RU2728340C1 - Method for producing a set of fluorescent rickettsial and query fever diagnosticums and use thereof for serological diagnosis of rickettsiosis and query fever, a serological diagnostic technique - Google Patents

Abstract

FIELD: biotechnology; microbiology.SUBSTANCE: group of inventions relates to biotechnology and medical microbiology and includes three objects: a method for producing a set of fluorescent rickettsial and query fever diagnosticums, use thereof for serological diagnostics of rickettsiosis and query fever, a method for serological diagnostics of rickettsiosis and query fever. Method for producing a set of fluorescent diagnosticums of rickettsiae of group of typhus (R.prowazekii, R. typhi), rickettsiae of groups of spotted fever (R.sibirica, R.conorii) and genus Coxiella (Coxiella burnetii) includes preparation of sorbent from formalized lactic acid bacilli with their labeling with inorganic fluorophores, extraction of active protein-lipopolysaccharide complex from corpuscular antigens Rickettsia prowazekii, R. typhi, R.sibirica, R.conorii, Coxiella burnetii combined methods of ultrasonic disintegration of corpuscles and chromatography, attachment of a protein-lipopolysaccharide complex to a fluorescent sorbent by means of a binding reagent and obtaining an end product – set of fluorescent rickettsial and query fever diagnosticums. Kit contains 5 lyophilically dried diagnosticums: fluorescent diagnosticum R.prowazekii, fluorescent diagnosticum R. typhi, fluorescent diagnosticum R.sibirica, fluorescent diagnosticum R.conorii, fluorescent diagnosticum C.burnetii. Obtained fluorescent diagnosticums are correlated by specificity and sensitivity with a reference preparation – "gold standard" of immunofluorescence – (indirect immunofluorescence reaction, IIFR) and are suitable for diagnosing said diseases. Method for serodiagnosis of rickettsiosis and query fever using a kit is carried out in the format of a single-step direct immunofluorescence method involving applying a test serum onto a slide, adding a fluorescent diagnosticum and incubating mixture at 37 °C for 60 minutes. Smear preparations are dried and viewed under the immersion lens of the fluorescent microscope, the results are evaluated by a four-cross system, documentation is carried out by preserving object glass with smears or preservation of photo from microscope on USB-carrier.EFFECT: proposed group of inventions enables accurate and effective serological diagnosis of rickettsiosis and query fever.9 cl, 10 dwg, 2 tbl, 9 ex

Description

A method for obtaining a set of fluorescent rickettsial and coxiellosis diagnostics and their use for serological diagnosis of rickettsial diseases and coxiellosis, a method for serological diagnostics

Technology area

The invention relates to the field of biotechnology and medical microbiology and relates to a method for producing a set of fluorescent preparations of rickettsia and coxiella for the detection of specific antibodies in human blood serum, the use of this set for the serological diagnosis of rickettsia and coxiellosis.

Prior art

The polymorphic picture of clinical manifestations in rickettsioses and coxiellosis, the subacute and asymptomatic course of the infectious process create great difficulties in recognizing diseases, therefore, laboratory, and primarily serological, research methods are of decisive importance in the diagnosis of these diseases.

The choice of a laboratory diagnostic method is determined by factors such as specificity, sensitivity, and ease of implementation.

Comparative analysis of the methods used for serodiagnosis of rickettsioses and coxiellosis: agglutination reaction (RA), indirect hemagglutination reaction (RHA), complement fixation reaction (CSC), indirect immunofluorescence reaction (RNIF), immunofluorescence analysis (ELISA) indicates that the most sensitive, specific and rather express of them are IFA and RNIF.

However, the diagnosis of rickettsioses and coxiellosis using the RNIF does not provide for the detection of the full spectrum of the antibodies formed, since the corpuscular antigens of rickettsia and coxiella used in immunofluorescence analysis, fixed on a glass slide, react with antibodies due to the available surface antigenic structures of cells and the results should be taken into account by morphological features pathogen and the presence of edge fluorescence of the microbial cell (Barban P.S., Pshenichnov R.A., Pantyukhina A.N., Ivshina I.B. Immunofluorescence analysis. Sverdlovsk: Ural Branch of the Academy of Sciences of the USSR. 1988, 176 p.; Patent RU 2557951. "Method of obtaining multiplex diagnosticum", IV Tarasevich, AN Pantyukhina, MS Vakstein, SV Dezhurov

The disadvantage of RNIF is its low specificity, due to both the interaction of protein-protein ingredients (induced nonspecific immunofluorescence), and the presence of antigenic structures on the outer membrane of rickettsial cells that cross-react with antigenic epitopes of other pathogens of the rickettsial group, as well as the presence of antigens, structures of other microorganisms such as: Proteus vulgaris OX_nineteen, Proteus vulgaris OX₂... The RNIF setup is difficult because of the multistage technique and the need to introduce additional reagents and washes. Another negative factor of RNIF is the high consumption of corpuscular antigens of rickettsia and coxiella, which determines the high cost of this research method.

It is known that soluble antigens of rickettsia and coxiella are widely used in diagnostic work due to the simplicity of their preparation, the detection of the entire spectrum of specific antibodies and higher serological activity in comparison with corpuscular antigens (Zdrodovsky P.F., Golinevich E.M., “The doctrine of rickettsia and rickettsioses ", M., 1972., S. 495).

However, soluble antigens of rickettsia and coxiella are not used in immunofluorescence analysis, since fixed soluble antigens are diffusely located on the glass and, when interacting with fluorescent antibodies, give homogeneous luminescence of the entire smear, not allowing to evaluate the research results...

Based on this, for the early diagnosis of rickettsial infections and coxiellosis, increasing the specificity and sensitivity of immunofluorescence analysis, it is necessary to obtain soluble antigens of rickettsia and coxiella in corpuscular form, which will allow evaluating the results of the reaction in the format of a one-step microimmunofluorescence analysis.

A new opportunity appeared with the development of developments for the production and use of sorbents in microbiology.

A known method of binding soluble antigens of rickettsia of the typhus group and the group of tick-borne spotted fever by treating the carrier with a sorbent - polyacrylamide gel or soluble starch or Sepharose 4B, a mixture consisting of a phospholipid antigenic complex of rickettsia and 0.5% solution in polymethylmethacrylate the specified mixture 1: 1. However, this sorbent is suitable for immobilization of only phospholipid antigenic complexes of rickettsia, and does not adsorb protein antigens of rickettsia, since the latter do not dissolve in chloroform. (Tupitsyn A. V. Timasheva O. A., Horowitz E. S., "Method of obtaining immunosorbent". Inventor's certificate SU No. 1007676, 1976).

The classic method of immobilization of soluble antigens of rickettsia and coxiella is their sensitization on formalized erythrocytes of a sheep (Misenzhnikov AV, Lazarevich SN, "On the issue of the preparation of hemosensitizing antigen from rickettsia siberian. Topical issues of theoretical and applied immunology. Perm 1994. - pp. 26-27; Tokarevich NK “Biological aspects of the relationship between Burnet's coxiella and the mammalian organism as the basis for the improvement and specific prevention of Q fever.” Abstract of Doctor of Medical Sciences, St. Petersburg, 1997, p. 46; ).

However, such a sorbent cannot be used in immunofluorescence analysis in the format of a microimmunofluorescence reaction, since erythrocytes loaded with antigens are distributed in an even layer on a glass slide both in the experiment and in the control, which does not allow evaluating the result of the reaction.

The closest to the proposed method in terms of technological essence is ELISA + MFA in the format of multiplex analysis (Wang L. et al. "Nanoparticles for multiplex diagnostics and imaging", Nanomedicine, 2006. V. 1, No. 4, p. 413-426).

With this type of ELISA + MFA, organic polymers or inorganic materials (magnoimmunosorbents, polystyrene microspheres, microspheres, latexes, etc.) are used as a matrix for the sorption of analytes (antigens, antibodies, synthetic peptides, oligonucleotides, etc.).

The method is carried out as follows: stained with fluorophores, for example, polystyrene microspheres of 5.6 μm; then attach the binding analytes (oligonucleoproteins, antibodies, peptides, etc.); add suspensions of microspheres to the test samples (plasma, serum, blood, etc.); a detection agent and a fluorescent label (a mixture of streptovidin and phycoerythrin) are added. During measurement in the flow cell of the Luminex analyzer in a flowing liquid, each microsphere is irradiated with two lasers (green and red) with different wavelengths and the fluorescence signal emitted by the fluorophores is recorded by the instrument's photodetectors. Thus, the type of microsphere and the presence (concentration) of the desired analyte on the corresponding type of particles are analyzed simultaneously.

The disadvantage of this method is the need to use special expensive equipment and hard-to-reach polystyrene microboards, as well as the loss of time for their sensitization, laundering, multi-stage method, creation of a research program and long-term interpretation of the results. This method is only suitable for highly specialized laboratories and not for practical laboratories.

