RU2726484C1 - Diagnostic technique for rickettsial diseases group of spotted fever, immunoassay diagnostic test system for implementation thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: group of inventions refers to clinical laboratory diagnostics, biotechnology and medicine. Disclosed is a method of producing inactivated soluble purified protein-lipopolysaccharide antigenic substancefor preparing a main component of a test system - an immunosorbent, comprising inactivated lyophilized fully solubleantigen resuspension, then the antigen suspension is subjected to ultrasonic treatment with subsequent centrifugation and the supernatant is extracted, then the supernatant is subjected to the chromatography on the chromatographic sorbent column, after which the fractions are evaluated for specific antigenic activity in the EIA and the fractions of the first peak are further combined and adjacent to it, which demonstrated in EIA best ratio of average values of optical density of OD of positive control samples to average values of optical density of OD of negative control samples. Also disclosed are a test system comprising said inactivated soluble purified protein-lipopolysaccharide antigenic substance, and a diagnostic technique for rickettsial diseases using said test system.EFFECT: group of inventions provides higher sensitivity, information value and specificity of the method for diagnosing rickettsial diseases, conducted by indirect solid-phase EIA technique.4 cl, 2 dwg, 3 tbl, 5 ex

Description

Technical field

The invention relates to the field of clinical laboratory diagnostics, biotechnology and medicine, and relates to a method for diagnosing rickettsioses of the group of tick-borne spotted fever and an enzyme immunoassay diagnostic test system for its implementation.

State of the art

For serological laboratory diagnostics of rickettsioses of the tick-borne spotted fever (LPF) group, well-known methods are used: complement fixation reaction (CSC), indirect immunofluorescence reaction (RNIF), immunofluorescence reaction (RIF), agglutination reaction, indirect hemagglutinated immunoassay test) blotting and others based, as a rule, on native rickettsial antigens. The starting material for the preparation of such antigens are pathogenic rickettsia infected with primary cultures, for example, well-characterized strains, ovocultures (accumulation is carried out in the yolk sac of developing chicken embryos), adapted cell cultures, rickettsial tissue material obtained from the organs of infected susceptible animals. Depending on the method of purification, corpuscular, soluble or whole-soluble antigens of rickettsia are obtained. The corpuscular antigen of rickettsia is a purified rickettsia (corpuscle) obtained, as a rule, by the method of combined ether treatment and differential centrifugation. Soluble rickettsial antigen is most often isolated by water-phenol extraction of rickettsial suspensions followed by centrifugation. The whole-soluble antigen, along with the soluble antigenic fraction, contains purified corpuscles [Zdrodovsky P.F., Golinevich E.M. The doctrine of rickettsia and rickettsioses. Moscow, "Medicine", 1972, 495 s].

It is known that ELISA has high sensitivity, specificity, the method is easily reproducible and available. A known method for laboratory diagnosis of tick-borne rickettsiosis using enzyme-linked immunosorbent assay to determine antibodies to the antigen Rickettsia sibirica according to patent RU 2477860 (prototype) (Rudakov N.V., Abramova N.V., Penievskaya N.A., Samoilenko I.E., Shpinov S.N., Reshetnikova T.A. Method for laboratory diagnosis of tick-borne rickettsiosis using enzyme-linked immunosorbent assay to determine antibodies to Rickettsia sibirica antigen Patent RU 2477860, priority from 13.08.2010), according to which for its implementation as an antigen for preparation immunosorbent is used "Diagnosticum rickettsial Sibirica dry for RSK" produced by NPO Microgen, Russia. In accordance with the description of the patent, the basis of this diagnosticum is the whole-soluble antigen of Rickettsia sibirica. According to the Instructions for use of the manufacturer NPO Microgen, Diagnosticum Rickettsial Sibirica dry for RSK is lyophilized antigenic complexes isolated by ether treatment from Siberian rickettsia grown (accumulated) in the yolk sacs of chicken embryos and inactivated with phenol. An enzyme-linked immunosorbent assay system based on it allows detecting antibodies of classes M and G to Rickettsia sibirica in human blood serum and performing serological diagnostics of tick-borne rickettsiosis with a sensitivity exceeding CSC. The disadvantage of the antigen used by the authors of the patent for the preparation of the immunosorbent of the enzyme-linked immunosorbent assay is that, despite the cleaning carried out in production conditions, it contains impurities of the tissues of the yolk sac shells of developing chicken embryos (ovocultures), which can lead to false positive results. Antibodies to the antigenic structures of ovoculture tissues can be found in individuals suffering from intolerance to the components of chicken eggs (allergic reactions, which usually develop to the main allergens - ovalbumin, ovamucoid, lysozyme, conalbumin), or for other reasons (for example, antibodies to lysozyme increase in patients with ulcerative colitis and rheumatoid vasculitis). Evidence of the specific immunoreactivity of antibodies in the test sample exclusively for the rickettsial antigen is the absence of reactivity of the sample with the control antigen. In the case of accumulation of rickettsiae (obtaining antigens) in ovocultures, a homogenizate of tissues of the yolk sac of a developing chicken embryo not infected with rickettsia can serve as such a control antigen. The use of a panel of blood sera from people suffering from allergic reactions to the components of chicken eggs showed that in ELISA, where the "Diagnosticum rickettsial Sibirica dry for RSK" was used for the preparation of immunosorbent, false positive values were revealed, confirmed by cross-immunoreactivity with a control antigen. A detailed proof of this is given in example 3. Thus, to increase the specificity of the method for diagnosing rickettsial diseases, a high degree of purification of the used rickettsial antigens from impurities of the cultivation medium (accumulation) is required.

