RU2769578C1 - Method for producing a fluorescent immunoglobulin diagnostic agent for detecting pathogens of rickettsioses and coxielloses, fluorescent immunoglobulin diagnostic agent and application thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and medical microbiology. Described is a method for producing a fluorescent immunoglobulin diagnostic agent for detecting pathogens of rickettsioses and coxielloses, consisting in preparing a fluorescent matrix by applying a fluorescent label to the matrix, then attaching analytes thereto by means of a binding reagent; according to the invention, formalinised lactic bacteria are used as a matrix, and inorganic fluorochromes are used as a fluorescent label, wherein an antibody-containing fraction of immunoglobulins to rickettsiae and coxiellae in the range of 0.1 to 0.5 mg/ml is used as analytes, combined by means of a binding reagent with the fluorescent matrix, resulting in the final product, a fluorescent immunoglobulin diagnostic agent to rickettsiae and coxiellae, wherein the antibody-containing fraction of immunoglobulins is produced from corresponding immune serums containing antibodies to rickettsiae and coxiellae by means of isolation thereof by combined methods: deposition with neutral salts, gel filtration, removal of cross-reacting and heterologous antibodies. Also described is an associated fluorescent immunoglobulin diagnostic agent for detecting pathogens of rickettsioses and coxielloses and an application thereof for testing samples in order to identify pathogens of rickettsioses and coxielloses.

EFFECT: invention is used for specific assessment of the activity of vaccines and for testing objects of epidemiological examination, being infected organs and tissues of animals, or samples of infected external environment, or carriers.

8 cl, 10 dwg, 3 tbl, 14 ex

Description

Technical field

The invention relates to the field of biotechnology and medical microbiology and relates to a method for obtaining fluorescent immunoglobulin diagnosticums to R.prowazekii or R.typhi, or C.burnetii phase I or C.burnetii phase II for the detection and identification of pathogens of epidemic lousy typhus or rat typhus , or coxiellosis in materials of various biological and non-biological origin.

Prior Art

The etiological agents of epidemic or lousy typhus and Brill-Zinsser disease are R.prowazekii, the causative agent of rat endemic flea typhus is R.typhi, and the causative agent of coxiellosis is C.burnetii phase I or C.burnetii phase II.

Initially, these pathogens were classified on the basis of morphobiological and immunological characters as representatives of Rickettsiaceae, order Rickettsiales, genus Rickettsia.

Dumler S., Walker DH, Class I. Order II. Rickettsiales Gieszczykiewicz, 1939, 25 ^AL emend.

Dumler, Barbet, Bekker, Dasch, Palmer, Ray. Rikihisa and Rurangirwa. 2001, 2156. Bergey' ^, s. Manual of Systematic ^Bacteriology , 2nd ed., Vol 2. The Proteobacteria. Pt C: The alfa-, beta-, delta-, and epsilonproteobacteria, eds Brenner DJ, Krieg NR, Stanley JT, Garrity GM New York, Springer, 2005, P.96.

Later, based on phenotypic characteristics in the analysis of the 16S ribosomal RNA gene, the classification of rickettsiae was revised. As a result of the analysis, the pathogen C.burnetii was moved from the family Rickettsiaceae to the family Coxillaceae, order Legionellales, class Gammaproteobacteria, phylum Proteobacteria, genus Coxiella. However, rickettsiologists are still studying coxiellosis.

Thompson C.C. et al. Microbial genetic taxonomy. BMC Genomics, 2013, 14, P.913.

All three pathogens belong to the II group of pathogenicity in accordance with the Decree of the Chief State Sanitary Doctor of the Russian Federation dated November 28, 2013 N 64 "On approval of the sanitary and epidemiological rules SP 1.3.3118-13. Safety of working with microorganisms of I - II pathogenicity groups (hazards )".

R.prowazekii and R.typhi are similar in morphological, biological, genetic, antigenic and immunological properties. This kind of proximity of pathogens is also reflected in the similarity of the clinical manifestations of diseases, which makes it difficult and practically impossible to clinically differentiate these diseases without appropriate epidemiological data and additional research methods, such as: biological samples or immunological studies to identify pathogens and their antigens.

The need to differentiate these pathogens is important from an epidemiological point of view, since anti-epidemic measures for these infections are fundamentally different.

The causative agent of epidemic typhus circulates in the human-louse-human chain and, therefore, anti-epidemic measures are aimed at identifying and eliminating infected R.prowazekii lice and emergency hospitalization of patients with typhus.

The circulation of the pathogen in rat typhus is carried out in the chain rat (mouse) - flea - rat (mouse). Proceeding from this, prevention consists in deratization and disinfestation in rooms inhabited by R.typhi-infected rats and their infected ectoparasites.

In natural foci, wild animals, birds and ticks parasitizing them are naturally infected with the causative agent of coxiellosis - C.burnetii. Such polyadaptation of C.burnetii contributes to the involvement of domestic animals in the epidemiological process, which are one of the main sources of human infection. For diagnostics, the phase variability of coxiella, which differ mainly in antigenic properties, is of particular importance. Under natural conditions, coxiella are in phase I, and with long-term cultivation in laboratory conditions in a different habitat (chicken embryos, cell culture), coxiella pass into phase II.

The most reliable method for diagnosing epidemic typhus, Brill-Zinsser disease, flea typhus, coxiellosis is the detection of the infectious agent and its identification.

For this purpose, the following microbiological and immunological methods have been and are being used: the classical method of biological assays (bioassays) with subsequent identification by serological methods, the immunofluorescence method (MFA), the indirect hemagglutination reaction (IHA), the complement fixation reaction (CFR), enzyme-linked immunosorbent assay (ELISA) .

The choice of a serological method for the detection of rickettsiae is determined by such factors as specificity, sensitivity, rapidity.

Classical bioassay method.

Accurate recognition of the pathogens of epidemic typhus and rat flea typhus and coxiellosis is ensured by isolating the pathogen from sick people or animals, or their ectoparasites using the classical bioassay method, followed by identification of pathogens.

For this purpose, in patients, if possible in the early period of infection, blood is taken from a vein or suspensions of ectoparasites are used and injected into the abdominal cavity of male guinea pigs at a dose of 3-5 ml. The appearance of fever in guinea pigs, accompanied by a scrotal reaction in the presence of cells inhabited by pathogens in scrapings from the vaginal membranes of the testicles, indicates the presence of a disease of rat typhus or epidemic typhus, or coxiellosis. To clarify the pathogen, pathogens are isolated from the organs and tissues of animals, followed by identification by serological methods using RA, RSK or MFA.

The bioassay method is a highly sensitive, specific and reliable test.

The disadvantages of the method include the duration of observation of animals (on average 10 days), the autopsy of animals to extract biological material, followed by identification of the pathogen by serological methods.

Currently, the bioassay method has not lost its diagnostic value and is used in specialized laboratories to identify known and newly emerging infections of rickettsial etiology.

Zdrodovsky P.F., Golinevich E.M. The doctrine of rickettsiae and rickettsiosis. M., Medicine, 1972, p.495.

Methods of immunofluorescence.

The method of immunofluorescence (MFA) is used both for scientific purposes to obtain data on the morphology, reproduction, antigenic specificity and intracellular parasitism of rickettsiae and coxiella, and in laboratory practice to detect rickettsiae and coxiella in different substrates.

MFA has high sensitivity, specificity, expressiveness and reproducibility. Allows for direct identification of corpuscles of rickettsia and coxiella in mixed materials: in tissues, cells and their derivatives, secrets and excretions, and other materials.

The disadvantages of MFA are associated with induced nonspecific (nonspecific adsorption of the conjugate) and specific (presence of heterologous antibodies or cross-reacting antibodies in the conjugates) fluorescence, as well as the impossibility of detecting soluble antigens.

To detect rickettsiae and their antigens, direct and indirect variants of MFA are used.

Direct fluorescent antibody method.

The direct method of fluorescent antibodies (pMFA) is based on the specific interaction of labeled antibodies directly with a homologous antigen fixed on a glass slide.

The preparation of fluorescent antibodies is a freeze-dried fraction of hyperimmune serum immunoglobulins, to which a fluorochrome (for example, fluorescein isothiocyanate) is chemically attached.

Direct processing of smears-prints from infected objects of epidemiological examination (carriers, samples of the infected environment), as well as living biological systems (smear-prints of infected organs and tissues, tissue culture cells, chicken embryos) allows you to quickly detect the causative agent of rickettsiosis and coxiellosis.

The method is specific, methodologically simple, less labor-intensive and the most economical in comparison with indirect modifications.

Barban P.S., 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.

The sensitivity of pMFA is lower than in the indirect modification of MFA. The detectability of the pathogen and its corpuscular antigens ranges from 100 - 500×10 ³ - cells per 1 ml.

Goldin R.B. Immunoluminescent analysis in the study and diagnosis of viral infections and rickettsiosis. Immunoluminescence in medicine. 1977, M., Medicine, S. 80-144.).

The disadvantages of pMFA include induced nonspecific fluorescence of heterologous structures of the mixed material of the object of study (preparation background) caused by fluorescent antibodies. Therefore, to detect rickettsia and coxiella in materials of various biological and non-biological origin, fluorescent immunoglobulins must be used in combination with a contrast agent - bovine albumin labeled with rhodamine.

Noskov F.S., Boldasov V.K., Goldin R.B., Ermakov N.V., Volkova L.A. Contrast method for immunofluorescent detection of adenoviruses in guinea pig kidney cell culture. Question. Virol., 1965, No. 5, S. 613-618.

Smith C., Marshall G., Eveland W. Use of contrasting fluorescent dyed as counterstrain in fixed tissue preparation. Proc.Soc.Exp.Biol. Med., 1958, V.102, P. 179-181.

In addition, for the use of pMPA, it is necessary to have fluorescent antibodies to each type of antigen, which limits the use of pMPA.

It should be noted that the use of fluorescent immunoglobulins of the typhus group (group antibodies) or the genus Coxiella does not allow differentiation of R.prowazekii, R.typhi, C.burnetii phase I and C.burnetii phase II due to the presence of cross-reacting and group-specific antibodies in preparation.

This implies the need to prepare species-specific fluorescent antibodies, which will increase the sensitivity and specificity of the method.

Indirect method of fluorescent antibodies (antigen detection).

The indirect method of fluorescent antibodies (nMFA) is based on the specific interaction of a homologous antigen fixed on a glass slide with complementary immunoglobulins of unlabeled immune serum, which then specifically reacts with fluorescent anti-species immunoglobulins.

It was noted that when using nMFA, it is sufficient to have only a few preparations of anti-species fluorescent antibodies (against human, mouse, rabbit, etc. immunoglobulins), but, as before, there is a need to have diagnostic unlabeled immune sera for each desired antigen.

The advantages of nMFA include its versatility, since with its help it is possible not only to identify the desired antigens, but also to identify and titrate complementary antibodies to it. According to the literature, nMPA is much (10-12 times) more sensitive than pMPA when detecting an antigen.

In contrast to direct MFA, indirect MFA is three-component, two-stage, more laborious, less economical, and requires more time to conduct the study.

The disadvantage of nMFA is the increase in the number of diagnostic errors in the detection of antigens in biological objects. This is caused by nonspecific sorption of the fluorescent conjugate on denatured cells, detritus, and other organic impurities in objects of biological and nonbiological material (induced nonspecific fluorescence). To level this fluorescence, it is necessary to apply another stage - contrasting with rhodamine-labeled bovine albumin.

A negative point is the presence on the outer membrane of rickettsial cells of antigenic structures that cross-react with common antigenic epitopes of other pathogens of the rickettsiosis group, as well as the presence of antigens that are common with the antigenic structures of other microbes, such as: Proteus OX ₁₉ , Proteus OX _2, which react with homologous immune sera. These reactions with heterologous antibodies further complicate the interpretation of specific staining.