Disclosure of invention

The problem to be solved by the present invention is to obtain a set of fluorescent rickettsial and coxiellosis diagnostics, namely, fluorescent diagnostics of rickettsiae of the typhus group (R. prowazekii, R. typhi), rickettsia of the tick-borne spotted fever group (R. sibirica) and R. conorii. genus coxiella (C. burnetii) based on their protein-lipopolysaccharide complex and, their use for the simultaneous one-stage serological diagnosis of rickettsioses and coxiellosis, as well as the creation of a method for serological diagnosis using the direct method of fluorescent antigens (pMPAg) while maintaining high sensitivity and specificity due to giving corpuscular form to the protein-lipopolysaccharide complex and increasing the number of antigenic determinants on its surface.

The technical result is achieved due to the fact that a method has been created for obtaining a set of fluorescent rickettsial and coxiellosis diagnosticums, including the preparation of a fluorescent sorbent by purifying formalinized lactobacilli from the components of a protective drying medium, followed by labeling them with inorganic fluorochromes, obtaining protein-lipopolysaccharide riciphogenic antigen complexes from or a group of tick-borne spotted fever or or of the genus Coxiella by isolating active protein-lipopolysaccharide complexes from them by combined methods of ultrasonic disintegration of antigen corpuscles and chromatography, after which the isolated protein-lipopolysaccharide complexes are combined with a fluorescent sorbent by means of a binder reagent and the final product of fluorescent and fluorescent products is obtained. coxiella diagnostics from the listed ones.

In this case, protein-lipopolysaccharide complexes are obtained from corpuscular antigens of rickettsia of the typhus group - R.prowazekii or R. typhi; or the R. sibirica or R. conorii tick-borne spotted fever group; or the genus Coxiella - C. burnetii. Lactobacilli with a concentration of at least 1x109 cells / ml, labeled with inorganic fluorochromes, are used as a fluorescent sorbent. As a binder reagent of the fluorescent sorbent with the protein-lipopolysaccharide complex, tannin or water-soluble carbodiamide is used in a volume ratio of the fluorescent sorbent and a solution of tannin or carbodiimide 1: 1. In this method, a fluorescent sorbent with a binding reagent is mixed with a solution of protein-lipopolysaccharide complexes with a protein concentration of at least 0.5 mg / ml at a volume ratio of 1: 1. In this case, the fluorescent sorbent is incubated with the protein-lipopolysaccharide complex for 1 hour at a temperature of 43 ° C. Each fluorescent rickettsial and coxiellosis diagnosticum from the kit is a freeze-dried suspension of fluorescent lactobacilli covalently linked by means of a binding reagent to protein-lipopolysaccharide complexes of rickettsiae of the rickettsia group of the typhus rickett ricketa R. .sibirica or R. conorii, or the genus Coxiella C. burnetii.

The application of the created set of fluorescent rickettsial and coxiella diagnostics for the serological diagnosis of rickettsial diseases and coxiellosis for the detection of diseases of the typhus group - epidemic typhus lice and endemic flea typhus, the group of tick-borne spotted fever of North Asia or aoxia strains - tick-borne fever Q fever, as well as for seroimmunological monitoring of the population in endemic foci.

A method for serological diagnosis of rickettsial infections and coxiellosis has also been created using a set of fluorescent rickettsial and coxiellosis diagnostics, which consists in the fact that the reaction of fluorescent diagnostics and tested sera is performed in the format of a direct method, on glass slides with wells located in two horizontal rows, while in all wells of the first horizontal row, the test serum is introduced in a diagnostic titer with a dilution of 1:40, and in all wells of the second row - purified water, then sequentially in the vertical rows of both horizontal rows, in the serum of the first horizontal row and purified water of the second horizontal row, add created fluorescent diagnostics, and in the 1st horizontal row - R.prowazekii, in the 2nd - R. typhi, in the third -R. sibirica, in the fourth - R. conorii and in the fifth - C. burnetii, and the interaction of diagnosticum antigens with antibodies of the studied serum is carried out by stirring by tapping on the substrate and further placing in a humid chamber with a temperature of 37 ° C for 60 minutes, then the smears are dried and viewed under an immersion lens of a fluorescent microscope, the results are assessed using a four-cross system and documentation is carried out by saving slides with smears or saving a photo from a microscope on a USB-carrier, if a positive reaction is detected in the wells, a semi-quantitative analysis is performed using a fluorescent diagnosticum with which the serum showed a positive reaction, while dilutions of the tested serum from 1:20 to 1: 1280 are introduced into the wells of the slides, starting with the maximum, then this fluorescent diagnosticum is added to the wells of the slides, then, as in the first experiment, the interaction of antigens fluorescent diagnostics godfathers with antibodies of the studied serum are carried out by stirring by tapping on the slide support and further placement in a humid chamber with a temperature of 37 ° C for 60 minutes, then the smears are dried and viewed under the immersion lens of a fluorescent microscope, the results are also evaluated using a four-cross system, documentation is also carried out by saving slides with smears or saving a photo from a microscope on a USB-carrier.

Brief description of the figures

FIG. 1.

The preparation of a fluorescent sorbent is presented.

A - formalized lactobacilli are presented, which are a matrix for the sorption of analytes - protein-lipopolysaccharide complexes of rickettsial and coxiellosis antigens;

B-formula of inorganic fluorochrome - fluorescein isothiocyanate (FITC), used to stain lactobacilli;

C-fluorescent sorbent obtained after staining with FITC (photo).

FIG. 2.

The preparation of a fluorescent sorbent with activated groups is presented.

A-fluorescent sorbent;

B-formula of the binder reagent (tannin) used to attach the protein-lipopolysaccharide complex to the fluorescent sorbent;

C-activated fluorescent sorbent containing active hydroxyl and carboxyl groups.

FIG. 3.

The preparation of a fluorescent diagnosticum is schematically shown.

A-activated fluorescent sorbent;

B-attachment of protein-lipopolysaccharide complexes of rickettsiae of the typhus group (R.prowazekii, R. typhi), rickettsiae of the tick-borne spotted fever group (R.sibirica, R.conorii) and the genus Coxiella (C.burnetii) to the activated fluorescent sorbent;

C-end products of fluorescent diagnostics:

- fluorescent diagnosticum R.prowazekii,

- fluorescent diagnosticum R. typhi,

- fluorescent diagnosticum R. sibirica,

- fluorescent diagnosticum R. conorii,

- fluorescent diagnosticum C. burnetii.

FIG. 4.

The result of the use of fluorescent dagnostikums from a set with a specific homologous serum has been demonstrated. The result of visual assessment of the reaction when viewed under a fluorescent microscope.

A - photo of a negative result - the absence of specific antibodies in the tested serum. The fields of view are filled with isolated corpuscles of fluorescent diagnosticum;

B - photo of a positive result - specific antibodies are present in the tested serum, in a titer of 1: 640.

In the field of view, agglutinates containing more than 50 corpuscles of fluorescent diagnosticum are a positive reaction, rated at 4+.

Similar positive and negative results were obtained when staging with any diagnostic kit from the kit.

Figure 5.

A typical chromatogram obtained at the final stage of processing and purification of corpuscular suspensions of rickettsia of the typhus group (Rickettsia prowazekii / R.typhi) is presented. Demonstrated the elution profile of supernatants after ultrasonic treatment of a suspension of rickettsia, showing the separation of the disintegrant into four fractions (I, II, III, IV). Serological specific activity was concentrated in the first fraction and corresponded to RNGA 1:64, in other fractions there was no serological activity. To determine the composition of the obtained first fraction, the content of protein and bacterial lipopolysaccharides was evaluated. The protein content was 0.9 mg / ml, the content of bacterial lipopolysaccharides was 42,920 EU / ml, the specific activity in RNGA was -1128. Based on the data obtained, the first fraction was named the protein-lipopolysaccharide complex.

The X-axis indicates the volume of the eluent passed through the column, in ml / ml; Y-axis - optical density (detection at a wavelength of 280 nm).

FIG. 6.

Protein profiles of the Rickettsia prowazekii protein-lipopolysaccharide complex obtained by electrophoresis in SDS-PAGE and staining with EZBlue ^tm GelStainingReagent (Sigma-Aldrich, USA). A molecular weight marker ab115832 (Abcam, UK) was used. Proteins with Mw 18, 29, 57, 105, 121 kDa were detected in the sample.

FIG. 7.

A typical chromatogram obtained at the final stage of processing and purification of corpuscular suspensions of rickettsia from the tick-borne spotted fever group (Rickettsia sibirica, R. conorii) is presented. Demonstrated the elution profile of supernatants after ultrasonic treatment of a suspension of rickettsia, showing the separation of the disintegrant into four fractions (I, II, III). Serological specific activity was concentrated in the first fraction and corresponded to RNGA 1: 128, in the remaining fractions there was no serological activity. To determine the composition of the obtained first fraction, the content of protein and bacterial lipopolysaccharides was evaluated. The protein content was 1.4 mg / ml, the bacterial lipopolysaccharide content was 60150 EU / ml.

Based on the data, the first fraction was named protein-derived lipopolysaccharide complex.

The X-axis indicates the volume of the eluent passed through the column, in ml / ml; Y-axis - optical density (detection at a wavelength of 280 nm).

FIG. 8.