It is known that for the serological diagnosis of rickettsioses, it is possible to use not the whole native antigen, but the lipopolysaccharide (LPS) or proteins isolated from it. For example, Fuller Laboratories, USA (http://www.fullerlabs.com) uses LPS for the production of enzyme-linked immunosorbent assays to determine IgG to antigens of the LP group, and OmpA surface antigens and / or OmpB. The Vircell company, Spain, uses a single inactivated native Rickettsia conorii antigen to detect antibodies of classes M and G to antigens of the Rickettsia conorii group (in particular, Rickettsia conorii), but the manufacturer does not disclose detailed information about this antigen.

Known commercial enzyme-linked immunosorbent assay systems for diagnosing rickettsioses of the LPL group are designed to detect specific antibodies of two classes in human serum or plasma - M (IgM) and G (IgG).

Disclosure of invention

The technical problem of the invention is to increase the sensitivity, information content and specificity of the method for diagnosing rickettsioses of the LP group, carried out by the method of indirect enzyme-linked immunosorbent assay.

The technical problem is solved due to the fact that the authors have developed a method for diagnosing rickettsioses of the group of tick-borne spotted fever by the method of enzyme-linked immunosorbent assay, which consists in the fact that separate differential detection of antibodies of classes M, G and A is carried out in human serum or plasma, and the immunosorbent is obtained on the basis of inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from a suspension of Rickettsia sibirica antigens.

A method has also been developed for obtaining inactivated soluble purified protein-lipopolysaccharide antigenic substance, which consists in ultrasonic treatment of rickettsial antigens previously resuspended in a buffer of 10 mM Tris-HCl and 0.5 M sodium chloride, pH-7.4-7.6, containing 10% isopropanol or 8 M urea, subsequent centrifugation and gel filtration of the supernatant on a column with a chromatographic sorbent, further combining the fraction of the first peak and those adjacent to it, demonstrating the best ratio of the optical density signal of positive human serum or plasma samples containing antibodies to antigens when checked in ELISA rickettsiae of the tick-borne spotted fever group, to the optical density of negative control samples of human serum or plasma that do not contain antibodies to the antigens of the rickettsiae of the tick-borne spotted fever group. In this case, ultrasonic treatment of previously resuspended rickettsial antigens is carried out in the frequency range of 15-23 kHz for 15-30 minutes, and centrifugation is carried out at 10,000 -12,000g.

An enzyme immunoassay diagnostic test system has also been created for implementing a method for diagnosing rickettsioses of the tick-borne spotted fever group, which includes:

immunosorbent obtained on the basis of inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from a suspension of rickettsial antigens of the tick-borne spotted fever group,

three conjugates: human anti-IgA, human anti-IgM and human anti-IgG, labeled with horseradish peroxidase,

auxiliary solutions for ELISA,

positive and negative control samples:

K + and K.