To eliminate cross-reactions, it is necessary to use specific antibodies free from cross-reacting and heterologous antibodies, as well as highly specific fluorescent conjugates, as immune sera.

Goldin R.B. Immunoluminescent analysis in the study and diagnosis of viral infections and rickettsiosis. Immunoluminescence in medicine. 1977, M., Medicine, C 80-144.

RNHA with immunoglobulin erythrocyte diagnosticum

RIHA with immunoglobulin erythrocyte diagnosticum (IED) is based on the agglutination of specific rickettsial or coxiella antibodies adsorbed on the surface of ram erythrocytes in the presence of homologous antigens. With the use of IED according to the standard method, it became possible to quickly detect pathogens and their antigens in various substrates.

The reaction is one-stage, two-component, technologically simple to set up, economical, and the stability of the diagnostics used ensures reproducibility of the results. Its main advantage is its high sensitivity and, according to this indicator, it is close to enzyme immunoassay and radiological methods. Threshold sensitivity of RNHA with immunoglobulin erythrocyte diagnosticum (IED) in the detection of antigens corresponds to 5×10 ⁴ - 1×10 ⁶ microbial bodies in 1 ml.

Levina E.N. Immunofluorescence method in bacteriology. Immunoluminescence in medicine. 1977, M., Medicine, S. 46-71.

Barban P.S., Misenzhnikov A.V. A comparative study of some modern methods for the rapid detection of Prowacek's rickettsiae in the vector. Microbiology. 1974, No. 2, pp. 17-20.

The disadvantage of the reaction is the appearance of non-specific reactions due to the interaction of cross-reacting antibodies and heterologous sensitin antibodies with other group antigens and antigen substrate proteins.

In order to eliminate non-specific reactions for sensitization of erythrocytes, pure or adsorbed specific antibodies with a high binding constant should be used.

Enzyme-linked immunosorbent assay for antigen detection (ELISA).

The method is based on the specific interaction of an antigen immobilized on a carrier with a homologous antibody, followed by its binding to anti-species antibodies conjugated with an enzyme, the latter, as a result of reaction with the corresponding chromogenic substrate, form a colored product, the amount of which is determined spectrophotometrically.

ELISA is a three-stage and multicomponent method. The wide application of the method is due to its ability to study a significant number of sera and automatic registration of results.

Shpynov S.N. Development of an enzyme immunoassay test system for the detection of antigens of rickettsia of the tick-borne spotted fever group and the rationale for its use in the system of epidemiological surveillance of tick-borne rickettsiosis, Abstract of the dissertation for the degree of candidate of medical sciences, Omsk, 1999. P. 1-23.

The method is distinguished by sufficient sensitivity - usually the presence of a soluble antigen at a concentration of 1 ng / ml is sufficient.

Egorov A.M., Osipov A.P., Dzantiev B.B., E.M. Gavrilova. Theory and practice of enzyme immunoassay. M., Higher School, 1991. S. 288.

The disadvantages of ELISA include the ambiguous sorption capacity of the plates, insufficient capacity of non-porous materials, the presence of cross-reacting and heterologous antibodies in immune sera, and the possibility of elution of part of the adsorbed antigens from the carrier during washings, as a result of which the sensitivity, accuracy, and reproduction of the analysis results deteriorate.

Immunochromatographic indicator element (IE).

The IE was built in the sandwich format using a multimembrane composite consisting of an analytical membrane with attached substrates of various types. To conduct studies in the analytical zone, a solution of specific antibodies to Burnet's rickettsia, labeled with gold sol nanoparticles, was applied to the membrane, and goat antibodies against rabbit immunoglobulins were applied to the control zone. Then the multimembrane composite was placed in a plastic frame with round holes for sample application and a rectangular hole for reading the reaction results. A suspension of Burnet's rickettsiae was applied to a substrate to absorb the sample, which, due to capillary forces, washed away the specific conjugate from the conjugate substrate. In this case, Burnet's rickettsiae interacted with labeled specific antibodies to form an immune complex, which was retained on the analytical membrane and appeared as a cherry-red line. The unbound immune complex and conjugate moved along the analytical membrane to the control zone, where a stained immune complex of anti-species antibodies with the conjugate was formed. If Burnet's rickettsiae were absent in the sample, then the colored line did not form in the analytical zone.

As a result of the studies, IEs were developed to detect Burnet's rickettsiae with a sensitivity of 1×10 ⁶ mc/ml and an analysis time of 20 minutes. IE is also specific for Provachek's rickettsiae, Provachek's rickettsia antigen complexes, and Sibirik's rickettsiae at a concentration of 8 AU/ml. The developed IEs can be used for express indication of Burnet's rickettsiae at various stages of laboratory research.

The registration of the analytical effect was carried out both visually and with the help of the domestic video-digital analyzer of immunochromatograms "Reflecom" (LLC "Sinteco-Complex"), included in the state register of measuring instruments of Russia. The device registered the staining of the analytical and control zones of IE in arbitrary units. Measurements for each concentration of microbial cells and negative controls were carried out five times, the arithmetic mean was calculated and the measurement error was determined using Student's coefficients (tp=2.78 at n=5) with 95% reliability.

Basharova L.A., Yarkov S.I., Tretyakov S.I., Zlobin V.N. Creation of indicator immunochromatographic elements for the detection of Burnet's rickettsiae. Problems of especially dangerous infections, 2013, No. 4, 80, pp. 79-81.

Advantages of the method: short analysis time, no additional technological operations, visual and/or instrumental registration, which allow us to consider the immunochromatographic method for detecting Burnet's rickettsiae as one of the options for detecting these microorganisms in the field, when monitoring environmental objects.

The limiting factors for the application of this method in practical laboratories are the presence of heterologous antibodies to the culture medium in fluorescent immunoglobulins, the detection of only corpuscular antigens, the expensive fluorochrome - colloidal gold nanoparticles, the complexity of setting up and taking into account the results of the IE reaction in instrumental research.

Fluorescent Resonant Energy Transfer.

(Fluorescence Resonance Energy Transfer (FRET)).

Rapid, highly sensitive and selective detection of pathogenic bacteria is essential for correct diagnosis and treatment. Most current bacterial detection methods are labor intensive and can only detect one bacterial pathogen in one assay. A method has been developed for sensitive multiplex monitoring of bacterial pathogens for 30 min using multicolor FRET (fluorescence resonance energy transfer) silica NPs (nanoparticles). By varying the ratio of three tandem dyes coencapsulated into NPs, NPs were obtained that emit unique colors when excited at a single wavelength. When these NPS were conjugated with monoclonal antibodies specific for pathogenic bacteria and then incubated with small concentrations of bacteria, simultaneous and sensitive detection of multiple bacterial targets was achieved.

The disadvantage of the method is the difficulty of obtaining labeled nanoparticles of different fluorescence colors, the use of expensive equipment and reagents (monoclonal antibodies).

Wang L., Zhao Wenjun, O'Donoghue M., Tan W. Fluorescent nanoparticles for multiplexed bacteria monitoring. Bioconjug Chem Mart-Apr 2007, 18(2). S.297-301.

Multiplex analysis of ELISA+MFA.

Closest to the proposed method in terms of technological essence is ELISA+MFA in the format of multiplex analysis.

With this type of ELISA + MFA, organic polymers or inorganic materials (magnoimmunosorbents, polystyrene

microspheres, microballoons, latexes, etc.) are used as a matrix for adsorption of analytes (antigens, antibodies, synthetic peptides, oligonucleotides, etc.).

The method is carried out as follows: stained with fluorophores, for example, polystyrene microspheres 5.6 μm; then binder analytes (oligonucleoproteins, antibodies, peptides, etc.) are attached; add suspensions of microspheres to the studied samples (plasma, serum, blood, etc.); add a detecting agent and a fluorescent label (a mixture of streptovidin and phycoerythrin). During measurement in the flow cell of the Luminex analyzer in the flowing liquid, each microsphere is exposed to irradiation with two lasers (green and red) with different wavelengths and the fluorescent 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 presence of heterologous antibodies in the preparations, the need to create a research program and long-term interpretation of the results, the use of special expensive equipment and hard-to-reach polystyrene microplates, the multi-stage method, as well as the loss of time for sensitization of microspheres, the introduction of reagents. This method is highly sensitive, high-tech, but suitable only for large specialized epidemiological centers, and not for clinical laboratory practice. An important limiting point is also the presence of cross-reactive and heterologous antibodies in the preparations.

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

Disclosure of invention

The objective of the present invention is to obtain a group of fluorescent immunoglobulin diagnosticums, namely a fluorescent immunoglobulin diagnosticum for R.prowazekii or a fluorescent immunoglobulin diagnosticum for R.typhi, or a fluorescent immunoglobulin diagnosticum for C.burnetii phase I, or a fluorescent immunoglobulin diagnosticum for C.burnetii phase II, the use which ensures the detection and differentiation of pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II in the test samples while maintaining high sensitivity and specificity.

The technical result is achieved due to the fact that a method has been created for obtaining a group of fluorescent immunoglobulin diagnosticums for differentiating pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II, which consists in preparing a fluorescent matrix by applying onto the matrix of a fluorescent label, then analytes are attached to it using a binding reagent, characterized in that formalinized lactobacilli with a concentration of at least 1 × 10 ⁹ cells/ml are used as a matrix, and inorganic fluorochromes are used as a fluorescent label, while analytes use antibody-containing immunoglobulins R.prowazekii, or R.typhi, or C.burnetii phase I, or C.burnetii phase II, which are connected by means of a binding reagent with each fluorescent matrix and receive end products - fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi, or to C.burnetii phase I, or to C.burneti i phase II, while antibody-containing immunoglobulins are obtained from the corresponding immune sera R.prowazekii or R.typhi, C.burnetii phase I, or C.burnetii phase II by their isolation by combined methods: precipitation with neutral salts, gel filtration, removal of cross- reacting and heterologous antibodies, while for each newly isolated series of antibody-containing immunoglobulins, before they are combined with a fluorescent matrix, the optimal sensitizing dose is empirically selected in the range of 0.1-0.5 mg/ml. Moreover, as a binding agent of the fluorescent matrix with antibody-containing immunoglobulins, tannin is used in the volume ratio of the fluorescent matrix and tannin solution 1:1, or a solution of water-soluble carbodiimide is used in the volume ratio of the fluorescent matrix and water-soluble carbodiamide solution 1:1. As inorganic fluorochromes, fluorescein isothiocyanate or nanocrystals in the form of semiconductor colloidal particles of different fluorescence are used. In this case, the removal of cross-reacting and heterologous antibodies is carried out with fluorescent antigenic diagnosticums to R.prowazekii or to R.typhi or to C.burnetii phase I, or to C.burnetii phase II. The peculiarity of the method lies in the fact that each fluorescent matrix after connection with the corresponding antibody-containing immunoglobulins is incubated for 60 minutes at a temperature of 37°C. At the same time, each fluorescent immunoglobulin diagnosticum from the group is a 1% suspension of fluorescent formalized lactobacilli covalently linked by a binding reagent with antibody-containing immunoglobulins R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II.

Created fluorescent immunoglobulin diagnosticums are used to differentiate the pathogens R.prowazekii or R.typhi, or C.burnetii of phase I, or C.burnetii of phase II, which is necessary for a specific assessment of the activity of vaccines, as well as for studying the objects of epidemiological examination, which are infected organs and animal tissues, or samples of the infected environment, or carriers of epidemic lousy typhus, or endemic flea typhus, or Q fever in the reaction of the interaction of the studied pathogens with fluorescent immunoglobulin diagnosticums to R. prowazekii or to R. typhi, or to C. burnetii I phase, or to C.burnetii II phase.