Protein profiles of the Rickettsia sibirica protein-lipopolysaccharide complex obtained as a result of SDS-PAGE and EZBlue ^tm GelStainingReagent staining (Sigma-Aldrich, USA). A molecular weight marker ab115832 (Abcam, UK) was used. Proteins with Mw 18, 29, 57, 105, 121 kDa were detected in the sample.

FIG. nine.

A typical chromatogram obtained at the final stage of processing and purification of particulate suspensions of Coxiella burnetii is presented. Demonstrated the elution profile of supernatants after ultrasonic treatment of a suspension of rickettsia, showing the separation of the disintegrant into two fractions (I, II). Serological specific activity was concentrated in the first fraction and corresponded to RNGA 1: 128, in the remaining fractions there was no serological activity. To determine the composition of the obtained first fraction, the content of protein and bacterial lipopolysaccharides was evaluated. The protein content was 1.1 mg / ml, the content of bacterial lipopolysaccharides was 26,990 EU / ml. Based on the data obtained, the first fraction was named protein-derived lipopolysaccharide complex.

The X-axis indicates the volume of the eluent passed through the column, in ml / ml; Y-axis - optical density (detection at a wavelength of 280 nm).

FIG. ten.

Protein profiles of the Coxiella burnetii protein-lipopolysaccharide complex obtained by SDS-PAGE and staining with EZBluetmGelStainingReagent (Sigma-Aldrich, USA) are presented. The used molecular weight marker is Wide Range Protein Molecular Weight Marker (cat. Number BSM0661, Bio Basic, Canada). Proteins with Mw 15, 65, 85, 120 kDa were detected in the sample.

Implementation of the invention

The claimed invention is aimed at creating a set of fluorescent diagnostics designed for the simultaneous detection of specific antibodies in human blood serum to different antigens of rickettsia and coxiella pathogenic for humans. The proposed method of serological diagnostics using a set of fluorescent diagnostics is less time consuming, is an affordable and methodologically simple method that does not require large costs for the analysis. The introduction of a set of fluorescent diagnostics into practice for clinical and diagnostic laboratories of medical institutions of the Ministry of Health of the Russian Federation and the Rospotrebnadzor Centers of the Russian Federation is a reliable way to diagnose rickettsioses of the typhus group (epidemic typhus and endemic typhus), tick-borne tick-borne fever and tick-borne fever (tick-borne fever) ) and coxiellosis. Diagnostics of infections of rickettsial etiology and coxiellosis was carried out in the relevant regulatory documents (see References 15-19).

The method of preparation of fluorescent rickettsial and coxiellosis diagnostics consists of the following stages:

1. Obtaining a fluorescent sorbent:

1.1. cleaning lactobacilli,

1.2. formalization of lactobacilli,

1.3. obtaining a fluorescent sorbent.

2. Obtaining a protein-lipopolysaccharide complex of rickettsia and coxiella:

2.1. preparation of lyophilized cultures of corpuscles of rickettsia of the typhus group (R.prowazekii, R. typhi), rickettsia of the group of tick-borne spotted fever (R.sibirica, R.conorii) and the genus coxiella (C.burnetii),

2.2. disintegration of cultures of rickettsia and coxiella using the ultrasonic disintegrator Soni Prep 150 Plus,

2.3. separation of Rickettsia and Coxiella disintegrates using an Akta Explorer 10 chromatograph and isolation of a specifically active protein-lipopolysaccharide complex,

3. Preparation of fluorescent diagnostics (Fig. 1):

3.1. obtaining fluorescent sorbents with activated groups,

3.2. sensitization of tanned fluorescent sorbents with a protein-lipopolysaccharide complex,

3.3. obtaining fluorescent diagnostics.

Example 1.

Obtaining a fluorescent sorbent.

1.1. Purification of lactobacilli.

Lactobacillus fermentum 90T-C4 was used as a matrix for the sorbent; however, other strains of lactobacilli can also be used in the work: Lactobacillus plantarum 8P-A3, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp., Brueckii subsp. lactis, Lactobacillus paracasei and Lactobacillus rhamnosus.

Lactobacilli are isolated from the Lactobacterin preparation by centrifugation three times at 3000 rpm for 25 minutes at 23 ° C to remove the protective medium for drying.

1.2. Formalization of lactobacilli.

The sediments of lactobacilli were resuspended in 50 ml of 0.9% sodium chloride solution, pH 7.0. In the resulting biomass of lactobacilli add neutral formalin to a final concentration of 10% and incubate for 7 days at a temperature of 6-8 ° C. The sedimentation of biomass is carried out in a centrifuge at 3000 rpm for 25 minutes at a temperature of 23 ° C. Washing of biomass is carried out in 0.9% sodium chloride solution at 3000 rpm for 25 minutes. The precipitate is resuspended in a working solution - 0.9% sodium chloride solution - 70%, carbonate-bicarbonate buffer pH 9.0 - 30%. The output of suspension of formalinized lactobacilli - sorbent 50 ml with a concentration of 10 ⁹ cells / ml.

1.3. Obtaining a fluorescent sorbent.

For staining, inorganic fluorescent dyes were used: fluorescein isothiocyanate, semiconductor colloidal nanoparticles of different fluorescence colors - nanocrystals. In the described experiment, for staining, a fluorescent dye, fluorescein isothiocyanate, was added to the obtained suspension of formalinized lactobacilli at the rate of 2 mg per 10x10 ⁹ lactobacilli.

The conjugation of the mixture was carried out for 18 hours at a temperature of 23 ° C with constant stirring. Labeling with nanocrystals was carried out in a similar way, except that the nanocrystals were added to formalinized lactobacilli 10 ml with a nanoparticle concentration of 5.4 x 10 ^-6 .

After the conjugation time elapsed, the fluorescent sorbent was precipitated and washed from unreacted fluorochrome by three-fold precipitation by centrifugation in 0.9% sodium chloride solution at 3000 rpm for 25 min. and the pellet was resuspended in 0.9% sodium chloride solution. The yield of the fluorescent sorbent is 45 ml. Stored at 6-8 ° C.

Example 2.

Obtaining a protein-lipopolysaccharide complex of rickettsia and coxiella.

2.1. Preparation of lyophilized cultures of rickettsiae of the typhus group (R.prowazekii, R. typhi), rickettsiae of the tick-borne spotted fever group (R.sibirica, R.conorii) and the genus Coxiella (C.burnetii).

The initial material for obtaining the protein-lipopolysaccharide complex was lyophilized corpuscular antigens of Rickettsia prowazekii, R. sibirica, which were obtained by a non-ether method, including the cultivation of R. prowazekii and R. sibirica in developing chick embryos, extraction of infected yolk membranes, from chick embryos. % formalin and homogenization at 5000 rpm for 3 min, the combined use of a low-speed -1500 rpm for 5 min and a high-speed -10000 rpm for 30 min0 centrifugation followed by the removal of fine particles by microfiltration.

Corpuscular antigens of R. typhi, R. conorii and Coxiella burnetii were obtained using ether technology (P.F. Zdrodovsky and E.M. Golinevich.

"The doctrine of rickettsia and rickettsioses", M., 1972, p. 114-117), including the cultivation of rickettsia and coxiella in developing chick embryos, extraction of infected vitelline membranes and grinding with glass beads, three times treatment of the resulting suspension with ether, and the separated aqueous phase was subjected to prolonged centrifugation for 2 hours at 3000 rpm min, followed by resuspension of the sediment in a buffered 0.9% sodium chloride solution with the addition of formalin to 0.2%.

2.2 Disintegration of rickettsia and coxiella cultures using an ultrasonic disintegrator Soni Prep 150 Plus.

For this purpose, the contents of ampoules with corpuscular antigens were resuspended in a buffer solution (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5) and processed using an ultrasonic disintegrator Soni Prep 150 Plus (Great Britain) at a frequency of 23 kHz and an amplitude of 112 six times for 5 min. The sedimentation of intact cells was carried out by centrifugation at 3000 rpm for 20 min at a temperature of 23 ° C.

2.3. Separation of Rickettsia and Coxiella disintegrates using an Akta Explorer 10 chromatograph and isolation of a specifically active protein-lipopolysaccharide complex.

The disintegrate was subjected to size exclusion chromatography using an Akta Explorer 10 chromatograph (Sweden) at a temperature of 4 ° C. On a column with a size of 10 x 300 mm, the disintegrate was fractionated in a buffer solution of pH 7.5 (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol).

Chromatograms of the separation of protein-lipopolysaccharide complexes of rickettsia and coxiella are shown in Figures 5, 7, 9.

A qualitative analysis of the isolated protein-lipopolysaccharide complex is presented in Figures 6, 8, 10.

The first fraction contained a serologically active protein-lipopolysaccharide complex, the remaining fractions had no serological activity.

The isolated protein-lipopolysaccharide complexes met the following requirements:

- the protein-lipopolysaccharide complex should have a pH of 7.2-7.4;

- it should not contain particles in reaction with fluorescent immunoglobulins of appropriate specificity;

- must have a specific activity in the RNGA with the corresponding diagnostics not lower than 1:32;

- the protein content in the protein-lipopolysaccharide complex is not less than 0.5 mg / ml;

- the amount of lipopolysaccharide (LPS) complex must be at least 25,000 EU / ml.

