula for calculating the number of structural particles (RNA molecules of the rabies virus) is the following ratio:

N _{RNA WB} = (0.97 × Na × F _RNAvb × K × (OD _{260 sample} - OD _260k- - OD _{320 sample} )) / (10 ^7.48 × L × Mw _{ribonucleoside} ). After performing a number of calculations and mathematical simplifications, we obtain the final formula for calculating the number of RNA molecules of the rabies virus in the eluate:

N _{RNA WB} = 4.3734 × 10 ⁹ × K × (ОВ _{260 samples} - OD _260K- - OD _{320 samples} ),

where: K is the dilution factor of the eluate,

OD ₂₆₀ is the extinction value of the eluate of the rabies virus RNA at a wavelength of 260 nm,

About _260K - - the extinction value of the negative control at a wavelength of 260 nm,

_Sample OD ₃₂₀ is the extinction value of the rabies virus RNA eluate at a wavelength of 320 nm.

Example 3. Determination of the dependence of the concentration of ribonucleoprotein in the raw material for rabies vaccine on the number of RNA molecules extracted from the rabies virus.

To determine the dependence of the concentration of the ribonucleoprotein of the rabies virus on the number of viral RNA molecules, a series of 30x dilutions of the RNA standard isolated from the suspension of the rabies virus strain PB-97 with a ribonucleoprotein content of 3.5 μg / ml corresponding to the concentration of RNP from 0.1 to 3.5 μg is obtained. / ml with a step of 0.2 μg / ml. Before determining the extinction of the obtained standard samples, the background values of the TE buffer are automatically subtracted. Spectral analysis of the prepared positive standards is carried out, determining the extinction values at wavelengths of 260 and 320 nm. The extinction value of the negative control is measured to detect the presence of nonspecific RNA molecules, the maximum absorption for which is also observed at a wavelength of 260 nm. The results of measurements and calculation of the number of RNA molecules isolated from full particles of the rabies virus are shown in Table 2, from which it can be seen that the values of N _{RNA wb} for all 30x dilutions of the standard with the concentration of rabies virus ribonucleoprotein from 0.1 to 3.5 μg / ml are in the range from 87468000 to 3940433400, respectively.

Based on the data obtained on the number of viral RNA molecules in standard dilutions and the concentration of ribonucleoprotein corresponding to them, a calibration graph of the dependence of C _{RNP wb} and N _{RNA wb was constructed} , which is presented as a linear function C _{RNP wb} = 9 × 10 ^-10 × N _RNPB + 0.022 s reliability of approximation R ² = 0.9991 (Fig. 1). No rabies virus was detected in the negative control.

The resulting model, based on the number of rabies virus RNA molecules isolated from full particles after their immune binding, makes it possible to determine the concentration of rabies virus ribonucleoprotein in viral culture suspensions.

Example 4. Determination of the concentration of the ribonucleoprotein of the rabies virus strain PB-97 in suspension for the attenuated rabies vaccine.

At this stage of the work, a suspension of the rabies virus strain PB-97 for an attenuated vaccine was tested. Manipulations on the strain-specific binding of complete rabies virus particles and the isolation of RNA from them, as well as the assessment of the purity of the resulting 30x RNA eluate using spectral analysis for the sample, positive and negative control was carried out as described in example 1. The results of the study are shown in Fig. 3.

The spectrogram data show that the average values of OD _205-259 and OD _261-325 do not exceed OD ₂₆₀ (0.001-0.640 <0.642 and 0.641-0.002 <0.642), therefore, the eluate of the isolated RNA of the rabies virus is characterized by a high degree of purity. The resulting preparation is not contaminated with impurities of phospholipids, polysaccharides and residues of GTZ, carbolic acid, polypeptides and large conglomerates, since there are no pronounced peaks on the graph at wavelengths 205, 235, 270, 280 and 320 nm, respectively. The extinction coefficient R ₁ is 2.000 (OD ₂₆₀ / OD ₂₈₀ = 0.642 / 0.321), which corresponds to the norm and means a high degree of purity of the RNA virus RNA eluate of strain PB-97 and an almost complete absence of protein and carbolic acid residues after the RNA isolation step. The extinction coefficient R ₂ is 1.994 (OD ₂₆₀ / OD ₂₃₅ = 0.642 / 0.322), which is close to 2.000 and provides a high degree of purity of the eluate. When replacing 1% of RNA with polysaccharide components, the value of R ₂ decreases by 0.002, therefore, in the resulting eluate, the presence of polysaccharide impurities is no more than 3%, which is permissible.