At the same time, the method for differentiating pathogens R.prowazekii or R.typhi, or C.burnetii of the I phase, or C.burnetii of the II phase, consists in setting up the reaction of the interaction of pathogens in the test samples with fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi, or C. burnetii phase I or C. burnetii phase II and further analysis and reporting of results using detection equipment. At the same time, the method for differentiating pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II is that the interaction reaction of pathogens in the test samples with fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi , or C.burnetii phase I, or C.burnetii phase II is carried out as a fluorescent microagglutination reaction, while on 4 glass slides with 8 wells each, located on the substrates, test samples are added to seven wells according to p 9., and in the 8th well - purified water, then the created fluorescent immunoglobulin diagnosticum for R. prowazekii according to paragraphs is added to all wells of the first glass. 1 and 7, and in all wells of the 2nd glass slide - created fluorescent immunoglobulin diagnosticum to R.typhi according to paragraphs. 1 and 7, in all wells of the 3rd glass slide - the created fluorescent immunoglobulin diagnosticum for C.burnetii phase I according to paragraphs. 1 and 7, in all wells of the 4th glass slide - the created fluorescent immunoglobulin diagnosticum for C.burnetii phase II according to paragraphs. 1 and 7, then the reaction ingredients are mixed by tapping on the substrate, after which the substrates with glasses are placed in a humid chamber at a temperature of 37°C for 60 minutes, then the slides are dried and analyzed using detection equipment, which is an immersion objective of a fluorescent microscope , while the results are evaluated according to a two-cross system, after which a positive reaction is observed on which of the four slides with fluorescent immunoglobulin diagnosticums and with the test samples, thus determining which pathogen R.prowazekii or R.typhi, or C.burnetii I phase, or C. burnetii phase II is in the test sample, while documentation is carried out by saving slides with contents or by saving a photo from a microscope on a USB drive.

Another way of differentiation is that the reaction of the interaction of the studied samples with fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi, or to C.burnetii phase I, or to C.burnetii phase II is carried out as a reaction of the interaction of antibody-containing immunoglobulins R.prowazekii or R.typhi, or C.burneti phase I, or C.burneti phase II, immobilized in the wells of the plates, with homologous pathogens of the test samples, followed by detection of attached pathogens using fluorescent immunoglobulin diagnosticums, as indicator reagents, followed by registration of the reaction results using detecting devices, for which strips of collapsible plates for immunofluorimetric analysis are used, in which antibody-containing immunoglobulins of R.prowazekii or R.typhi or C.burneti phase I or C.burneti phase II are immobilized, and to control fluorescent immunoglobulin diagnosticums according to paragraphs. 1 and 7, pure strips are used for spontaneous agglutination, and 5 strips of 8 holes are used for the reaction, in four of which, from the 1st to the 8th, antibody-containing immunoglobulins R.prowazekii, R.typhi are sequentially immobilized; C.burneti phase I, C.burneti phase II, and the 5th control strip is used clean, then test samples are added to all wells from the 1st to the 4th of each of the 7 horizontal rows of strips in series, that is, 7 test samples, and pathogens R.prowazekii, R.typhi are introduced into the wells of the 8th horizontal row of 4 strips sequentially from the 1st hole to the 4th one; C.burneti phase I, C.burneti phase II, while purified water is added to all wells from the 1st to the 8th of the 5th strip, after which the tablet with strips is closed with a lid and placed in a thermostat at a temperature of 37 ° C for 60 min with periodic stirring and after this time, the plate with strips is washed, then sequentially fluorescent immunoglobulin diagnosticums for R.prowazekii, for R.typhi, for C.burnetii phase I are introduced into all wells of the strips from the 1st to the 4th, to C.burneti phase II, and in every two wells of the 5th control strip, fluorescent immunoglobulin diagnosticums are introduced sequentially to R.prowazekii, then fluorescent immunoglobulin diagnosticums to R.typhi are added to the next two wells, then to C.burnetii I phase, and then to C.burneti phase II, after which the tablet with strips is closed with a lid and placed in a thermostat at a temperature of 37 ° C for 60 minutes with occasional stirring, then the tablet is washed, dried and analyzed, while fluorescence occurs in that l unke, which contains the pathogen R.prowazekii, or R.typhi; or C.burneti phase I, or C.burneti phase II, in the test sample, and fluorescence appears in the well of the strip, and the luminescence intensity is detected and recorded using detecting equipment, which is a special hardware-software complex-fluorimeter.

Another way to differentiate pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II is that the reaction of the interaction of the studied samples with fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi, or to C.burnetii of phase I, or to C.burnetii of phase II is carried out as a reaction of interaction of molecular probes - antibody-containing immunoglobulins of R.prowazekii or R.typhi, or C.burneti of phase I, or C.burneti of phase II, immobilized in gel microcells of biochips , with homologous pathogens of the test sample and with subsequent detection and registration of the resulting complex using fluorescent immunoglobulin diagnosticums as indicator reagents, followed by registration of the reaction results using detection devices, for which the required number of biochips with gel microcells is taken, while the number of necessary gel microcells is determined the number of molecular probes, and as well as the number of fluorescent immunoglobulin diagnosticums used, with each gel microcell containing immobilized intramolecular probes - antibody-containing immunoglobulins R.prowazekii or R.typhi, or C.burneti phase I, or C.burneti phase II, and 8 biochips are selected for the study of one sample , carry out the introduction of the test sample on the biochips, and the reaction on the biochips is carried out in 2 stages: first, on the gel microcells of biochips 1, 2, 3, 4, inside which are immobilized antibody-containing R.prowazekii or R.typhi, or C.burneti I immunoglobulins phase, or C.burneti phase II, the test sample is applied, and on the control gel microcells of biochips 5, 6, 7, 8, inside which there are immobilized antibody-containing immunoglobulins R.prowazekii or R.typhi, or C.burneti phase I, or C. burneti phase II, apply - a solution of sodium chloride, after which the biochips are placed in a humid chamber and incubated for 60 minutes at a temperature temperature of 37°C, after this time, the biochips are washed with distilled water three times for 1 min and dried, while at the second stage, fluorescent immunoglobulin diagnosticums according to paragraphs 1 and 7, namely: in all gel microcells of biochips 1 and 5 fluorescent immunoglobulin diagnosticums to R.prowazekii; - in all gel microcells of biochips 2 and 6 - fluorescent immunoglobulin diagnosticums for R.typhi; in all gel microwells of biochips 3 and 7 - fluorescent immunoglobulin diagnosticums to C.burneti phase I, in all gel microwells of biochips 4 and 8 fluorescent immunoglobulin diagnosticums of C.burneti phase II, then all biochips after the introduction of fluorescent immunoglobulin diagnosticums are placed in a humid chamber and incubated for 60 min at a temperature of 37°C, after which the biochips are washed and dried, while in the presence of pathogens R.prowazekii or R.typhi, or C.burneti phase I, or C.burneti phase II in the test sample, they interact with corresponding fluorescent immunoglobulin diagnosticums and antibody-containing immunoglobulins in the probes, after which fluorescence appears in the gel microcell of the biochip, while the intensity of the luminescence of the gel microcells of the biochip is detected and recorded using detecting devices, which are special hardware-software complexes of analyzers, thus obtaining data on the presence in the test sample of homologous pathogens, and when using biochips, one test sample is tested for several pathogens simultaneously, with each subsequent sample being examined in a similar way.

Brief description of the figures

Figure 1

a scheme for obtaining a fluorescent matrix is presented

A - matrix - formalized lactobacilli.

B - fluorescent label.

C - fluorescent matrix.

Figure 2

schematically shows the connection of a fluorescent matrix with a binding reagent.

C - fluorescent matrix.

D - binding agent.

E - fluorescent matrix with a binding reagent.

Figure 3

the receipt of a fluorescent diagnosticum is schematically presented.

E - fluorescent matrix with a binding reagent.

F - antibody-containing immunoglobulin.

G - fluorescent immunoglobulin diagnosticum.

Figure 4

shows an immunogram of antibody-containing immunoglobulins isolated from immune serum by combined methods: precipitation with neutral salts, gel filtration, removal of cross-reacting and heterologous antibodies.

A - rabbit immune serum:

1 - albumin,

2 - beta globulin,

3 - alpha globulin,

4 - gamma globulin.

B5 - antibody-containing immunoglobulins.

Figure 5

detection of pathogens R.prowazekii, R.typhi, C.burnetii phase I and C.burnetii phase II using fluorescent immunoglobulin diagnosticums for R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii II phase (PID) in the reaction of fluorescent microagglutination.

A1 - positive PID reaction to R.prowazekii with the pathogen R.prowazekii and B1, C1, D1 - negative reactions with pathogens R.typhi, C.burnetii phase I and C.burnetii phase II and in the control for spontaneous agglutination.

B2 - positive PID reaction to R.typhi with the pathogen R.typhi and A2, C2, D2 - negative reactions with pathogens R.prowazekii, C.burnetii phase I and C.burnetii phase II and in the control for spontaneous agglutination.

C3 - positive PID reaction to C.burnetii phase I with the pathogen C.burnetii phase I and A3, B3, D3 - negative reactions with pathogens R.prowazekii, R.typhi, and C.burnetii phase II and in the control for spontaneous agglutination.

D4 - positive PID reaction to C.burnetii phase II with the pathogen C.burnetii phase II and A4, B4, C4 - negative reactions with pathogens R.prowazekii, R.typhi, and C.burnetii phase I and in the control for spontaneous agglutination.

A5, B5, C5, D5 - PID control for spontaneous agglutination.

Figure 6A

the detection of R.prowazekii and R.typhi using fluorescent immunoglobulin diagnosticums for R.prowazekii or R.typhi is presented.

A - negative control of the fluorescent immunoglobulin diagnosticum to R.prowazekii for spontaneous agglutination.

B - positive reaction of the fluorescent immunoglobulin diagnosticum to R.prowazekii with the pathogen R.prowazekii.

C - negative reaction of the fluorescent immunoglobulin diagnosticum to R.prowazekii with the pathogen R.typhi.

D - negative control of the fluorescent immunoglobulin diagnosticum to R.typhi for spontaneous agglutination.

E - positive reaction of the fluorescent immunoglobulin diagnosticum to R.typhi with the pathogen R.typhi.

F - negative reaction of the fluorescent immunoglobulin diagnosticum to R.typhi with the pathogen R.prowazekii.

Figure 6 B

the identification of pathogens C.burnetii phase I and C.burnetii phase II using fluorescent immunoglobulin diagnosticums for C.burnetii phase I and C.burnetii phase II is presented.

A - negative reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase I for spontaneous agglutination.

B - positive reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase I with the pathogen C.burnetii phase I.

C - negative reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase I with the pathogen C.burnetii phase II.

D - negative reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase II for spontaneous agglutination.

E - positive reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase II with the pathogen C.burnetii phase II.

F - negative reaction of the fluorescent immunoglobulin diagnosticum to C.burnetii phase II with the pathogen C.burnetii phase I.

Figure 7

the principle of interaction of a fluorescent immunoglobulin diagnosticum with pathogens present in the test samples is presented.

G - fluorescent immunoglobulin diagnosticum.

H - test samples with homologous pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II.

K - interaction of the fluorescent immunoglobulin diagnosticum with pathogens present in the samples under study.

On the figure. eight

the principle of the distribution of ingredients in the strips of the tablet for immunofluorimetric analysis (IFLA) for setting up the reaction is presented.

The figure shows strips of a collapsible tablet for IFLA, in which antibody-containing immunoglobulins R. prowazekii, R. typhi, C. burnetii phase I and C. burnetii phase II are immobilized in vertical wells of strips 1, 2, 3, 4.

In the wells of strip 5, the distribution of sequentially fluorescent immunoglobulin diagnosticums (PID) into every two wells is shown to control for spontaneous agglutination.

The figures in the wells of the horizontal rows - A, B, C, D, E, F, G indicate the order of applying the test samples.

In the wells of the 8th horizontal row H - PID control was placed with standard pathogens R.prowazekii, R.typhi, C.burnetii phase I and C.burnetii phase II.

Figure 9

schematically depicts the principle of interaction of antibody-containing immunoglobulins with pathogens on biochips and the manifestation of the reaction by a fluorescent immunoglobulin diagnosticum

9A. Positive test result.