Example 3.

Preparation of fluorescent diagnostics.

3.1. Obtaining fluorescent sorbents with activated groups (Fig. 2).

Hydrolyzed tannins (esters of gallic acid or related digalic and trigallic acids with a polyhydric alcohol - glucose) or water-soluble carbodiimides ( N - (3-Dimethylaminopropyl) - N ′ -ethylcarbodiimide hydrochloride) were used as a binder reagent.

For this, 2 ml of a 1% suspension of a fluorescent sorbent was precipitated and washed in 2 ml of 0.9% sodium chloride solution pH 7.0 twice by centrifugation for 25 min at 3000 rpm. The precipitate was resuspended in 2 ml of 0.9% sodium chloride solution, pH 7.2. Then, 2 ml of tannin solution in 0.9% sodium chloride solution pH 7.2 was added to 2 ml of washed fluorescent sorbent to a concentration of 1: 20,000, the mixture was left for 15 min at 23 ° C with periodic stirring. The mixture was sedimented by centrifugation at 3000 rpm¹ within 25 minutes at a temperature of 23 ° C. The mixture was washed three times in 0.9% sodium chloride solution pH 7.0 at 3000 rpm within 25 minutes at a temperature of 23 ° C. The output of the tanned fluorescent sorbent is 2 ml.

The preparation of fluorescent sorbents with activated groups using water-soluble carbodiimide ( N - (3-Dimethylaminopropyl) - N '-ethylcarbodiimide hydrochloride) was carried out in a similar way, only the concentration of carbodiimide was 1: 2000.

3.2. Coupling of fluorescent sorbents with activated groups of protein-lipopolysaccharide complexes.

For this, 2 ml of a tanned fluorescent sorbent was combined with 2 ml of a protein-lipopolysaccharide complex solution with a protein concentration of 0.5 mg / ml and incubated at 43 ° C for 1 hour with periodic stirring.

3.3. Obtaining fluorescent diagnostics (Fig. 3).

After the incubation time, the mixture was centrifuged for 25 min at 3000 rpm and washed 0.9% sodium chloride solution pH 7.0...After that, the final concentration of fluorescent diagnostics was prepared equal to 0.5%. Stored at 6-8 ° C.

Example 4.

Production of fluorescent diagnostics for rickettsia of the typhus group (Rickettsia prowazekii and R. typhi).

4.1. Obtaining a fluorescent diagnosticum Rickettsia prowazekii was carried out in accordance with the above steps of the technological process.

We used lyophilized corpuscular antigen Rickettsia prowazekii (strain "Brainl", registration no. 1, taken from the "Collection of pathogens of borreliosis, brucellosis, clostridiosis, pertussis, leptospirosis, legionellosis, mycoplasmosis, rickettsiosis and Microbiology named after Honorary Academician NF Gamaleya "of the Ministry of Health of the Russian Federation), which was obtained by the non-ether method, including the cultivation of R. prowazekii and R. sibirica in developing chicken embryos, the extraction of infected yolk membranes from chicken embryos, inactivation of 0.4% formalin and homogenization at 5000 rpm for 3 min, combined use of low-speed (1500 rpm for 5 min) and high-speed (10000 rpm for 30 min 0 centrifugation followed by removal of fine particles by microfiltration.

The contents of ampoules with corpuscular antigen were resuspended in a buffer solution (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5) and treated with an ultrasonic disintegrator at a frequency of 23 kHz and an amplitude of 112 six times for 5 min. The sedimentation of intact cells was carried out by centrifugation at 10,000 rpm for 10 min at a temperature of 23 ° C. To isolate the protein-lipopolysaccharide complex, the disintegrate was applied to a column (10 x 300 mm) filled with Superdex 200 sorbent and equilibrated with a buffer solution of 10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5.

The protein-lipopolysaccharide complex was eluted in the first fraction with a protein concentration of 0.5-1.4 mg / ml.

The protein antigen met the following requirements: fraction - pH 7.3, no corpuscles were detected when viewed with fluorescent immunoglobulins to rickettsia of the typhus group; specific activity in RNGA with erythrocytic typhus antigenic diagnosticum corresponded to 1: 64.

Previously, 50 ml of a fluorescent sorbent was prepared. For this purpose, the contents of 10 vials with "Lactobacterin" were resuspended in 50 ml of 0.9% sodium chloride solution pH 7.0, precipitated by centrifugation at 3000 rpm for 25 minutes at 23 ° C to remove the protective medium for drying. Then they were washed three times in 0.9% sodium chloride solution pH 7.0 at the same parameters. The sediments were resuspended in 50 ml of 0.9% sodium chloride solution pH 7.0, neutral formalin was added to the resulting biomass of lactobacilli to a final concentration of 10% and kept for 7 days. The biomass was sedimented in a centrifuge at 3000 rpm for 25 min at a temperature of 23 ° C. The biomass was washed in 0.9% sodium chloride solution at 3000 rpm for 25 min. The precipitate was resuspended in a working solution (0.9% sodium chloride solution - 70%, carbonate-bicarbonate buffer pH 9.0 - 30%). The output of suspension of formalinized lactobacilli is 45 ml with a concentration of 10^nine / ml.

A fluorescent dye, fluorescein isothiocyanate, was added to the resulting suspension at a rate of 2 mg per 10x10 ⁹ sorbent corpuscles. The conjugation of the mixture was carried out for 18 hours at a temperature of 23 ° C with constant stirring. After the conjugation time elapsed, the fluorescent sorbent was precipitated and the unreacted fluorochrome was washed off by 3-fold precipitation by centrifugation in 0.9% sodium chloride solution at 3000 rpm for 25 min and the precipitate was resuspended in a calculated amount of 0.9% chloride solution sodium required to obtain 1% concentration of the fluorescent sorbent. The output of the fluorescent sorbent is 40 ml. Stored at 6-8 ° C.

The fluorescent sorbent was labeled with nanocrystals in a similar manner with the difference that the concentration of nanocrystals was taken at the rate of 10 ml per 10^nine corpuscles of a fluorescent sorbent. To prepare the fluorescent diagnosticum R. prowazekii, 2 ml of a 1% suspension of the fluorescent sorbent were taken and washed in 2 ml with a 0.9% sodium chloride solution pH 7.0 twice by centrifugation for 25 min at 3000 rpm. The precipitate was resuspended in 2 ml of 0.9% sodium chloride solution pH 7.2. To 2 ml of washed fluorescent sorbent was added 2 ml of tannin solution in 0.9% sodium chloride solution pH 7.2 to a concentration of 1: 20,000, the mixture was left for 15 min at 23 ° C with periodic stirring. The mixture was sedimented by centrifugation at 3000 rpm for 25 min at 23 ° C. The mixture was washed three times in 0.9% sodium chloride solution pH 7.0 at 3000 rpm within 25 minutes at a temperature of 23 ° C. The yield of the tanned fluorescent sorbent is 1.8 ml. Then 1.8 ml of the tanned fluorescent sorbent was combined with 1.8 ml of a protein-lipopolysaccharide complex solution with a protein concentration of 0.5 mg / ml and incubated at 43 ° C for 1 hour. After incubation, the mixture was centrifuged for 25 min at 3000 min.¹and washed 0.9% sodium chloride solution pH 7.0...Then the final concentration of fluorescent diagnostics was prepared at 0.5%. Stored at a temperature of 6-8 ° C (Fig. 4).

Characteristics of the isolated protein-lipopolysaccharide complex of R.prowazekii: the protein antigen met the following requirements: fraction - pH 7.3; corpuscles were not detected when viewed with fluorescent immunoglobulins to rickettsiae of the typhus group; specific activity in RNGA with erythrocyte typhus antigenic diagnosticum corresponded to 1:64. The chromatogram of the protein-lipopolysaccharide complex is shown in Figure 5.

The qualitative characteristics of the protein-lipopolysaccharide complexes of the typhus group in SDS-PAGE are shown in Fig. 6.

A qualitative assessment of the obtained antigenic substances of rickettsia of the typhus group (R. prowazekii and R. typhi) was carried out by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate according to the known method (OFS 1.2.1.0023.15. "Electrophoresis in polyacrylamide gel"). Then the gel was stained using EZ Blue Gel Staining Reagent (Sigma-Aldrich, USA).

A typical variant of the protein profile of the protein-lipopolysaccharide complex of the typhus group is shown in figure 6.

The quantitative determination of the protein in the obtained antigenic complexes was assessed spectrophotometrically by the Bradford method (OFS.1.2.3.0012.15. "Protein determination"). The quantitative determination of bacterial lipopolysaccharides was assessed using the turbidimetric kinetic LAL test (OFS.1.2.4.0006.15. "Bacterial endotoxins"). The protein content in the obtained antigenic substance, determined by the Bradford method, ranged from 0.9 to 1.4 mg / ml; the content of bacterial lipopolysaccharides, determined using the method of kinetic turbidimetric LAL test, was 42,920 EU / ml.