According to the results of the spectrometric study, the average values of OD ₂₆₀ are 0.642 ± 0.001, OD ₃₂₀ - 0.002 ± 0.000, OD _260k- - 0.004 ± 0.000. Based on the data obtained, the number of RNA molecules in the eluate is calculated using the formula:

N _{RNA WB} = 4.3734 × 10 ⁹ × K × (OD ₂₆₀ sample _-OD 260K- -OD _{320 sample} )

The dilution factor (K) is taken as 1, since the whole 30x eluate was not diluted. The arithmetic mean number of RNA molecules isolated from full particles of the rabies virus strain PB-97 is 2781482400 ± 2211. Using the developed linear model of the relationship with _{RNP VB} and N _RNVB , reflected by the function C _{RNP wb} = 9 × 10 ^-10 × N _PNVB + 0.022, we determined the concentration of ribonucleoprotein of the rabies virus strain PB-97, which was 2.525 ± 0.002 μg / ml, which correlates with the results of the Bradford method (2.50 ± 0.10 µg / ml) and ELISA (2.52 ± 0.04 µg / ml) (Table 3). Hence, it follows that the linear model developed using spectral analysis data makes it possible to estimate the concentration of the ribonucleoprotein of the rabies virus strain PB-97 in the raw material for the attenuated rabies vaccine.

Example 5. Determination of the concentration of ribonucleoprotein of the rabies virus strain ARRIAH in suspension for inactivated rabies vaccine.

Investigated a suspension of the rabies virus strain ARRIAH for inactivated rabies vaccine. Manipulations on the strain-specific binding of complete rabies virus particles and the isolation of RNA from them, as well as the assessment of the purity of the resulting 30x RNA eluate using spectral analysis for the sample, positive and negative control was carried out as described in example 1. The results of the study are shown in Fig. four.

The spectrogram data show that the average values of OD _205-259 and OD _261-325 do not exceed OD ₂₆₀ (0.001-0.544 <0.546 and 0.545-0.002 <0.546), therefore, the eluate of the isolated RNA of the rabies virus strain ARRIAH is characterized by a high degree of purity. The resulting preparation is not contaminated with impurities of phospholipids, polysaccharides and residues of GTZ, carbolic acid, polypeptides and large conglomerates, since there are no pronounced peaks on the graph at wavelengths 205, 235, 270, 280 and 320 nm, respectively. The extinction coefficient R ₁ is 2.000 (OD ₂₆₀ / OD ₂₈₀ = 0.546 / 0.273), which corresponds to the norm and means a high degree of purity of the RNA virus RNA eluate of the ARRIAH strain and a complete absence of protein and carbolic acid residues after the stage of RNA isolation. The extinction coefficient R ₂ is 2.007 (OD ₂₆₀ / OD ₂₃₅ = 0.546 / 0.272), which is close to 2.000 and provides a high degree of purity of the eluate. When replacing 1% of RNA with polysaccharide components, the value of R ₂ decreases by 0.002, therefore, the presence of polysaccharide impurities was not detected in the resulting eluate.

According to the results of the spectrometric study, the average values of OD ₂ 6o are 0.546 ± 0.002, OD ₃₂₀ - 0.002 ± 0.000, OD _260k- - 0.004 ± 0.000. Based on the data obtained, the number of RNA molecules in the eluate is calculated using the formula:

N _{RNA VB} = 4.3734 × 10 ⁹ × K × (ОВ ₂₆₀ samples _{-OD 260k -} -OD _{320 samples} ) The dilution factor (K) is taken as 1, since the whole 30x eluate was not diluted. The arithmetic mean number of RNA molecules isolated from full particles of the rabies virus strain ARRIAH is 2366009400 ± 2829. Using the developed linear model of the relationship C _{RNP wb} and N _RNKVB , reflected by the function C _{RNP WB} = 9⋅10 ^-10. N _RHKVB +0.022, we determined the value of the concentration of the ribonucleoprotein of the rabies virus strain ARRIAH, which was 2.100 ± 0.003 μg / ml, which correlates with the results of the Bradford method (2.14 ± 0.09 μg / ml) and ELISA (2.11 ± 0, 05 μg / ml) (Table 3). It follows that the linear model developed using spectral analysis data makes it possible to estimate the concentration of the ribonucleoprotein of the rabies virus strain ARRIAH in the raw material for inactivated rabies vaccine.

Example 6. Revealing the correlation of the results of determining the concentration of ribonucleoprotein of the rabies virus using the developed method and previously used methods.

The proposed method was tested to determine the concentration of rabies virus ribonucleoprotein of the vaccine strains PB-97 and ARRIAH in 355 virus-containing suspensions. The studies were carried out in parallel by the Bradford method and in the direct sandwich ELISA in three repetitions. The coincidence of the actual results obtained using the Bradford method and the expected results for determining the concentration of RNP of the rabies virus by the spectrometric method by the number of RNA molecules was 95.4-99.0%. When compared with the data of the ELISA method, the coincidence of the actual and expected results corresponded to the values of 96.0-99.7%. For the positive control, the coincidence of actual and expected results was 99.2-99.7%. In the negative control sample, no rabies virus RNA or full ribonucleoprotein particles were detected, which was also in line with expectations. Thus, according to the results of the study, the correlation between the method of spectrometric determination of the concentration of ribonucleoprotein of the rabies virus by assessing the amount of viral RNA molecules in the raw material for rabies vaccines with the methods according to Bradford and direct sandwich ELISA was 95.4-99.7%.