1 - Antibody-containing immunoglobulin immobilized on a biochip.

2 - Homologous pathogen in the test sample.

3 - Fluorescent immunoglobulin diagnosticum.

4 - Complex of an antibody-containing immunoglobulin with a homologous pathogen and a fluorescent immunoglobulin diagnosticum.

9B - Negative result of the analysis.

1 - Antibody-containing immunoglobulin immobilized on a bichip.

5 - Heterogeneous pathogen in the test sample.

3 - Fluorescent immunoglobulin diagnosticum.

The complex was not formed: 1 - antibody-containing immunoglobulin immobilized on the biochip remained, and 5 and 3 - were removed by washing.

On figure 10.

schematic representation of the reaction setup on biochips.

10A and 10B - eight biochips each with 4 gel microwells, in which molecular probes are immobilized - antibody-containing immunoglobulins R. prowazekii, R. typhi, C. burnetii phase I and C. burnetii phase II.

C - test sample.

G - 0.9% sodium chloride solution.

H, I, J, K - fluorescent immunoglobulin diagnosticums (PID): H - PID for R.prowazekii, I - PID for R.typhi,

J - PID to C.burnetii phase I, K - PID to C.burnetii phase II.

Disclosure of invention

The created fluorescent immunoglobulin diagnosticums can be used for a specific assessment of the activity of vaccines, as well as for the study of objects of epidemiological examination, which are infected organs and tissues of animals, or samples of an infected environment, or carriers of epidemic typhus, or endemic flea typhus, or Q fever in interaction reactions

flea typhus, or Q fever in the interaction of pathogens with complementary fluorescent immunoglobulin diagnosticums with registration of reaction results using hardware and software analyzers.

Fluorescent immunoglobulin diagnosticums can be used in the practice of clinical diagnostic laboratories of institutions of the Ministry of Health of the Russian Federation and the Centers of Rospotrebnadzor to detect pathogens of epidemic typhus and endemic typhus and coxiellosis during epidemiological examination.

The method for preparing fluorescent immunoglobulin diagnosticums consists of the following steps

1. Obtaining a fluorescent matrix.

1.1. Formalization of lactobacilli.

1.2. Fluorescent label of lactobacilli.

1.3 Obtaining a fluorescent matrix with a binding reagent.

2. Obtaining purified antibody-containing immunoglobulins.

2.1. Isolation of immunoglobulins from immune sera by precipitation with neutral salts, in particular, ammonium sulphate.

2.2. Removal of salts of ammonium sulfate from the antibody-containing fraction of immunoglobulins by gel filtration.

2.3. Removal of cross-reacting and heterologous antibodies from the antibody-containing fraction of immunoglobulins by adsorption.

3. Preparation of fluorescent immunoglobulin diagnosticums.

3.1. Sensitization of a fluorescent matrix by adsorbed antibody-containing immunoglobulins.

3.2. Obtaining fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi, or to C.burnetii phase I, or C.burnetii phase II

Example 1

1. Obtaining a fluorescent matrix.

Obtaining a fluorescent matrix was carried out according to the following method.

1.1. Formalization of lactobacilli.

Lactobacilli isolated from the Lactobacterin preparation (manufactured by Microgen, Perm NPO Biomed) were used as a matrix by centrifugation three times at 2500 rpm for 25 min at a temperature of 23°C in order to remove the protective medium for drying.

The resulting precipitates of lactobacilli were resuspended in 0.9% sodium chloride solution pH 7.2, neutral formalin was added to the resulting biomass of lactobacilli to a final concentration of 10% and kept for 10 days at a temperature of 6 - 8°C. Precipitation of the formalized biomass was carried out by centrifugation at 2500 rpm for 25 min at a temperature of 23°C. The biomass was washed twice in 0.9% sodium chloride solution at 2500 rpm for 25 min. The sediment was resuspended in a working solution - 0.9% sodium chloride solution - 70%, carbonate-bicarbonate buffer pH 9.0 - 30% to a concentration of 1×10 ⁹ cells/ml.

Received a formalized suspension of lactobacilli.

1.2. Fluorescent label of lactobacilli.

Inorganic fluorochromes - fluorescein isothiocyanate or nanocrystals in the form of semiconductor colloidal particles of different fluorescence colors were used as a label. In the described experiment, for staining, a fluorescent dye, fluorescein isothiocyanate, was added to the resulting suspension of formalized lactobacilli at the rate of 2 mg per 1×10 ⁹ lactobacilli.

The conjugation of the mixture was carried out for 18 hours at a temperature of 23°C with constant stirring (Fig. 1).

The label was carried out in a similar way using nanocrystals in the form of semiconductor colloidal particles of different fluorescence colors, except that 10 ml of nanocrystals with a concentration of 5.4×10 ^-6 were added per 1×10 ⁹ cells/ml of formalized lactobacilli.

After the conjugation time, the fluorescent matrix was precipitated and washed from the unreacted fluorochrome by 3-fold centrifugation in 0.9% sodium chloride solution at 2500 rpm for 25 min. and the pellet was resuspended in 0.9% sodium chloride solution.

A 1% suspension of the fluorescent matrix was obtained, which was stored at a temperature of 6-8°C.

1.3 Obtaining a fluorescent matrix with binding reagents (Fig. 2).

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

For tanization, 2 ml of a 1% suspension of the fluorescent matrix was precipitated and the precipitate was washed in 10 ml of 0.9% sodium chloride solution pH 7.0 twice by centrifugation for 25 min at 2500 rpm. The precipitate obtained was resuspended in 0.9% sodium chloride solution pH 7.2. Then, an equal volume of tannin solution at a concentration of 1:20000 in 0.9% sodium chloride solution pH 7.2 was added to the volume of the washed fluorescent matrix, the mixture was left for 15 min at a temperature of 23°C with occasional stirring. The sedimentation of the suspension was carried out by centrifugation at 2500 rpm ¹ for 25 min at a temperature of 23°C. WITH. The pellet was resuspended in phosphate buffer pH 6.4.

The production of a fluorescent matrix with activated groups using a water-soluble carbodiimide (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) was carried out in a similar way, except that an equal volume of a carbodiimide solution at a concentration of 1:2000 was added to the volume of the washed fluorescent matrix.

Received a suspension of the fluorescent matrix with a binding reagent (Fig. 2).

Example 2. Obtaining purified antibody-containing immunoglobulins.

2.1. Isolation of immunoglobulins from immune sera by precipitation with neutral salts, in particular ammonium sulphate (Fig. 4).

Isolation of the immunoglobulin antibody-containing fraction against R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II was performed from standard immune sera by double salt fractionation with ammonium sulfate at 34% saturation.

½ volume of purified water was added to one volume of rabbit serum, and the calculated (up to 34% saturation) amount of saturated ammonium sulfate solution was added dropwise, with constant stirring.

Calculation formula:

(NH ₄ ) ₂ SO ₄ \u003d (A + B) x34

100-34; where

A is the volume of the original serum,

B - the volume of purified water, equal to half the volume of whey,

34 - the required percentage of serum saturation with ammonium sulfate.

The mixture was kept for 60 min at 23°C with occasional stirring. The formed precipitate was separated by centrifugation at 2500 rpm for 25 min. The supernatant was removed, and the precipitate was rehydrated in purified water to the volume of the original serum. During the second salting out, a saturated solution of ammonium sulfate was added to the resulting volume, the volume of which was calculated by the formula:

(NH ₄ ) ₂ SO ₄ \u003d Ax34

100-34; where

A is the volume of rehydrated serum.

The mixture, periodically stirring, was kept for 60 min at a temperature of 23°C. The formed precipitate was separated by centrifugation at 2500 rpm for 25 min. The precipitate was dissolved in a buffered 0.9% sodium chloride solution, taken in a volume equal to 0.1 volume of serum.

Received a solution of immunoglobulins, which was subject to release from salts of ammonium sulphate.

2.2. Removal of salts of ammonium sulphate from the antibody-containing fraction of immunoglobulins by gel filtration.

The resulting solution of immunoglobulins was freed from ammonium sulphate by gel filtration on a column filled with Sephadex G-25 medium. Protein was collected from a sample with sulfosalicylic acid, the absence of ammonium sulfate was controlled using Nessler's reagent.

The purity of the obtained immunoglobulin was controlled by immunoelectrophoresis in agar.

Immunoelectrophoresis of immune sera to R.prowazekii or R.typhi or C.burnetii phase I or C.burnetii phase II, immunoglobulins isolated from them was carried out according to the modified method of Grabar P., Williams C.A. 1% Merck agar using veronal-medinal buffer solution pH 8.6 with ionic strength 0.05. The test samples were added to the wells cut in the agar layer and electrophoresis was performed at a voltage of 40 V for 2.5 hours. At the end of this procedure, sera against rabbit serum proteins were added to the cut trenches and incubated in a humid chamber for 48 hours. The result was evaluated visually.

Grabar P., Williams C.A. Methode permettant l'etude conjuguee des proprieties electrophoretigues d'un meiange de proteins. Application au serumsanguin. biochim. Biophys. acta. 1953.V.10. No. 1. P. 193-194.

Immunoglobulins that did not contain albumin and had one precipitation line in the gamma globulin zone were selected for work.

Received a solution of purified antibody-containing fraction of immunoglobulins, not containing salts of ammonium sulphate.

2.3. Removal of cross-reacting and heterologous antibodies from the antibody-containing fraction of immunoglobulins by adsorption.

Removal of cross-reacting and heterologous antibodies from the antibody-containing fraction of immunoglobulins was carried out using adsorbents - fluorescent antigenic diagnosticums R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II, prepared as described in patent No. 2728340 dated 12/20/2019

To obtain fluorescent antigenic diagnosticums according to the method specified in the patent, lyophilized corpuscular antigens of R. prowazekii or R. typhi, or C. burnetii phase I, or C. burnetii phase II were used.

To do this, the contents of the ampoules 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 Prep150 Plus (UK) at a frequency of 23 kHz and an amplitude of 112 six times 5 min. Sedimentation of intact cells was carried out by centrifugation at 3000 rpm for 20 min, at a temperature of 23°C. An Akta Explorer 10 chromatograph (Sweden) was used to isolate the active protein-lipopolysaccharide complex from the resulting disintegrate. The disintegrate was fractionated from a column (10×300 mm in size) in a buffer solution (10 mM Tris-HCl, 0.5 M sodium chloride, 10% isopropanol, pH 7.5).

The first serologically active fraction of the BLPS complex was collected and combined with a fluorescent matrix by means of a binding agent (tannin). The mixture was incubated at 43°C for 60 min. with occasional stirring. After the incubation time, the suspension was precipitated at 2500 rpm for 25 min, the precipitate was washed from the unreacted BLPS complex by double sedimentation by centrifugation at 2500 rpm for 25 min, at a temperature of 23°C. The pellet was resuspended in 0.9% sodium chloride solution to a final concentration of 1%. Stored at 6-8°C.

To remove cross-reactive and heterologous antibodies from the antibody-containing fraction of immunoglobulins, the optimal adsorbing dose of diagnosticum was preliminarily selected by adsorption.

The optimal adsorbing dose of fluorescent antigenic diagnosticums was selected empirically in the range from 0.1 to 0.5 ml per 0.1 ml of purified immunoglobulins. The selected dose of fluorescent antigenic diagnosticums was added to the immunoglobulin solution, kept for 30 min with occasional stirring, and removed from the immunoglobulin solution by centrifugation at 2500 rpm for 25 min.

The resulting adsorbed antibody-containing immunoglobulins were evaluated for immunofluorescent activity in nMFA using fluorescent anti-rabbit immunoglobulins as an indicator (FIG. 4).

Received a solution of purified antibody-containing immunoglobulins R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II.

Example 3

Preparation of fluorescent immunoglobulin diagnosticums.