Thus, the results obtained confirmed the presence of both proteins and bacterial lipopolysaccharides in soluble purified protein-lipopolysaccharide substances of Rickettsia prowazekii.

The results of experiments on the study of sera to rickettsiae of the typhus group using the fluorescent diagnosticum R. prowazekii are shown in Table 1 and 2.

Fluorescent antigens from other rickettsiae of the typhus group (R. typhi), rickettsiae of the tick-borne spotted fever group (R. sibirica and R. conorii), and Burnet's coxiella (C. burnetii) were prepared and studied in a similar manner.

Example 4.2.

Manufacturing of fluorescent diagnosticum R. typhi.

Lyophilized R. typhi corpuscular antigen was used.

(strain "Isakhanyan" registration No. 37, taken from the same "Collection" as the strain with registration number No. 1), obtained by the method of P.F. Zdrodovsky. and Golinevich E.M. “The doctrine of rickettsia and rickettsioses. Corpuscular antigens of rickettsia ", M., 1972. p. 114-117. Corpuscular antigens of R. typhi were obtained by ether technology (PF Zdrodovsky and EM Golinevich. "The doctrine of rickettsia and rickettsioses", M., 1972, pp. 114-117), including the cultivation of rickettsiae and coxiella in developing chicken embryos, extraction of infected vitelline membranes and grinding with glass beads, three times treatment of the resulting suspension with ether, and the separated aqueous phase was subjected to prolonged centrifugation for 2 hours at 3000 rpm, followed by resuspension of the sediment in a buffered 0.9% sodium chloride solution with the addition of formalin up to 0.2%.

Contents of ampoules with lyophilized R. typhi antigen

resuspended in buffer solution (10mM Tris-HCl, 0.5M sodium chloride, 10% isopropanol, pH 7.5) and processed using an ultrasonic disintegrator followed by exclusion chromatography on a Superdex 200 column.

R. typhi fluorescent diagnosticum was prepared as described in example 1.

Characteristics of the R. typhi protein-lipopolysaccharide complex: pH 7.2, did not contain corpuscles, specific activity in PHA 1: 64-1: 128.

The chromatogram and protein profiles of R. typhi in SDS PAGE were similar to those of the chromatogram and protein profiles of R. prowazekii.

It was found that the protein content in the obtained antigenic substance, determined by the Bradford method, ranged from 0.9 to 1.4 mg / ml; the content of bacterial lipopolysaccharides, determined using the kinetic turbidimetric LAL test, was more than 42,950 EU / ml.

A qualitative analysis confirmed the presence of both proteins and bacterial lipopolysaccharides in the purified protein-lipopolysaccharide substances of Rickettsia typhi.

The results of experiments on the study of sera to rickettsiae of the typhus group using the fluorescent diagnosticum R. typhi are shown in Table 1 and 2.

Example 5.

Production of a fluorescent diagnosticum for rickettsia of the tick-borne spotted fever group (R. sibirica and R. conorii).

5.1. Obtaining a fluorescent diagnosticum R. sibirica.

We used lyophilized corpuscular antigen of R. sibirica (strain "Netsvetaev", registration No. 52, taken from the same "Collection" as the strain with registration No. 1), obtained by the non-ether method, including the cultivation of R. sibirica in developing chicken embryos, extraction of infected yolk membranes from chicken embryos, inactivating them with 0.4% formalin and homogenizing at 5000 rpm for 3 min, combined use of low-speed (1500 rpm for 5 min) and high-speed (10000 rpm for 30 min), centrifugation with subsequent removal of fine particles by microfiltration.

The contents of ampoules with lyophilized antigen of Siberian rickettsia were resuspended in a buffer solution (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5) and processed using an ultrasonic disintegrator, followed by exclusive chromatography on a Superdex 200 column. The fluorescent diagnosticum of Siberian rickettsia was prepared as described in example 1.

The isolated protein-lipopolysaccharide complexes of R. sibirica had a pH of 7.3, did not contain corpuscles, the specific activity in RNGA was 1:32 - 1:64. The qualitative analysis of the isolated protein-lipopolysaccharide complex of R. sibirica was carried out as described in example 1. The protein content in the obtained antigenic substances, determined by the Bradford method, ranged from 0.5 to 1.4 mg / ml; the content of bacterial lipopolysaccharides, determined using the kinetic turbidimetric LAL test, was more than 60 150 EU / ml.

Thus, the results obtained confirmed the presence of both proteins and bacterial lipopolysaccharides in the soluble purified protein-lipopolysaccharide substances of R. sibirica.

Typical profiles of chromatograms and protein composition of R. sibirica are shown in Figures 7 and 8.

The results of experiments on the study of sera to rickettsia of the group of tick-borne spotted fever using the fluorescent diagnosticum R. sibirica are shown in Table 1 and 2.

Example 5.2.

Receiving fluorescent diagnosticum R. conorii.

We used a lyophilized corpuscular antigen R. conorii (strain "Sevastopol", registration No. 122, taken from the same "Collection" as the strain with registration No. 1)), obtained by the method of P.F. Zdrodovsky. and Golinevich E.M. “The doctrine of rickettsia and rickettsioses. Corpuscular antigens of rickettsia ", M., 1972. p. 114-117. The corpuscular antigens of R. conorii were obtained by the ether technology of P.F. Zdrodovsky and E.M. Golinevich. "The doctrine of rickettsia and rickettsioses", M., 1972, p. 114-117, including the cultivation of rickettsia and coxiella in developing chick embryos, extraction of infected vitelline membranes and grinding with glass beads, three times treatment of the resulting suspension with ether, and the separated aqueous phase was subjected to prolonged centrifugation for 2 hours at 3000 rpm, followed by resuspending the sediment in a buffered 0.9% sodium chloride solution with the addition of formalin to 0.2%.

The contents of ampoules with lyophilized R. conorii antigen were resuspended in a buffer solution (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5) and processed using an ultrasonic disintegrator followed by exclusive chromatography on a Superdex 200 column Conorii fluorescent diagnosticum was prepared as described in Example 1.

The isolated protein-lipopolysaccharide complex of R.conorii has a pH of 7.3, does not contain corpuscles, the specific activity in RNGA is 1:32 - 1:64. The qualitative analysis of the isolated protein-lipopolysaccharide complex of R. conorii was carried out as described in example 1. The protein content in the obtained antigenic substances, determined by the Bradford method, ranged from 0.5 to 1.4 mg / ml; the content of bacterial lipopolysaccharides, determined using the method of the kinetic turbidimetric LAL test, was higher than 60 150 EU / ml.

Thus, the results obtained confirmed the presence of both proteins and bacterial lipopolysaccharides in the soluble purified protein-lipopolysaccharide substances of R. sibirica and R. conorii.

The results of experiments on the study of sera to rickettsia of the group of tick-borne spotted fever using the fluorescent diagnosticum R. conorii are shown in table 1 and 2.

Example 6.

Production of C. burnetii fluorescent diagnosticum.

Used lyophilized corpuscular antigen C. burnetii (strain "Grita, registration No. 13", taken from the same "Collection" as the strain with registration No. 1)). Corpuscular antigens of C. burnetii were obtained using ether technology (PF Zdrodovsky and EM Golinevich. The doctrine of rickettsiae and rickettsioses. Corpuscular antigens of rickettsia M., 1972, p. 114-117), including the cultivation of rickettsiae and coxiella in developing chicken embryos, extraction of infected vitelline membranes and grinding with glass beads, three times treatment of the resulting suspension with ether, and the separated aqueous phase was subjected to prolonged centrifugation for 2 hours at 3000 rpm, followed by resuspension of the sediment in a buffered 0.9% solution sodium chloride with the addition of formalin up to 0.2%.

The contents of vials with lyophilized Burnet's Coxiella antigen were resuspended in a buffer solution (10mM Tris-HCl, 0.5M sodium chloride, 10% isopropanol, pH 7.5) and processed using an ultrasonic disintegrator, followed by exclusive chromatography on a Superdex 200 column. Fluorescent diagnosticum C. burnetii was prepared as described in example 1.

The isolated protein-lipopolysaccharide complex of C. burnetii has a pH of 7.2, contained corpuscles and a soluble antigen, specific activity in PHA - 1: 128 - 1: 256. The qualitative analysis of the isolated protein-lipopolysaccharide complex C. burnetii was carried out as described in example 1. The protein content in the resulting antigenic substance, determined by the Bradford method, ranged from 0.9 to 1.1 mg / ml; the content of bacterial lipopolysaccharides, determined using the method of kinetic turbidimetric LAL test, was above 26990 EU / ml.

Thus, the results obtained confirmed the presence of both proteins and bacterial lipopolysaccharides in the purified protein-lipopolysaccharide substance of Coxiella burnetii .

A typical version of the profiles of chromatograms and electrophoresis in PAGE is shown in Figures 9 and 10.

The results of experiments on the study of sera using C. burnetii fluorescent diagnosticum are given below in table No. 1 and 2.