Example 7. Evaluation of the specificity of the method for spectrometric determination of the concentration of ribonucleoprotein of the rabies virus by assessing the number of viral RNA molecules in the raw material for rabies vaccines.

To assess the specificity of the proposed method for the indirect determination of the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccines spectrometrically to assess the amount of viral RNA, we studied suspensions of the rabies virus strain PB-97 (C _{RNP WB} = 2.70 μg / ml), the rabies virus strain ARRIAH ( With _{RNP WB} = 3.30 μg / ml), FMD virus strain O / Zabaikalsky / 2016, infectious necrosis of hematopoietic tissue of salmonids, spring viremia of cyprinids, infectious necrosis of the pancreas. The number of infectious doses of viruses in suspensions was 10 ^6.0 TCD ₅₀ / cm ³ . The studies were carried out in triplicate.

The stages of RNA isolation, assessment of their purity and calculations of the number of viral nucleic acid molecules, the concentration of rabies virus ribonucleoprotein were carried out as described in example 4. The isolated RNA extracts were characterized by high purity rates, since the extinction coefficients R ₁ and R ₂ were in the range of 1.995- 2.001, which met the requirements [6].

For the eluates of the RNA virus of rabies strains PB-97 and ARRIAH isolated after strain-specific binding of complete virus particles, at a wavelength of 260 nm, the optical densities of the samples were 0.700 and 0.845, which corresponded to the declared concentrations of 2.70 and 3.30 μg / ml. For samples containing other viruses, no peaks were observed on the spectrograms and no rabies virus was detected, which was true. In other words, the proposed method is specific for the rabies virus strains PB-97 and ARRIAH when using specific polyclonal antibodies at the stage of strain-specific binding.

The main advantage of the present invention is the ability to study rabies virus suspensions to determine the concentration of ribonucleoprotein in raw materials for rabies vaccines within 2-3 hours. In the proposed invention, between the concentration of ribonucleoprotein and the number of RNA molecules isolated from complete viral particles after strain-specific binding and determined by the spectrometric method according to the formula:

N _{RNA wb} = 4.3734 × 10 ⁹ × K × (OD ₂₆₀ sample _-OD 260k- -OD _{320 sample} ),

the dependence is established, which is presented in the form of a linear function with a high accuracy of approximation R ² = 0.9998. The developed model has the form C _{RNP WB} = 9⋅10 ^-10 ⋅N _RNKVB +0.022 and makes it possible to assess the value of the concentration of rabies virus ribonucleoprotein in raw materials for the production of antirabies vaccines.

The proposed invention makes it possible to quickly and with a high degree of reliability determine the value of the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccines based on the spectrometric method with subsequent application of the proposed linear model.

Information sources

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Claims (13)

1. A method for spectrometric determination of the concentration of rabies virus ribonucleoprotein by assessing the amount of viral RNA molecules in raw materials for rabies vaccines, including the following stages: - carrying out strain-specific binding of full particles of rabies virus from raw materials for rabies vaccine; - isolation of RNA from the resulting bound particles; - assessment of the degree of purity of RNA extracts using spectral analysis; - preparation of a series of dilutions of the positive standard of the RNA virus of rabies with the number of molecules corresponding to concentrations of ribonucleoprotein from 0.1 to 3.5 μg / ml with a dilution step of 0.2 μg / ml; - calculation of the number of RNA molecules isolated from complete rabies virus particles using absorption spectrum data; - Establishing the relationship between the concentration of ribonucleoprotein and the number of RNA molecules extracted from full particles of the rabies virus, as a linear function; - determination of the degree of reliability of the approximation (R ² ) of the developed model; - calculation of the concentration of rabies virus ribonucleoprotein in raw materials for rabies vaccines based on the developed model. 2. The method according to claim 1, characterized in that the processes of strain-specific binding of full particles of rabies virus from raw materials for rabies vaccines are combined with subsequent extraction of highly purified viral RNA and assessment of its purity by spectral analysis. 3. The method according to claim 1, characterized in that, based on the spectrometric study of the eluate of the rabies virus RNA, the extinction value at a wavelength of 260 nm (OD ₂₆₀ ) is determined and the number of RNA molecules extracted from the complete rabies virus particles (N _{RNA VV} ) is calculated, using the proposed function N _{RNA WB} = 4.3734 × 10 ⁹ × K × (OD ₂₆₀ samples _-OD 260k- -OD _{320 samples} ). 4. The method according to claim 1, characterized in that on the basis of the data on the number of RNA molecules isolated from the complete particles of the rabies virus, and determined in the spectrometric study, the concentration of the ribonucleoprotein of the rabies virus in the raw material for rabies vaccines is calculated using the developed linear function C _{rnp wb} = 9 × 10 ^-10 × N _RNKVB +0.022.

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