3.1. Sensitization of a fluorescent matrix by adsorbed antibody-containing immunoglobulins.

To do this, an equal volume of a solution of adsorbed antibody-containing immunoglobulins, taken in an empirically selected concentration, was added to the volume of the fluorescent matrix and incubated at a temperature of 37°C for 60 min with occasional stirring every 15 min.

3.2. Obtaining fluorescent immunoglobulin diagnosticums (Fig. 3).

After the incubation time, to remove unreacted antibody-containing immunoglobulins, the suspension was centrifuged for 25 min at 2500 rpm, and 10 ml of 0.9% sodium chloride solution pH 7.0 was added to the sediment and washed once in a 10-fold volume of 0 .9% sodium chloride solution. The washed precipitate was resuspended in 0.9% sodium chloride solution pH 7.0 to a final concentration of 1% and stored at a temperature of 6-8°C. Received a 1% suspension of fluorescent immunoglobulin diagnosticum to R.prowazekii or R.typhi, or S.burnetii phase I, or S.burnetii phase II.

Example 4

Obtaining and application of fluorescent immunoglobulin diagnosticum R.prowazekii.

Getting fluorescent immunoglobulin diagnosticum to R.prowazekii was carried out in accordance with the above steps of the process.

Lactobacilli isolated from the preparation "Lactobacterin" by triple centrifugation at 2500 rpm for 25 min at a temperature of 23°C were used as a matrix to remove the protective medium for drying.

The obtained sediment of lactobacilli was resuspended in 0.9% sodium chloride solution pH 7.2 to the initial volume, neutral formalin was added to the resulting biomass of lactobacilli to a final concentration of 10% and kept for 10 days at a temperature of 6 - 8°C. The sedimentation of the suspension was carried out by centrifugation at 2500 rpm for 25 min at a temperature of 23°C. The suspension was washed three times in a 0.9% sodium chloride solution pH 7.0 at 2500 rpm for 25 min at a temperature of 23°C. The sediment was resuspended in a working solution - 0.9% sodium chloride solution - 70%, carbonate-bicarbonate buffer pH 9.0 - 30% to a concentration of 1×10 ⁹ cells / ml.

An inorganic fluorescent dye, fluorescein isothiocyanate, was used to label lactobacilli with fluorochrome. In the described experiment, for labeling, a fluorescent dye, fluorescein isothiocyanate, was added to the obtained suspension of formalized lactobacilli at the rate of 2 mg per 1×10 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 manner, while 10 ml of nanocrystals with a nanoparticle concentration of 5.4×10 ^-6 were added per 1×10 ⁹ cells/ml of formalized lactobacilli.

After the conjugation time, the suspension of the fluorescent matrix was precipitated and washed from the unreacted fluorochrome by 3-fold sedimentation by centrifugation in 0.9% sodium chloride solution at 2500 rpm for 25 min, and the precipitate was resuspended in 0.9% chloride solution sodium to a final concentration of 1%.

A 1% suspension of the fluorescent matrix was obtained, which was stored at a temperature of 6-8°C.

A tannin solution in 0.9% sodium chloride solution pH 7.2 was used as a binding agent. To do this, 2 ml of a suspension of the fluorescent matrix was precipitated and the precipitate was washed by centrifugation for 25 min at 2500 rpm in 10 ml of 0.9% sodium chloride solution pH 7.0 twice. The pellet was resuspended in 2 ml of 0.9% sodium chloride solution pH 7.2. Then, 2 ml of a tannin solution at a concentration of 1:20,000 was added to 2 ml of the washed fluorescent matrix; the mixture was left for 15 min at a temperature of 23°C with occasional stirring. After the time elapsed, the suspension was precipitated by centrifugation at 2500 rpm ¹ for 25 minutes at a temperature of 23°C. for 25 minutes at 23°C. The pellet was resuspended in 2 ml of phosphate buffer pH 6.4.

Received 2 ml of a fluorescent matrix with a binding reagent.

To obtain antibody-containing immunoglobulins, standard immune sera for RNHA were used, obtained by immunizing rabbits with Rickettsia prowazekii, the Brainle strain. 10 ml of purified water was added to 20 ml of sera, and 15.45 ml of a saturated solution of ammonium sulphate pH 7.2 was added dropwise with constant stirring. The mixture was kept for 60 min at 23°C with occasional stirring. The formed precipitate was separated by centrifugation at 2500 rpm for 25 min. The supernatant was removed and the precipitate was rehydrated in 20 ml of purified water and the immunoglobulins were re-isolated. During the second salting out, 10.3 ml of a saturated solution of ammonium sulphate was added to 20 ml of the immunoglobulin solution. The mixture, periodically stirring, was kept for 60 min at a temperature of 23°C. The formed precipitate was separated by centrifugation at 2500 rpm for 25 min. The precipitate of immunoglobulins was dissolved in 2.0 ml of buffered 0.9% sodium chloride solution.

The release of immunoglobulins from ammonium sulphate was carried out by gel filtration on a 2×15 cm column filled with Sephadex G-25 medium. Protein was collected from a sample with sulfosalicylic acid, the absence of ammonium sulfate was controlled using Nessler's reagent. Received 2.3 ml of an immunoglobulin solution with a protein concentration of 21 mg/ml.

The complete removal of albumin and the purity of the resulting immunoglobulin were judged by agar immunoelectrophoresis.

We selected antibody-containing immunoglobulins that did not contain albumin and, according to the data of immunophoresis in agar, had one precipitation line in the zone of serum gamma globulins (Fig. 4).

Adsorption of the obtained antibody-containing immunoglobulins from cross-reacting and heterologous antibodies was carried out using fluorescent R.typhi antigen diagnosticums prepared as described in step 2.3. The optimal dose of the adsorbent was selected empirically. To do this, a fluorescent antigenic diagnosticum R.typhi was added to a series of test tubes with 0.1 ml of a 1% solution of antibody-containing immunoglobulins in the range from 0.1 to 0.5 ml. The mixture was held for 30 minutes and then subjected to centrifugation at 2500 rpm for 25 minutes. The supernatant, which is purified antibody-containing immunoglobulins, was tested for the completeness of the removal of cross-reacting and heterologous antibodies, as well as for species-specific immunofluorescent activity with R.prowaztkii and R.typhi antigens in nMFA.

Immunoglobulins to R.prowazekii, purified by adsorption of R.typhi fluorescent antigen diagnosticum in nMFA, did not react with R.typhi, which indicated complete adsorption of cross-reacting and heterologous antibodies.

Characterization of the isolated antibody-containing immunoglobulin to R.prowazekii: the protein concentration of the antibody-containing immunoglobulin ranged from 20 to 25 mg/ml. Specific activity according to nMFA was 1:320-1:640 with R.prowazekii antigen, 1:2.5-1:5 with R.typhi antigen.

The isolated fractions of antibody-containing immunoglobulins, according to immunoelectrophoresis, did not contain albumin, were homogeneous and gave one line of precipitation in the zone of serum gamma globulins (Fig. 4).

The obtained antibody-containing immunoglobulins to R.prowazekii were combined with a fluorescent matrix using a binding reagent.

The connection of the fluorescent matrix with binding reagents c adsorbed immunoglobulins was carried out as follows (Fig. 2 and 3).

To 2 ml of a fluorescent matrix with a binding reagent, 2 ml of an antibody-containing immunoglobulin solution with a protein concentration of 0.2 mg/ml was added and incubated at a temperature of 37°C for 60 minutes with occasional stirring every 15 minutes. After the incubation time, the suspension was centrifuged for 25 min at 2500 rpm and the pellet of the suspension was washed with 0.9% sodium chloride solution pH 7.0. After that, a final concentration of fluorescent diagnosticums equal to 1% was obtained and stored at a temperature of 6-8°C.

Received a 1% suspension of fluorescent immunoglobulin diagnosticum to R.prowazekii. The final product - fluorescent immunoglobulin diagnosticum R.prowazekii, which was stored at a temperature of 6-8°C, for long-term storage diagnosticum stabilized by freeze drying.

The results of experiments on the use of the created fluorescent immunoglobulin diagnosticum to R.prowazekii for the differentiation of pathogens R.prowazekii and R.typhi are shown in Tables 1 and 2.

Example 5

Obtaining and application of fluorescent immunoglobulin diagnosticum R.prowazekii using nanocrystals as a fluorescent dye.

Obtaining a fluorescent immunoglobulin diagnosticum to R.prowazekii was carried out in accordance with the above example 4, with the only difference that nanocrystals in the form of semiconductor colloidal particles were used as fluorochrome.

The prepared fluorescent immunoglobulin diagnosticum for R.prowazekii based on nanocrystals did not differ in biochemical and immunofluorescent characteristics from the fluorescent immunoglobulin diagnosticum for R.prowazekii obtained in example 4.

Example 6

Obtaining and application of fluorescent immunoglobulin diagnosticum to R.typhi.

Obtaining a fluorescent immunoglobulin diagnosticum to R.typhi was carried out in accordance with the steps of the technological process described in example 4, with the exception of immune sera, from which antibody-containing immunoglobulins and adsorbent were obtained. In these experiments, standard immune sera obtained by immunizing rabbits with R.typhi, the Isakhanyan strain, were used. Isolation of immunoglobulins from serum was performed as described in example 4.

Antibody-containing immunoglobulins were selected for work, which, according to the data of immunophoresis in agar, formed one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Adsorption of antibody-containing immunoglobulins from cross-reacting and heterologous antibodies was carried out using fluorescent antigenic diagnosticums to R. prowazekii, prepared as described in step 2.3.

The optimal dose of the adsorbent was selected empirically as shown in example 4.

Antibody-containing immunoglobulins were tested for completeness in the removal of cross-reacting and heterologous antibodies, as well as for species-specific immunofluorescent activity with R. prowaztkii and R. typhi antigens in nMFA.

Antibody-containing immunoglobulins to R.typhi, purified by adsorption of R.prowazekii fluorescent antigen diagnosticum in nMFA, did not react with R.prowazekii, which indicated complete adsorption of cross-reacting and heterologous antibodies.

Characterization of antibody-containing immunoglobulin to R.typhii: the concentration of immunoglobulin protein ranged from 20 to 23 mg/ml. Specific activity according to nMFA was 1:160 - 1:320 with R.typhi antigen, 1:2.5-1:5 with R.prowazekii antigen. The isolated fractions of antibody-containing immunoglobulins, according to immunoelectrophoresis, did not contain albumin, were homogeneous and gave one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Received antibody-containing immunoglobulins to R.typhi using a binding reagent - tannin was combined with a fluorescent matrix according to the method described in example 4 and received a 1% suspension of fluorescent immunoglobulin diagnosticum R.typhi.

The final product - fluorescent immunoglobulin diagnosticum R.typhi was stored at a temperature of 6-8°C, for long-term storage the drug was stabilized by lyophilization.

The results of experiments on the use of a fluorescent immunoglobulin diagnosticum to R.typhi for the differentiation of pathogens R.prowazekii and R.typhi are shown in Tables 1 and 2.

Example #7

Obtaining and application of a fluorescent immunoglobulin diagnosticum to C. burnetii phase I.

Obtaining a fluorescent immunoglobulin diagnosticum to S.burnetii phase I was carried out in accordance with the steps of the technological process described in example 4, with the exception of immune sera, from which antibody-containing immunoglobulins and adsorbents were obtained.

In these experiments, standard immune sera obtained by immunizing rabbits with C.burnetii phase I, strain "Grita" were used. Isolation of immunoglobulins from serum was performed as described in example 4.

Antibody-containing immunoglobulins were selected for work, which, according to the data of immunophoresis in agar, formed one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Adsorption of antibody-containing immunoglobulins from cross-reacting and heterologous antibodies was carried out using fluorescent phase I C. burnetii antigenic diagnosticums prepared as described in step 2.3.

The optimal dose of the adsorbent was selected empirically as shown in example 4.