Thus, according to the developed method, a set of fluorescent diagnosticums was prepared: R.prowazekii, R. typhi, R. sibirica, R. conorii and C. burnetii, studies were carried out with standard and test sera, in which their high activity and specificity were established. , which served as the basis for the use of fluorescent diagnostics as a kit for the detection of antibodies in rickettsioses and coxiellosis in the format of a simultaneous, one-stage microimmunofluorescence reaction.

Example 7.

Method for performing serological diagnostics using the created set of fluorescent diagnostics.

All obtained fluorescent diagnostics were used to carry out a one-step microimmunofluorescence reaction.

The reaction of microimmunofluorescence (direct method of fluorescent antigens - pMFAag) is based on the specific interaction of fluorescent diagnosticums directly with homologous antibodies of blood serum on a glass slide. For this purpose, human blood serum in dilutions is introduced into the wells of the slides and a fluorescent diagnosticum is added. The interaction of antigen with antibodies occurs within 60 minutes at a temperature of 37 ± 1 ° C in a humid chamber. After this time, the preparations are dried at room temperature and viewed and evaluated under the immersion objective of a fluorescence microscope (objective 100x, eyepiece 10x).

The method consists of two stages.

7.1. Qualitative analysis (screening of sera) followed by determination of the antibody titer (semi-quantitative analysis).

7.2. Registration and interpretation of results.

7.1. Qualitative analysis (screening of sera) followed by determination of the antibody titer (semi-quantitative analysis).

7.1.1. Reaction setting

The required number of slides with wells were placed on plastic supports with rims (cover from plates for immunological reactions).

It was prepared from sera studied for rickettsia and coxiellosis, dilutions 1: 40 in sterile 0.9% sodium chloride solution.

Prepared dilutions of control positive and negative sera 1:40 in sterile 0.9% sodium chloride solution.

To prepare a 1:40 dilution of sera, add 10 ml of whole serum to 390 ml of sterile 0.9% sodium chloride solution.

At the same time, when screening for one glass slide with 10 wells, 55 ml of fluorescent diagnosticum of one type is required.

Adding test and control serum samples.

One test serum was examined on one microscope slide with the setting of diagnosticum control.

1 row of glass slide:

- 5 ml of the test serum at a dilution of 1:40 was introduced into all the wells of the first row under No. 1-5,

- 5 ml of purified water was added to all wells of the second row under No. 6-10.

Thereafter:

- in both the wells of the first vertical row of the slide were added 5 ml of fluorescent diagnosticum R.prowazekii,

- in both wells of the second vertical row of the glass slide - 5 ml of fluorescent diagnosticum R.typhi,

- in both wells of the third vertical row of the glass slide - 5 ml of fluorescent diagnosticum R. sibirica,

- in both wells of the fourth vertical row of the slide - 5 ml of fluorescent diagnosticum R.conorii,

- in both wells of the fifth vertical row of the glass slide - 5 ml of C.burnetii fluorescent diagnosticum.

7.1.2. Ingredient interaction conditions

The reaction ingredients were mixed by gently tapping the plastic backing. Then the substrates with glass slides were placed in a humid chamber at a temperature of (37 ± 1) ° С for 60 min. The interaction of specific antibodies with fluorescent diagnosticum took place in a humid chamber. After the expiration of the time, the substrates with the preparations were removed from the humid chamber and dried in a thermostat or at room temperature (20 ± 2) ° С. The dried preparations were evaluated under an immersion objective of a fluorescence microscope (objective 100 x, eyepiece 10 x), viewing at least 10 fields of view.

In positive sera detected in the screening, the titer of specific antibodies to rickettsia and coxiella was determined.

7.1.3. Determination of antibody titer (semi-quantitative analysis)

For this, the number of microscope slides with 8 wells, corresponding to the number of positive sera detected, were placed on plastic supports with rims.

In a plate for immunological reactions, two-fold serial dilutions of the selected test sera were prepared from 1:20 to 1: 1280 in purified water. For this purpose, 95 ml of purified water was added to the first horizontal well of the plate for immunological reactions, and 50 ml of purified water were added to the remaining free wells of the horizontal row. The test serum in a volume of 5 ml was added to the first well of the plate. After thorough mixing, 50 ml of the diluted test serum was transferred from the first well to the second with an automatic pipette, 50 ml was transferred from the second to the third, etc.

5 ml of each dilution of the test serum, starting from 1: 1280, were added to the wells of an 8-well glass slide, and 5 ml of purified water was added to the 8th well to control the absence of spontaneous agglutination of the fluorescent diagnosticum. Then, 5 ml of fluorescent diagnosticum was added to all wells, with which positive results were obtained. A separate slide was used for control with positive and negative sera.

7.1.4. Conditions for the interaction of ingredients.

The reaction ingredients were mixed by gently tapping the plastic backing. Then the substrates with glass slides were placed in a humid chamber at a temperature of (37 ± 1) ° С for 60 min. The interaction of specific antibodies with fluorescent diagnosticum took place in a humid chamber. After the expiration of the time, the substrates with the preparations were removed from the humid chamber and dried in a thermostat or at room temperature (20 ± 2) ° С. The dried preparations were evaluated under an immersion objective of a fluorescence microscope (objective 100 x, eyepiece 10 x), viewing at least 10 fields of view.

7.2. Registration and interpretation of results.

The results were recorded either visually or using photographic documentation based on an assessment of the intensity of specific luminescence and the size of agglutinates, viewing at least 10 fields of view under the immersion objective of a fluorescent microscope (objective 100 x, eyepiece 10 x).

The results were assessed visually using a modified 4-cross system:

- four crosses (4+) - all viewed fields of view are filled with large and large agglutinates, containing from 30 rickettsiae or coxiella and more in agglutinate, among which there are single, separately located labeled rickettsiae or coxiella;

- three crosses (3+) - all viewed fields of view are filled with agglutinates of medium size (from 15 to 29 rickettsiae or coxiella in agglutinate), between them are smaller agglutinates and isolated labeled rickettsiae or coxiella;

- two crosses (2+) - all fields of view are filled with small agglutinates (from 5 to 14 rickettsiae or coxiella in agglutinate), and isolated labeled rickettsiae or coxiella;

- one cross (1+) - all viewed fields of view are filled with small agglutinates (from 2 to 4 rickettsiae or coxiella in agglutinate) and many isolated labeled rickettsiae or coxiella;

- minus - isolated labeled rickettsia or coxiella are observed in all the fields examined.

The highest dilution of the studied serum to rickettsiae or coxiella, at which the intensity of agglutination is estimated not lower than 3+, was taken as the serum titer in pMFag. It was taken into account that by adding an equal volume of fluorescent diagnosticum, the serum dilution is doubled.

The results of studies of sera in pMFA were documented by photographing the preparations in a fluorescent microscope and displayed on a personal computer screen and saved on a USB drive.

Example 8.

Comparative evaluation of the results of the indirect fluorescence reaction (RNIF) and the direct method of fluorescent antigens (pMFAg).

The study was carried out with sera of people who had rickettsioses of the typhus group (epidemic typhus, endemic typhus), rickettsioses of the tick-borne spotted fever group (tick-borne rickettsiosis of North Asia, Astrakhan spotted fever). coxiellosis.

The research results are summarized in Tables 1 and 2.

Results of a study of the immunofluorescent activity of fluorescent diagnostics R.prowazekii, R.typhi, R. conorii, R.sibirica, C. burnetii with sera of people who had previously had rickettsioses and coxiellosis.

As can be seen from the data presented in Table 1, fluorescent diagnostics of the typhus group and interacted in pMFag with the sera of people who had had Brill-Zinsser disease and endemic typhus in higher titers (1.3 times) than in the RNIF, which indicated a higher sensitivity. Table 1 shows the results of a study of the immunofluorescent activity of fluorescent diagnosticums R.prowazekii, R.typhi, R. conorii, R.sibirica, C. burnetii with sera of people who had previously had rickettsioses and coxiellosis.

Table 1

Test sera amount
Fluorescent diagnostics Geometric mean titer of antibodies in serological reactions pMFAg RNIF Sera from people who have had Brill-Zinsser disease 13 Fluorescent diagnosticum R.prowazekii 1: 275.4
X = 5.8 ± 0.2 1: 212.3
X = 5.42 ±
0.17 Sera from people who have had endemic (rat) typhus eleven Fluorescent
diagnosticum
R. typhi 1: 266.1
X = 5.75 ± 0.4 1: 223.9
X = 5.5 ± 0.16 Serums of people who have had Astrakhan spotted fever nine Fluorescent diagnosticum R. conorii 1: 133
X = 4.75 ± 0.5 1: 120
X = 4.6 ± 0.32 Sera from people who have had tick-borne rickettsiosis in North Asia 7 Fluorescent diagnosticum R. sibirica 1: 169.8
X = 5.1 ± 0.6 1: 158.5
X = 5.0 ± 0.12 Sera from people who have had Q fever 12 Fluorescent diagnosticum C. burnetii 1: 173 X = 5.13 ±
0.38 1: 173 X = 5.13 ±
0.38

Comparable results of immunofluorescence testing of the studied sera of patients who had Brill-Zinsser disease and endemic typhus (flea) using fluorescent diagnostics for R. also the presence of common proteins among R. typhi and R.

(Walker D.H. and Xue-jie yu (2005). Progress in rickettsial genome analysis from pioneering of rickettsia prowazekii to the recent rickettsia typhi. Ann. N.Y. Acad. Sci. 1063: 13-25.)

In the study of sera to rickettsia of the group of tick-borne spotted fever (sera of people who had had tick-borne typhus of North Asia and Astrakhan spotted fever) using fluorescent diagnostics, similar patterns were noted. Fluorescent diagnosticums R. sibirica and R. conorii reacted only with sera homologous to group sera, but did not interact with heterologous sera.

When comparing the results of immunofluorescence testing of sera from patients who have had tick-borne typhus of North Asia and Astrakhan spotted fever using fluorescent diagnostics for R. sibirica and R. conorii, a significant degree of serological affinity is shown, manifested in partial intragroup differentiation of sera from the tick-borne spotted fever group. Intra-group cross-reactions are due to the presence on the outer surface of R. sibirica and R. conorii of the total rОmpA protein, as well as cross-reactivity of LPS (Fournier P.-E., Roux V. and Raoult D. Phylogenetic analysis of spotted fever group rickettsia by study of the outer surface protein rOmpA. International Jornal of Systematic Bacteriology, 1998,48, pp. 839-849).

In the study of sera of people who had had Q fever, titers comparable to those of RNIF were established in pMFag with the fluorescent diagnosticum of Burnet's Coxiella.

Thus, it was found that fluorescent diagnostics R.prowazekii, R.typhi, R. conorii, R.sibirica, C. burnetii are sensitive and in activity they exceed or correspond to the reference drugs - RNIF, allow one-step method to identify significant for Russia rickettsioses and coxiellosis.

The specificity of fluorescent diagnostics R.prowazekii, R.typhi, R. conorii, R.sibirica, C. burnetii was assessed with rickettsial sera of different specificity, as well as sera from donors and people with various somatic diseases. The research results are shown in Table 2.

table 2

Investigation of the specificity of fluorescent diagnosticums R.prowazekii, R.typhi, R. conorii, R.sibirica, C. burnetii with human sera.

No. of pp Test blood sera and the amount of sera Geometric mean antibody titer in sera Fluoro diagnosis
ticum R.prowazekii Fluoro
diagnostic stickum
R. typhi Fluoro diagnosticum R. sibirica Fluoro-diagnosticum R.conorii Fluoro diagnosticum C. burnetii 1 Serums of people who have had vesicular rickettsiosis - 58 - - - - - 2 Sera of conventionally healthy people (donors) -38 - - - - - 3 Sera of people with various somatic diseases -41 - - - - - 4 Provaceka Standard Rickettsia Serum 1: 640 1: 320 - - - five Standard serum for rickettsia typhi 1: 160 1: 320 - - 6 Standard serum for rickettsia siberica - - 1: 160 1: 160 - 7 Conori Rickettsia Standard Serum - - 1: 160 1: 320 - 8 Burnet Coxiella Standard Serum - - - - 1: 640

From the data presented in Table 2, it follows that fluorescent diagnostics R.prowazekii, R.typhi, R.sibirica, R. conorii, C. burnetii do not react in pMPAg in a diagnostic titer (1:40) with either heterologous sera or with sera of donors and people with various somatic diseases, which indicates their high specificity.

Thus, it has been shown that the created fluorescent diagnosticums from the set: R.prowazekii or R. typhi or R.conorii or R. sibirica or C. burnetii have high sensitivity and specificity, which indicates a complete correlation with the data of the "gold standard" immunofluorescence - RNIF ( Fournier PE, Marrie TJ, Raoult D (1998) Diagnosis of Q fever. J Clin Microbiol 36: 1823-1834; van der Hoek W. et al. (2012). Epidemic Q Fever in Humans in the Netherlands. In: Toman R., Heinzen R., Samuel J., Mege JL. (Eds) Coxiella burnetii: Recent Advances and New Perspectives in Research of the Q Fever Bacterium. Advances in Experimental Medicine and Biology, vol 984, section 17.5.6. Springer , Dordrecht) and, therefore, can be applied for use in practical health care in the form of a kit for diagnosing diseases of the typhus group (epidemic (lousy) typhus and endemic (flea) typhus), tick-borne spotted fever (tick-borne ricketts) group iosis of North Asia, Astrakhan spotted fever) and coxiellosis (Q fever).

As the studies have shown, the use of the proposed fluorescent diagnostics makes it possible to detect a wide range of specific antibodies formed in the body (protein-lipopolysaccharide complex of rickettsial or coxiellosis cells), is one-stage, reduces the analysis time by speeding up manipulations and eliminating a number of analysis stages (fixing drugs for RNIF , multistage, introduction of reagents at different stages of production and removal of unreacted ingredients by washing smears), as well as an important point is the absence of induced fluorescence of the reaction.

Example 9.

The composition of the claimed set.

The kit contains the following components, packed in a box:

1.Liophilized fluorescent diagnosticum R. prowazekii - 1 ampoule in a volume of 0.5 ml;

2. Lyophilized fluorescent diagnosticum R. typhi - 1 ampoule in a volume of 0.5 ml;

3. Lyophilized fluorescent diagnosticum R. sibirica - 1 ampoule in a volume of 0.5 ml;

4. Lyophilized fluorescent diagnosticum R.conorii - 1 ampoule in a volume of 0.5 ml;

5. Lyophilized fluorescent diagnosticum C. burnetii - 1 ampoule per volume;

The formulation of pMFAag with the proposed kit for the diagnosis of rickettsioses and coxiellosis is practically available for clinical diagnostic laboratories of medical institutions and laboratories of the Rospotrebnadzor Centers, since it is technologically simple to use, demonstrative and can be documented on USB media or saved as a preparation.

All the above examples confirm that the problem posed by the present invention, namely, obtaining a set of fluorescent rickettsial and coxiella diagnostics, namely fluorescent diagnostics of rickettsiae of the typhus group (R. prowazekii, R. typhi), rickettsiae of the tick-borne spotted fever group (R. sibirica, R. conorii) and the genus coxiella (C. burnetii) based on their protein-lipopolysaccharide complex and, their use for the simultaneous one-stage serological diagnosis of rickettsioses and coxiellosis, as well as the creation of a method for serological diagnosis using the direct method of fluorescent antigens ( pMFAg) while maintaining high sensitivity and specificity due to giving a corpuscular form to the protein-lipopolysaccharide complex and increasing the number of antigenic determinants on its surface, has been resolved.

The technical result and effect is achieved through the use of fluorescent lactobacilli as a sorbent, and as an active substance of a protein-lipopolysaccharide complex, by combining the latter through a binder reagent with a fluorescent sorbent to form corpuscular forms, which leads to an increase in the specificity and sensitivity, the standard of fluorescent rickelles and diagnostics, carrying out diagnostics in the format of a one-step microimmunofluorescence reaction, reducing the time of analysis by speeding up manipulations and eliminating a number of analysis steps (fixing drugs for MFA, multistage, adding reagents at different stages of preparation and removing unreacted ingredients by washing smears), as well as increasing the specificity reactions due to the lack of induced fluorescence.

In addition, the use of the kit in the developed reaction will make it possible to carry out differential studies in mixed infections: for example, coxiellosis and Astrakhan spotted fever, tick-borne rickettsiosis of North Asia and coxiellosis, tick-borne rickettsiosis of Siberia and borreliosis (Joris Koetsveld et all. Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands.Ticks Tick Borne Dis 2016 Mar 17; 7 (2): 371-7.Epub 2015 Dec 17).

The result of solving the technical problem is also the early detection of specific antibodies in the reaction of microimmunofluorescence, the expansion of the range of studies due to the simultaneous diagnosis with one set of rickettsioses and coxiellosis, which is necessary when implementing the decisions and recommendations of the Chief Sanitary Doctor of the Russian Federation and the Minister of Health of the Russian Federation (Order of the Ministry of Health of the Russian Federation of November 26 .1998 № 312 "On strengthening measures for the prevention of epidemic typhus and the fight against pediculosis" with Appendices 1 and 2 - MU "Identification of patients with epidemic typhus or Brill-Zinsser disease, MU" Clinic, diagnosis and treatment of epidemic typhus and Brill's disease -Zinsser "; SP 3.1.7.2811-10" Prevention of coxiellosis (Q fever) ", approved by the decree of the Chief State Sanitary Doctor of the Russian Federation G.G. Onishchenko dated 28.12.2010 No. 181, registered with the Ministry of Justice of Russia on 21.03.2014 No. 20201; MUK 4.2.3115-13 "Laboratory diagnosis of community-acquired pneumonia", approved and "vve given in action by the Chief Sanitary Doctor of the Russian Federation G.G. Onishchenko on October 21, 2013; MU 3.1.2.3047-13 "Epidemiological surveillance of community-acquired pneumonia", approved and put into effect by the Chief Sanitary Doctor of the Russian Federation G.G. Onishchenko 10.01.2013).