Antibody-containing immunoglobulins were tested for the completeness of the removal of cross-reacting and heterologous antibodies, as well as for species-specific immunofluorescent activity with C.burnetii phase I and C.burnetii phase II antigens in nMFA.

Antibody-containing immunoglobulins to R.typhi, purified by the method of adsorption by a fluorescent antigenic diagnosticum of C.burnetii phase II in nMFA, did not react with C.burnetii phase II, which indicated complete adsorption of cross-reacting and heterologous antibodies.

Characterization of antibody-containing immunoglobulin to C.burnetii phase I: the concentration of immunoglobulin protein ranged from 20 to 23 mg/ml. Specific activity according to nMFA was 1:160 - 1:320 with C.burnetii phase I antigen, 1:2.5 with C.burnetii phase II antigen. The isolated fractions of antibody-containing immunoglobulins, according to immunoelectrophoresis, did not contain albumin, were homogeneous and gave one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Received antibody-containing immunoglobulins to C.burnetii phase I using a binding reagent - tannin was combined with a fluorescent matrix according to the method described in example 4 and received a 1% suspension of fluorescent immunoglobulin diagnosticum C.burnetii phase I.

The final product - fluorescent immunoglobulin diagnosticum C.burnetii phase I was stored at a temperature of 6-8°C, for long-term storage, the drug was stabilized by lyophilization.

The results of experiments on the use of a fluorescent immunoglobulin diagnosticum C.burnetii phase I for the differentiation of pathogens C.burnetii phase I and C.burnetii phase II are shown in table 3.

Example 8

Obtaining and application of a fluorescent immunoglobulin diagnosticum to C. burnetii phase II.

Obtaining an immunoglobulin diagnosticum for fluorescent C. burnetii phase II was carried out in accordance with the steps of the process described in example 4, with the exception of immune sera, from which antibody-containing immunoglobulins and adsorbent were obtained.

In these experiments, we used standard immune sera obtained by immunizing rabbits with C. burnetii phase II, strain "Grita". Isolation of immunoglobulins from serum was performed as described in example 4.

Antibody-containing immunoglobulins were selected for work, which, according to the data of immunophoresis in agar, formed one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Adsorption of antibody-containing immunoglobulins from cross-reacting and heterologous antibodies was carried out using fluorescent phase I C. burnetii antigenic diagnosticums prepared as described in step 2.3.

The optimal dose of the adsorbent was selected empirically, as shown in example 4.

Antibody-containing immunoglobulins were tested for the completeness of the removal of cross-reacting and heterologous antibodies, as well as for species-specific immunofluorescent activity with C.burnetii phase I and C.burnetii phase II antigens in nMFA.

Antibody-containing immunoglobulins to R.typhi, purified by the method of adsorption of phase I fluorescent antigenic diagnosticum C.burnetii in nMFA, did not react with C.burnetii phase I, which indicated complete adsorption of cross-reacting and heterologous antibodies.

Characterization of antibody-containing immunoglobulin to C.burnetii phase II: the concentration of immunoglobulin protein ranged from 20 to 23 mg/ml. Specific activity according to nMFA was 1:160 - 1:320 with C.burnetii phase II antigen, 1:2.5 with C.burnetii phase I antigen. The isolated fractions of antibody-containing immunoglobulins, according to immunoelectrophoresis, did not contain albumin, were homogeneous and gave one line of precipitation in the zone of serum gamma globulins (Fig. 4).

Received antibody-containing immunoglobulins to C.burnetii phase II using a binding reagent - tannin was combined with a fluorescent matrix according to the method described in example 4 and received a 1% suspension of fluorescent immunoglobulin diagnosticum C.burnetii phase II.

The final product - fluorescent immunoglobulin diagnosticum C.burnetii phase II was stored at a temperature of 6-8°C, for long-term storage, the drug was stabilized by lyophilization.

The results of experiments on the use of a fluorescent immunoglobulin diagnosticum C.burnetii phase II to differentiate pathogens C.burnetii phase I and C.burnetii phase II are shown in table 3.

Example 9

Method of using fluorescent immunoglobulin diagnosticums in the reaction of fluorescent microagglutination (RFMA) for the detection of R.prowazekii or R.typhi or C.burneti phase II or C.burneti phase II.

All obtained fluorescent diagnosticums were used for carrying out a one-step reaction of fluorescent microagglutination (RFMA).

RFMA is based on the specific interaction of fluorescent immunoglobulin diagnosticums directly with homologous antigens of the studied samples on a glass slide. For this purpose, test samples were added to the wells of slides and a fluorescent immunoglobulin diagnosticum was added. The interaction of antigen with antibodies occurred for 60 min at a temperature of 37°C in a humid chamber. After this time, the preparations were dried at room temperature, viewed and evaluated under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x).

9.1. Reaction setting.

The use of the created fluorescent immunoglobulin diagnosticums for differentiation of pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II for the specific assessment of the activity of vaccines, as well as the study of objects of epidemiological examination, which are infected organs and tissues of animals, or samples of an infected environment, or carriers of epidemic lousy typhus, or endemic flea typhus, or Q fever in the reaction of the interaction of antigens with fluorescent immunoglobulin diagnosticums with the registration of results using hardware-software analyzers.

To do this, the required number of glass slides with wells was placed on plastic substrates with sides (the lid of plates for immunological reactions).

Introduction of test and control samples of the studied materials.

On 4 glass slides with 8 wells, seven different test samples were examined, and the 8th well was used as a diagnosticum control.

For this, 5 ml of seven different test materials were added to 4 slides in 7 wells, and 5 ml of purified water was added to the 8th well. Then, 5 ml of fluorescent immunoglobulin diagnosticums for R.prowazekii were added to all wells of the first slide, and 5 ml of fluorescent immunoglobulin diagnosticums for R.typhi were added to all wells of the second slide, and 5 ml of fluorescent immunoglobulin diagnosticums for R.typhi were added to all wells of the third slide. immunoglobulin diagnosticums to C.burnetii phase I, and 5 ml of fluorescent immunoglobulin diagnosticums to C.burnetii phase II were added to all wells of the fourth slide.

9.2. Conditions for the interaction of ingredients.

The reaction ingredients were mixed by gently tapping on the plastic support. Then the substrates with glass slides were placed in a humid chamber at 37°C for 60 min. The interaction of specific antibodies with fluorescent diagnosticums took place in a humid chamber. After the time had elapsed, the substrates with preparations were taken out of the humid chamber and dried in a thermostat or at room temperature of 23°C. The dried preparations were evaluated under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x).

9.3. Registration and interpretation of results.

The results were recorded either visually or with the help of photodocumentation based on the assessment of the intensity of specific luminescence and the presence of agglutinates, viewing at least 10 fields of view (detecting equipment) under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x). The presence of pathogens in the test material was indicated by the formation of agglutinates and the absence of agglutinates in the control.

The evaluation of the results was carried out according to the two-cross system:

- positive result - all viewed fields of vision are filled with agglutinates of different sizes and isolated labeled corpuscles; negative result - isolated marked corpuscles are observed in all examined fields.

The results of antigen studies in RFMA were documented by photographing preparations in a fluorescent microscope and displayed on a personal computer screen and stored on a USB drive.

Example 10

The method of using fluorescent immunoglobulin diagnosticums in the reaction of fluorescent microagglutination for the differentiation of R.prowazekii or R.typhi.

Fluorescent immunoglobulin diagnosticums for R. prowazekii or R. typhi were used to differentiate pathogens in the fluorescent microagglutination test (RFMA), both separately and together. Both fluorescent immunoglobulin diagnosticums were used in the RFMA setting (Fig. 6A).

RFMA is based on the specific interaction of fluorescent immunoglobulin diagnosticums directly with homologous pathogens of the studied samples on a glass slide (Fig. 7). For this purpose, test samples were added to the wells of slides and a fluorescent immunoglobulin diagnosticum was added. The interaction of the pathogen with antibodies occurred for 60 min at a temperature of 37°C in a humid chamber. After this time, the preparations were dried at room temperature, viewed, and recorded under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x).

Statement of the reaction

The created fluorescent immunoglobulin diagnosticums were used to differentiate the pathogens R.prowazekii or R.typhi, to specifically assess the activity of the typhus vaccine, as well as to study the objects of epidemiological examination, which are infected organs and tissues of animals, or samples of the infected environment, or carriers of epidemic lousy typhus. , or endemic flea typhus, in the reaction of the interaction of antigens with fluorescent immunoglobulin diagnosticums with the registration of results using hardware and software analyzers.

To do this, the required number of glass slides with wells was placed on plastic substrates with sides (the lid of plates for immunological reactions).

Introduction of test and control samples of the studied materials.

On 2 glass slides with 8 wells, seven different test samples were examined, and the 8th well was used to control the diagnosticum.

For this, 5 ml of seven different test materials were added to 7 wells on 2 glass slides, and 5 ml of purified water was added to the 8th well. Then, 5 ml of fluorescent immunoglobulin diagnosticums for R.prowazekii were added to all wells of the first glass slide, and 5 ml of fluorescent immunoglobulin diagnosticums for R.typhi were added to all wells of the second glass slide.

Conditions for the interaction of ingredients

The reaction ingredients were mixed by gently tapping on the plastic support. Then the substrates with glass slides were placed in a humid chamber at 37°C for 60 min. The interaction of specific antibodies with fluorescent diagnosticums took place in a humid chamber. After the time had elapsed, the substrates with preparations were taken out of the humid chamber and dried in a thermostat or at room temperature of 23°C. The dried preparations were evaluated under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x).

Registration and interpretation of results

The results were recorded either visually or using photodocumentation based on an assessment of the intensity of a specific glow and the presence of agglutinates, looking through at least 10 fields of view (detecting device) under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x). The presence of pathogens in the test material was indicated by the formation of agglutinates and the absence of agglutinates in the control.

The evaluation of the results was carried out according to the two-cross system:

- positive result - all viewed fields of vision are filled with agglutinates of different sizes and isolated labeled corpuscles; negative result - isolated marked corpuscles are observed in all examined fields.

The results of studies of pathogens in RFMA were documented by photographing preparations in a fluorescent microscope and displayed on a personal computer screen and stored on a USB drive.

Example 11.

The method of using fluorescent immunoglobulin diagnosticums in the reaction of fluorescent microagglutination for the differentiation of C.burnetii phase I or C.burnetii phase II.

The resulting fluorescent diagnosticums were used to carry out a one-step fluorescent microagglutination (RFMA) reaction both separately and together (Fig. 6B).

RFMA is based on the specific interaction of fluorescent immunoglobulin diagnosticums directly with homologous antigens of the studied samples on a glass slide. For this purpose, test samples were added to the wells of slides and a fluorescent immunoglobulin diagnosticum was added. The interaction of the pathogen with antibodies occurred for 60 min at a temperature of 37°C in a humid chamber. After this time, the preparations were dried at room temperature, viewed and evaluated under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x).

Statement of the reaction

The use of the created fluorescent immunoglobulin diagnosticums for the differentiation of pathogens C.burnetii phase I, or C.burnetii phase II for the specific assessment of the activity of the vaccine against Q fever, as well as the study of objects of epidemiological examination, which are infected organs and tissues of animals, or samples of the infected environment, or carriers of Q fever in the reaction of the interaction of pathogens with fluorescent immunoglobulin diagnosticums with the registration of results using hardware and software analyzers.

To do this, the required number of glass slides with wells was placed on plastic substrates with sides (the lid of plates for immunological reactions).

Introduction of test and control samples of the studied materials.

On 2 glass slides with 8 wells, seven different test samples were examined, and the 8th well was used to control the diagnosticum.

For this, 5 ml of seven different test materials were added to 7 wells on 2 glass slides, and 5 ml of purified water was added to the 8th well. Then, 5 ml of fluorescent immunoglobulin diagnosticums for C. burnetii of phase I were added to all wells of the first slide, and 5 ml of fluorescent immunoglobulin diagnosticums for C.burnetii of phase II were added to all wells of the second slide.

Conditions for the interaction of ingredients.

The reaction ingredients were mixed by gently tapping on the plastic support. Then the substrates with glass slides were placed in a humid chamber at 37°C for 60 min. The interaction of specific antibodies with fluorescent diagnosticums took place in a humid chamber. After the time had elapsed, the substrates with preparations were removed from the humid chamber and dried in a thermostat or at room temperature 23°C.

Registration and interpretation of results.

The results were recorded either visually or using photodocumentation based on the assessment of the intensity of specific luminescence and the presence of agglutinates, viewing at least 10 fields of view under the immersion objective of a fluorescent microscope (objective 100x, eyepiece 10x). The presence of pathogens in the test material was indicated by the formation of agglutinates and the absence of agglutinates in the control.

The evaluation of the results was carried out according to the two-cross system:

- positive result - all viewed fields of vision are filled with agglutinates of various sizes and isolated labeled corpuscles

- negative result - isolated marked corpuscles are observed in all scanned fields.

The results of studies of pathogens in RFMA were recorded and documented by photographing preparations in a fluorescent microscope (detecting device) and displayed on a personal computer screen and stored on a USB drive.

Example 12

Method for using fluorescent immunoglobulin diagnosticums for differentiation of pathogens R.prowazekii or R.typhi or C.burneti phase I or C.burneti phase II in immunofluorimetric analysis.

The method is based on the interaction of antibody-containing immunoglobulins immobilized in the wells of the tablets with homologous pathogens of the samples under study, followed by the determination of attached pathogens using fluorescent immunoglobulin diagnosticums (Fig. 8).

For immunofluorimetric analysis, strips of collapsible plates for immunofluorimetric analysis were used, in which antibody-containing immunoglobulins of R.prowazekii or R.typhi, or C.burneti phase I, or C.burneti phase II are immobilized. Pure strips were used to control fluorescent immunoglobulin diagnosticums for spontaneous agglutination.

12.1. Statement of the reaction

To set up the reaction, 5 strips of 8 wells each were taken, which were inserts into a collapsible plate.

In the wells of the strips, antibody-containing immunoglobulins were immobilized:

- antibody-containing R.prowazekii immunoglobulins are immobilized in the wells of the 1st strip;

- R.typhi antibody-containing immunoglobulins are immobilized in the wells of the 2nd strip;

- in the wells of the 3rd strip immobilized antibody-containing immunoglobulins C.burneti phase I;

- in the wells of the 4th strip immobilized antibody-containing immunoglobulins C.burneti phase II;

- The 5th control strip was used clean.

The strips were placed in the plate frame, with each horizontal row of wells from the 1st to the 8th of each of the 5 strips designated sequentially in Latin letters: A, B, C, D, E, F, G, H vertically, and each strip is marked horizontally with numbers: 1, 2, 3, 4, 5.

10% suspensions were prepared from the test samples in 0.9% sodium chloride solution.

The immunofluorimetric analysis was performed in two stages.

Stage I. Introduction into the wells of each horizontal row of test samples.

7 test samples were introduced into all wells from the 1st to the 4th of each of the 7 horizontal rows of strips in succession:

- in the wells of row A1, A2, A3, A4, 100 μl of the first test sample were added;

- 100 µl of the second test sample was added to the wells of row B1, B2, B3, B4;

- in the wells of the row C1, C2, C3, C4, 100 μl of the third test sample were added;

- 100 µl of the fourth test sample was added to the wells of row D1, D2, D3, D4;

- 100 μl of the fifth test sample was added to the wells of the row E1, E2, E3, E4;

- in the wells of the row F1, F2, F3, F4, 100 μl of the sixth test sample were added;

- in the wells of row G1, G2, G3, G4, 100 µl of the seventh test sample was added.

In the wells of the 8th horizontal row H of 4 strips, suspensions of standard pathogens were added sequentially from the 1st to the 4th wells: R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II in dilution 1:10 (at a concentration of 2×10 ⁹ cells/ml).

Namely:

- 100 μl of a suspension of the pathogen R. prowazekii was added to the well of the horizontal row H1;

- 100 μl of a suspension of the pathogen R.typhi was added to well H2;

- 100 μl of a suspension of the pathogen C.burneti phase I was added to well H3;

- 100 μl of phase II suspension of C.burneti was added to well H4.

In all vertical wells from the 1st to the 8th of the 5th strip, 100 µl of purified water were added.

After that, the plate with strips was covered with a lid and placed in a thermostat at a temperature of 37°C for 60 min with occasional stirring every 15 min. After this time, the plate was taken out and the strips were washed by adding 200 μl of 0.9% sodium chloride solution with the addition of 5% phosphate buffer pH 7.2 to each well three times for 3 minutes to remove unreacted ingredients.

At this stage, there was an interaction of homologous epitopes of the pathogen with immobilized antibody-containing immunoglobulins.

Stage II. Introduction of fluorescent immunoglobulin diagnosticums into the wells of the strips.

Fluorescent immunoglobulin diagnosticums (PID) of R.prowazekii or R.typhi, or C.burnetii of phase I, or C.burnetii of phase II were introduced into all 8 wells of each strip from the 1st to the 8th:

- in all 8 wells of the 1st strip, 200 μl PID to R. prowazekii was added;

- in all 8 wells of the 2nd strip, 200 μl of PID to R.typhi were added;

- in all 8 wells of the 3rd strip, 200 μl PID to C.burnetii phase I was added;

- in all 8 wells of the 4th strip, 200 μl PID to C.burneti phase II was added;

- 200 µl of fluorescent immunoglobulin diagnosticum to R.prowazekii was added to every 2 wells of the 5th control strip, then 200 µl of fluorescent immunoglobulin diagnosticum to R.typhi was added to the next 2 wells, after which 200 µl of fluorescent immunoglobulin diagnosticum to C .burnetii phase I, followed by 200 µl of fluorescent immunoglobulin diagnosticum to C.burnetii phase II to assess spontaneous agglutination.

Thus, 200 µl PID to R. prowazekii was added to wells A5, B5;

- 200 µl PID to R.typhi was added to wells C5, D5;

- in wells E5, F5, 200 μl PID to C.burneti phase I was added;

- wells G5, H5 were filled with 200 µl PID to C.burneti phase II.

After the addition of PID, the plate with strips was closed with a lid and placed in a thermostat at 37°C for 60 min with occasional stirring every 15 min. After this time, the plate was taken out and the strips were washed by adding 200 μl of 0.9% sodium chloride solution with the addition of 5% phosphate buffer pH 7.2 to each well three times for 3 minutes to remove unreacted ingredients. The strips were dried and analyzed and recorded.

12.2. Registration and interpretation of results

At the second stage, in the case of binding pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II with antibody-containing immunoglobulins in each test sample, their epitopes interact with homologous corresponding fluorescent immunoglobulin diagnosticums and fluorescence appears in the well of the strip . The luminescence intensity of the strip wells was recorded using detector devices, which are special hardware-software analyzer complexes, which provided data on the presence of homologous pathogens in the test sample.

The use of fluorescent immunoglobulin diagnosticums to differentiate pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii II in immunofluorimetric analysis allows you to test each of the 7 studied samples with 4 fluorescent immunoglobulin diagnosticums at once.

Example 13.

A method of using fluorescent immunoglobulin diagnosticums for the differentiation of R.prowazekii or R.typhi or C.burneti phase I or C.burneti phase II on biological chips.

The reaction is based on the specific interaction of molecular probes - antibody-containing immunoglobulins immobilized in gel microcells of biochips with homologous pathogens, followed by the manifestation of the resulting complex using fluorescent immunoglobulin diagnosticums (Fig. 9, 10A and 10 B)

13.1 Statement of the reaction

The required number of biochips with gel microcells was selected. The number of required gel microcells in each biochip for setting up the main reaction was determined by the number of molecular probes, and the number of biochips was determined by the number of fluorescent immunoglobulin diagnosticums used. For the study of one sample, 8 biochips were used: 4 biochips (I) for setting up the experiment with the studied samples and 4 biochips (II) for setting the control of fluorescent immunoglobulin diagnosticums for spontaneous agglutination (Fig. 10A and 10B).

Each of the 8 biochips contained 4 gel microcells with molecular probes - immobilized antibody-containing immunoglobulins: R.prowazekii - in the first gel cell, R.typhi - in the second, C.burneti I phase - in the third, C.burneti II phase - in the fourth.

A 10% suspension (v/v) in 0.9% sodium chloride solution was prepared from the test sample. Next, the suspension was applied to all gel microwells of biochips from the 1st to the 4th, while the introduction was carried out using automatic dispensers for 200 μl.

The reaction was set up on biochips in 2 stages.

Stage 1. Application of the test sample to the gel microcells of the biochips.

On each biochip with gel microcells, inside which there are molecular probes - immobilized antibody-containing immunoglobulins: R.prowazekii, R.typhi, C.burneti phase I, C.burneti phase II, the test sample was applied.

The scheme of setting up the experiment at the 1st stage included:

- application of biochips on gel microcells from the 1st to the 4th - 200 µl of the test sample;

To control fluorescent immunoglobulin diagnosticums for spontaneous agglutination, similar control biochips (II) with gel microcells were used, inside of which there were molecular probes - immobilized antibody-containing immunoglobulins R.prowazekii, R.typhi, C.burneti phase I, C.burneti phase II.

The setting of the control at the 1st stage included applying 200 μl of 0.9% sodium chloride solution to the gel microcells of biochips from the 5th to the 8th. The staging was carried out as follows:

After that, all biochips were placed in a humid chamber and incubated for 60 min at 37°C. After this time, the biochips were washed with distilled water three times for 1 min and dried.

Stage II. Introduction of fluorescent immunoglobulin diagnosticums onto dried biochips.

For stage II, the corresponding fluorescent immunoglobulin diagnosticums were applied to experimental (I) and control (II) biochips.

200 μl of fluorescent immunoglobulin diagnosticum to R.prowazekii were added to all gel microcells of the 1st and 5th biochips;

- 200 μl of fluorescent immunoglobulin diagnosticum to R.typhi were added to all gel microcells of the 2nd and 6th biochips;

- on all gel microwells of biochips of the 3rd and 7th, 200 μl of a fluorescent immunoglobulin diagnosticum to C.burneti phase I were added;

- 200 μl of fluorescent immunoglobulin diagnosticum C.burneti phase II were added to all gel microwells of the 4th and 8th biochips.

All biochips after the introduction of fluorescent immunoglobulin diagnosticums were placed in a humid chamber and incubated for 60 min at 37°C. After this time, the biochips were washed with distilled water three times for 1 min, dried and analyzed.

13.2. Registration and interpretation of results.

At the second stage, in the case of binding pathogens R.prowazekii or R.typhi, or C.burnetii phase I, or C.burnetii phase II of the test sample with molecular probes - antibody-containing immunoglobulins, the epitopes of pathogens interacted with homologous corresponding fluorescent immunoglobulin diagnosticums and in the well of the strip fluorescence appeared (FIG. 9A).

No fluorescence was observed when staged with control samples, due to the absence of a homologous pathogen (Fig. 9B).

The intensity of the luminescence of the gel microcells of the biochip with the contents was recorded using detecting equipment, which was used as special hardware-software analyzer complexes, with their help a report was obtained on the presence of homologous pathogens in the test sample.

Thus, the use of biochips to differentiate pathogens R.prowazekii or R.typhi, or C.burnetii of phase I, or C.burnetii of phase II allows you to test one test sample at once with 4 fluorescent immunoglobulin diagnosticums simultaneously.

Example 14

Comparative evaluation of the results of the indirect method of fluorescent antibodies (nMFA) and the reaction of fluorescent microagglutination (RFMA) with fluorescent immunoglobulin diagnosticums.

The study was carried out with the studied samples, corpuscular and soluble pathogens (antigens) R.prowazekii and R.typhi.

The results of one typical series of experiments on the study of commercial pathogens (antigens) using fluorescent immunoglobulin diagnosticums R. prowazekii and R. typhi are presented in tables 1 and 2.