Industrial applicability

All examples given also confirm the industrial applicability of this invention.

List of references

1. Barban PS, Pshenichnov R.A., Pantyukhina A.N., Ivshina I.B. "Immunofluorescent analysis", Sverdlovsk: Ural Branch of the Academy of Sciences of the USSR, 1988, p. 176.

2. Patent RU 2557951. Method of obtaining multiplex diagnosticum / Authors: Tarasevich IV, Pantyukhina AN, Vakstein MS, Dezhurov SV.

3. Zdrodovsky P.F., Golinevich E.M. "The doctrine of rickettsia and rickettsioses", M., 1972. 495 p.

4. Tupitsyn A.V. Timasheva O.A., Horowitz E.S. "Method of obtaining immunosorbent", Ed. certificate No. 1007676.

5. Misenzhnikov AV, Lazarevich SN, “On the issue of the preparation of hemosensitizing antigen from Siberian rickettsia”, “Topical issues of theoretical and applied immunology”, Perm, 1994. - p. 26-27.

6. Tokarevich N.K. "Biological aspects of the relationship between Burnet's coxiella and the mammalian organism as the basis for improving the diagnosis and specific prevention of Q fever", Abstract of MD, St. Petersburg, 1997, p. 46.

7. Wang L. et al. "Nanoparticles for multiplex diagnostics and imaging", Nanomedicine, 2006, V. 1, No. 4. R. 413-426.

8. Patent RU 2062110 C1 No. 2062110. Method of obtaining diagnosticum of rickettsial Provachek, corpuscular / Authors: Petrov V.F., Pantyukhina A.N., Aleksandrova L.V., application No. 93039399/14, 1993.08.03.

9. Application for the grant of patent RU No. 94020953 "Method for obtaining diagnosticum of rickettsial Siberian, corpuscular" Pantyukhina A.N., Aleksandrova L.V., Petrov V.F., Tarasevich I.V., Yablonskaya V.A.,

10. Fournier PE, Marrie TJ, Raoult D, 1998. "Diagnosis of Q fever. J Clin Microbiol, 36: 1823-1834.

11. Van der Hoek W. et al., 2012, "Epidemic Q Fever in Humans in the Netherlands". Toman R, Heinzen R, Samuel J, Mege JL. (eds) Coxiella burnetii: Recent Advances and New Perspectives in Research of the Q Fever Bacterium. Advances in Experimental Medicine and Biology, vol 984, section 17.5.6 Springer, Dordrecht.

12. Walker D.H. and Xue-jie, 2005, "Progress in rickettsial genome analysis from pioneering of rickettsia prowazekii to the recent rickettsia typhi", Ann. N.Y. Acad. Sci. 1063, 13-25.

13. Fournier P. E., Roux V. and Raoult D., 1998, "Phylogenetic analysis of spotted fever group rickettsia by study of the outer surface protein rOmpA," International. Journal of Systematic Bacteriology, 48, 839-849.

14. Joris Koetsveld et all, "Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands" Ticks Tick Borne Dis, 2016, Mar 17; 7 (2): 371-7. Epub, 2015, Dec 17.

15. Order of the Ministry of Health of the Russian Federation of 11/26/1998 No. 312 "On strengthening measures to prevent epidemic typhus and combating head lice" with Appendices 1 and 2 - MU "Identification of patients with epidemic typhus or Brill-Zinsser disease", MU "Clinic , diagnosis and treatment of epidemic typhus and Brill-Zinsser disease ”.

16. SP 3.1.7.2811-10 "Prevention of coxiellosis (Q fever)", approved by the decree of the Chief State Sanitary Doctor of the Russian Federation G.G. Onishchenko dated 28.12.2010. No. 181, registered with the Ministry of Justice of Russia, 21.03.2014, No. 20201.

17. MUK 4.2.3115-13, "Laboratory diagnostics of community-acquired pneumonia", approved and "put into effect by the Chief Sanitary Doctor of the Russian Federation G.G. Onishchenko, 21.10.2013

18. MU 3.1.2.3047-13, "Epidemiological surveillance of community-acquired pneumonia", approved and put into effect by the Chief Sanitary Doctor of the Russian Federation G.G. Onishchenko, 10.01.2013

19. MU 3.1.1755-03, “Organization of epidemiological surveillance of tick-borne rickettsiosis. Prevention of infectious diseases "

Date of introduction 2003-09-28, approved and put into effect by the Chief State Sanitary Doctor of the Russian Federation - First Deputy Minister of Health of the Russian Federation G.G. Onishchenko, September 28, 2003

Claims (9)

1. A method of obtaining a set of fluorescent rickettsial and coxiellosis diagnostics, including the preparation of a fluorescent sorbent by purifying formalinized lactobacilli from the components of a protective drying medium, followed by labeling them with inorganic fluorochromes, then obtaining protein-lipopolysaccharide complexes from corpuscular antigens of the rickets syphilis group genus Coxiella by isolating active protein-lipopolysaccharide complexes from them by combined methods of ultrasonic disintegration of antigen corpuscles and chromatography, after which the isolated protein-lipopolysaccharide complexes are combined with a fluorescent sorbent by means of a binder reagent and the final product is obtained - a set of fluorescent rickettsial and coxyellosis diagnostics. 2. The method according to claim 1, characterized in that the protein-lipopolysaccharide complexes are obtained from corpuscular antigens of the rickettsia of the typhus group - R.prowazekii or R. Typhi; or the R. sibirica or R. conorii tick-borne spotted fever group; or the genus Coxiella - C. burnetii. 3. The method according to claim 1, characterized in that lactobacilli with a concentration of at least 1 x 109 cells / ml, labeled with inorganic fluorochromes, are used as the fluorescent sorbent. 4. The method according to claim 1, characterized in that tannin or water-soluble carbodiamide in a volume ratio of the fluorescent sorbent and a solution of tannin or carbodiimide 1: 1 is used as a binding agent of the fluorescent sorbent with the protein-lipopolysaccharide complex. 5. The method according to claim 1, characterized in that the fluorescent sorbent with a binder is mixed with a solution of protein-lipopolysaccharide complexes with a protein concentration of at least 0.5 mg / ml at a volume ratio of 1: 1. 6. A method according to claim 1, characterized in that the sorbent is incubated with a fluorescent protein-lipopolysaccharide complex for 1 hour at 43 ° ^C. 7. The method according to claim 1, characterized in that each fluorescent rickettsial and coxiellosis diagnosticum from the kit is a freeze-dried suspension of fluorescent lactobacilli from a volume of 0.5 ml, covalently linked by means of a binding reagent to protein-lipopolysaccharide complexes of rickettsia of the typhus R group. prowazekii, or R. typhi, or rickettsia of the group of tick-borne spotted fever R. sibirica, or R. conorii, or the genus Coxiella - C. burnetii. 8. The use of a set of fluorescent rickettsial and coxiella diagnostics obtained by the method according to claim 1. for serological diagnosis of rickettsiosis and coxiellosis for the detection of diseases of the typhus group - epidemic lousy typhus and endemic flea typhus, tick-borne spotted fever group - tick-borne rickettsiosis of North Asia or Astrakhan spotted fever and the genus of seroxymellus, and Q fever for the population in the population endemic foci. 9. A method for serological diagnosis of rickettsial infections and coxiellosis using a set of fluorescent rickettsial and coxiellosis diagnostics according to claim 1, which consists in the fact that the reaction of fluorescent diagnostics and tested sera is performed in the format of a direct method, on glass slides with wells located in two horizontal rows , while in all wells of the first horizontal row, the test serum is introduced in a diagnostic titer with a dilution of 1:40, and purified water is added to all wells of the second row, then sequentially into the vertical rows of both horizontal rows into the serum of the first horizontal row and purified water of the second horizontal row make created fluorescent diagnostics, and in the 1st horizontal row - R.prowazekii, in the 2nd - R. typhi, in the third -R. sibirica, the fourth - R.conorii and fifth - C.burnetii, wherein the interaction of antigens with antibodies diagnostics test serum is carried out by stirring the substrate and the tapping of the further placement in a moist chamber at 37 ^{° C} for 60 minutes, then dried smears and browsing under an immersion lens of a fluorescence microscope, the results are assessed using a four-cross system and documentation is carried out by saving slides with smears or saving a photo from a microscope on a usb-carrier, if a positive reaction is detected in the wells, a semi-quantitative analysis is performed using a fluorescent diagnosticum, with which the serum showed positive reaction, while in the wells of the slides, a two-fold sequential dilution of the test serum is introduced from 1:20 to 1: 1280, starting with the maximum, then this fluorescent diagnosticum is added to the wells of the slides, then, as in the first experiment, the interaction of antigen s fluorescent diagnostics antibody assayed is carried out with stirring by tapping the substrate slide and further placement in a moist chamber at 37 ^{° C} for 60 min, then smears were dried and viewed in the immersion lens of the fluorescent microscope, the evaluation results are also conducted by chetyrehkrestovoy system, documentation is carried out also by saving slides with smears or saving a photo from a microscope on a USB-carrier.

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