Table 1
Study of commercial pathogens (antigens) of rickettsiae of the typhus group with fluorescent immunoglobulin diagnosticums for R.typhi and R.prowazekii Researched
pathogens
(antigens) Series of pathogens (antigens) Reciprocal values of titers of rickettsia antigens in the test material FID to
R.prowa-zekii PID to R.typhi nMFA with polyvalent serum R.prowaze-kii R.typhi Corpuscular
pathogens
(antigens)
R.prowazekii 24 128 - 128 128 27 256 - 128 64 43 64 - 256 128 46 32 - 64 64 52 128 - 512 256 Soluble
pathogens
(antigens)
R.prowazekii fifteen
22
35
45
55 1024 - - - 2048 - - - 1024 - - - 4096 - - - 2048 - - - Corpuscular
pathogens
(antigens)
R.typhi
(experimental series) thirteen - 256 256 128 23 - 128 128 64 37 - 64 256 128 47 - 128 64 64 2 - 512 512 256 Soluble
pathogens
(antigens)
R.typhi eleven - 2048 - - sixteen - 1024 - - 25 - 512 - - 33 - 1024 - - 42 - 2048 - - Immunoglobulins to R.prowazekii (positive control) caption 1:256 Titer
1:128 R.typhi immunoglobulin (positive control) caption 1:128 Titer
1:256

As can be seen from the data presented in Table 1, the studied series of commercial pathogens (antigens) R.prowazekii and R.typhi contained both types of antigens, which was shown in nMFA with polyvalent serum to rickettsia of the typhus group. When staging the reaction of fluorescent microagglutination with a fluorescent immunoglobulin diagnosticum to R.prowazekii, positive results were observed only with pathogens (antigens) of R.prowazekii and were absent with pathogens (antigens) of R.typhi. On the contrary, in the reaction of fluorescent microagglutination with fluorescent immunoglobulin diagnosticum to R.typhi, positive results were found only with R.typhi and were absent with R.prowazekii.

The experimental data obtained indicate that fluorescent immunoglobulin diagnosticums prepared according to the claimed method provide a clear differentiation of rickettsiae of the typhus group.

The next stage of research was to study the possibility of using fluorescent immunoglobulin diagnosticums to R.prowazekii or R.typhi to identify rickettsiae in various materials of biological origin.

To identify rickettsiae of the typhus group, materials prepared from corpuscular pathogens (antigens), ovo- and tissue cultures of rickettsiae and entomological cultures of R.prowazekii and R.typhi were used.

To detect corpuscular and soluble rickettsia antigens prepared directly from the test material (chicken embryos, laboratory animals, carriers), a 5–10% suspension was preliminarily prepared.

The results of rickettsia differentiation in various sources of accumulation are shown in Table 2.

From the data presented in Table 2, it can be seen that the studied materials of various biological origin contain pathogens (antigens) of both R.prowazekii and R.typhi, as evidenced by positive results (fluorescence intensity 3+) in nMFA with polyvalent serum of the typhus group .

In objects containing R.prowazekii, when using fluorescent immunoglobulin diagnosticums for R.prowazekii, agglutination was observed (positive result), and when using fluorescent immunoglobulin diagnosticums for R.typhi, negative reactions were observed.

The reverse picture was observed in the study of objects containing R.typhi. In these materials of various origins, positive results were obtained when reacting with fluorescent immunoglobulin diagnosticums to R.typhi, and when using fluorescent immunoglobulin diagnosticums to R.prowazekii, negative reactions were obtained.

The results of these studies indicate the possibility of a clear identification of R.prowazekii or R.typhi using prepared fluorescent immunoglobulin diagnosticums for R.prowazekii or R.typhi.

table 2
Differentiation of R.prowazekii and R.typhi in various rickettsia-containing materials with fluorescent immunoglobulin diagnosticums (PID)
to R.prowazekii and to R.typhi Investigated rickettsia-containing material The presence of an immunofluorescent specific reaction in rickettsia-containing materials with Rickettsia fluorescence intensity in nMFA with polyvalent typhus serum PID R.prowazekii PID R.typhi causative agent R.prowazeki causative agent R.typhi Corpuscular pathogen R.prowazekii + - 3+ 3+ Soluble
pathogen
R.prowazekii + - 3+ 3+ Ovoculture R.prowazekii + - 3+ 3+ Entomological culture of R.prowazekii + - 3+ 3+ Corpuscular pathogen R.typhi - + 3+ 3+ Soluble
pathogen
R.typhi - + 3+ 3+ Ovoculture R.prowazekii - + 3+ 3+ Spleens of white mice infected with R.typhi - + 3+ 3+ Yolk membrane uninfected - - - - Spleen emulsion uninfected - - - - Suspension from intact lice - - - -

Designations: "+" the presence of rickettsia,

"-" absence of rickettsiae

"3+" intensity of rickettsia luminescence in nMPA

Studies of the differentiating ability of fluorescent immunoglobulin diagnosticums to C.burnetii phase I or C.burnetii phase II were carried out on C.burnetii pathogens (antigens) of different phase variability.

Table 3
Evaluation of the differentiating ability of fluorescent immunoglobulin diagnosticums to C.burnetii phase I or C.burnetii phase II Material under study The results of the reaction of fluorescent microagglutination with fluorescent immunoglobulin diagnosticum to C.burnetii phase I fluorescent immunoglobulin diagnosticum to C.burnetii phase II Corpuscular pathogen C.burnetii phase I + - Corpuscular pathogen C.burnetii phase II - + Ovoculture C.burnetii phase II - + Testicular pathogen C.burnetii phase I + - Vaccine Ku M-44 - +

As shown in Table 3, fluorescent immunoglobulin diagnosticums for C.burnetii phase I interact with the corpuscular pathogen (antigen) C.burnetii phase I and the testicular pathogen (antigen) C.burnetii phase I and do not react with the pathogen (antigen) C.burnetii II phases obtained from different biological materials. On the contrary, fluorescent immunoglobulin diagnosticums for C.burnetii phase II in the reaction of fluorescent microagglutination react positively with pathogens (antigens) of C.burnetii phase II and show a negative result with pathogens (antigens) of C.burnetii phase I. The data obtained indicate a clear differentiating ability of the pathogens C.burnetii phase I and C.burnetii phase II in the reaction of fluorescent microagglutination using fluorescent immunoglobulin diagnosticums to C.burnetii phase I or C.burnetii phase II.

The use of these diagnosticums in the production of coxiella vaccine will allow tracking the transition of C.burnetii phase I to C.burnetii phase II and evaluating the effectiveness of the vaccine preparation.

Thus, a clear differentiating ability of the developed fluorescent immunoglobulin diagnosticums to R.prowazekii or to R.typhi or C.burnetii of phase I or C.burnetii of phase II was established when indicating pathogens in materials of various biological origins.

All the above examples confirm that the task to be solved by the present invention, namely, the development of fluorescent immunoglobulin diagnosticums for R.prowazekii or for R.typhi or C.burnetii phase I or C.burnetii phase II based on adsorbed immunoglobulins and the use them for intraspecific differentiation of rickettsiae of the typhus group and coxiella in the reaction of fluorescent microagglutination (RFMA) while maintaining high sensitivity and specificity due to the adsorption of sensitin from cross-reacting and heterologous antigens and an increase in the number of active species-specific antibody determinants on the surface of the fluorescent matrix, has been solved.

The proposed variant of the use of fluorescent immunoglobulin diagnosticums in the fluorescent agglutination reaction was supplemented by their use in other serological methods: immunofluorimetric analysis and biological chips as indicator systems for the detection of rickettsiae and coxiella associated with immobilized antibody-containing immunoglobulins.

All the above examples confirm that the set technical problem has been solved.

The technical result and effect was achieved through the use of fluorescent lactobacilli as a matrix, and adsorbed antibody-containing immunoglobulins as an active substance, by combining the latter with a fluorescent matrix by means of a binding agent to form corpuscularized forms, which leads to an increase in specificity and sensitivity, standardization of fluorescent immunoglobulin diagnosticums, carrying out diagnostics in the format of a single-stage fluorescent microagglutination reaction, reducing the time of analysis by speeding up manipulations and eliminating a number of analysis steps (fixing drugs, multi-stage, adding reagents at different stages of formulation and removing unreacted ingredients by washing smears), as well as increasing the specificity of the reaction due to the absence induced non-specific and specific fluorescence.

In addition, the technical simplicity of the methods of differentiation, stability and standardization of the preparations used were noted, which ensures reliable reproducibility of the results and the possibility of working without constant communication with the rickettsiological laboratory.

Industrial Applicability

All the above examples also confirm the possibility of industrial application of this invention.

List of abbreviations

Biochip - biological chip

ELISA - enzyme-linked immunosorbent assay

IED - immunoglobulin erythrocyte diagnosticum

IE - immunochromatographic indicator element

IFLA - immunofluorimetric analysis

MFA - method of fluorescent antibodies

nMFA - indirect method of fluorescent antibodies

pMFA - direct method of fluorescent antibodies

pMFAG - direct method of fluorescent antigens

RNGA - reaction of indirect hemagglutination

RNA - ribonucleic acid

RSK - complement fixation reaction

RFMA - fluorescent microagglutination reaction

PID - fluorescent immunoglobulin diagnosticums

FITC - fluorescein isothiocyanate

C.burnetii - Coxiella burnetii

FRET - Fluorescent Resonance Transfer

R.prowazekii - Rickettsia prowazekii

R.typhi - Rickettsia typhi

Bibliography

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Claims (8)

1. A method for obtaining a fluorescent immunoglobulin diagnosticum for detecting causative agents of rickettsiosis and coxiellosis, which consists in preparing a fluorescent matrix by applying a fluorescent label to the matrix, then attaching analytes to it using a binding reagent, characterized in that formalinized lactobacilli are used as a matrix, and inorganic fluorochromes are used as a fluorescent label, while the antibody-containing fraction of immunoglobulins to rickettsia and coxiella in the range of 0.1-0.5 mg/ml is used as analytes, which are combined with a fluorescent matrix by means of a binding reagent and the final product is obtained - a fluorescent immunoglobulin diagnosticum to rickettsia and coxiella, while the antibody-containing fraction of immunoglobulins is obtained from the corresponding immune sera containing antibodies to rickettsia and coxiella by their isolation by combined methods: precipitation with neutral salts mi, gel filtration, removal of cross-reacting and heterologous antibodies. 2. The method according to p. 1, which consists in the fact that tannin is used as a binding reagent of the fluorescent matrix with the antibody-containing fraction of immunoglobulins in the volume ratio of the fluorescent matrix and tannin solution 1:1. 3. The method according to claim 1, which consists in using a solution of water-soluble carbodiimide in a volume ratio of fluorescent matrix and a solution of water-soluble carbodiimide 1:1 as a binding reagent of a fluorescent matrix with an antibody-containing fraction of immunoglobulins. 4. The method according to claim 1, which consists in using fluorescein isothiocyanate or nanocrystals in the form of semiconductor colloidal particles of different fluorescence as inorganic fluorochromes. 5. The method according to p. 1, which consists in the fact that the removal of cross-reacting and heterologous antibodies is carried out with a fluorescent antigenic diagnosticum for rickettsiae and coxiella. 6. The method according to p. 1, which consists in the fact that each fluorescent matrix after connection with antibody-containing fractions of immunoglobulins is incubated for 60 minutes at a temperature of 37°C. 7. Fluorescent immunoglobulin diagnosticum for the detection of causative agents of rickettsiosis and coxiellosis, obtained by the method according to claim 1, which is fluorescent formalized lactobacilli associated by means of a binding reagent with an antibody-containing fraction of immunoglobulins to rickettsiae and coxiella. 8. The use of a fluorescent immunoglobulin diagnosticum according to claim 7 for examining samples in order to identify pathogens of rickettsiosis and coxiellosis.

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