Moreover, in the immunoassay diagnostic test system, a solution for diluting samples, a solution for diluting conjugates, chromogen 3 ', 5' - tetramethylbenzidine, stop reagent, concentrate of phosphate-saline solution with tween 20 - FSR-T are used as auxiliary solutions. And as positive and negative control samples - K + and K - respectively - use human serum or plasma or a pool of human serum or plasma containing and, respectively, not containing specific antibodies of classes A, M, G to the antigens of rickettsia of the LP group, inactivated by heating ... A method has also been created for the diagnosis of rickettsioses of the tick-borne spotted fever group using an enzyme immunoassay diagnostic test system, which consists in the fact that 100 μl are added to the wells of parallel rows of the immunosorbent into the well of the dilution of the test samples of human serum or plasma and control samples K + and K- prepared using a solution for diluting samples in initial dilutions of 1: 10 to detect specific antibodies of class A; 1:20 for detecting class M antibodies; 1: 40 to detect specific antibodies of class G, then the diluted test and control samples are incubated at 37-38 ° C for 45-60 minutes, the immunosorbent is washed with a working solution of FSR-T 5-6 times, and 100 times are added to the corresponding rows of the immunosorbent μl per well, working dilutions of human anti-IgA, human anti-IgM and human anti-IgG conjugates labeled with horseradish peroxidase are incubated at 37-38 ° C for 30-60 minutes, the immunosorbent is washed with FSR-T 5-6 times, 100 μl of TMB chromogen is added to each well of the immunosorbent and kept for 10-30 minutes at room temperature, protecting the plate from direct light, then the enzymatic reaction is stopped by adding 50-100 μl of stop reagent and spectrometric measurement of optical density of OP is carried out at a wavelength of 450 nm with the possible use of a reference wave of 620-680 nm, the OPcrit threshold value for each class of antibodies is calculated separately using the formula:

ODcrit = average value of OD K- +0.2,

where OD K- is the average value of optical density in the wells of the corresponding rows with a negative control sample K-.

Description of figures

FIG. 1

shows a typical chromatogram obtained at the final stage of processing and purification of a suspension of primary antigens of Rickettsia sibirica. The elution profile of the supernatant after ultrasonic treatment of a suspension of the Rickettsia sibirica whole-soluble antigen was demonstrated. Inactivated soluble purified protein-lipopolysaccharide antigenic substance is obtained as a result of combining the fractions of the first peak and some adjacent to it, which demonstrated in ELISA the best ratio of the mean values of optical density (OD) of positive control samples containing antibodies to antigens of rickettsia from the LP group, to the average values of OD negative control samples that do not contain antibodies to the antigens of the rickettsia group of the LP.

Fractions 1-30 are marked on the X-axis (the volume of each fraction is 1.5 ml), the total volume of the phase passing through the column is 45 ml; Y-axis - optical density (detection at two wavelengths 260 nm and 280 nm).

FIG. 2

shows electrophoresis of inactivated soluble purified protein-lipopolysaccharide antigenic substance in a polyacrylamide gel, stained with EZBlue ^tm GelStainingReagent (Sigma-Aldrich, USA). To characterize the obtained inactivated soluble purified protein-lipopolysaccharide, the content of protein and bacterial lipopolysaccharides is estimated.

1 - marker, 2 - inactivated soluble purified protein-lipopolysaccharide substance.

DISCLOSURE OF THE INVENTION

The technical result of the invention is achieved by additional processing and purification of primary suspensions of whole-soluble or corpuscular or soluble antigens of Rickettsia sibirica. For this, inactivated rickettsial antigens are resuspended in a buffer, for example, 10 mM Tris-HCl and 0.5 M sodium chloride, pH 7.4-7.6, containing 10% isopropanol or 8 M urea, and subjected to ultrasonic treatment in the frequency range 15 -23 kHz, for 15-30 minutes with periodic cooling using a suitable model of ultrasonic disintegrator. The resulting suspension is centrifuged at 10,000 - 12,000 g, the supernatant is taken, and the supernatant (soluble antigen) is gel-filtered on a column filled with a chromatographic sorbent, for example, Superdex-200 sorbent. Fractions are assessed for specific antigenic activity in ELISA using control human blood sera, containing and not containing specific antibodies to the antigens of rickettsiae of the LP group. The fractions of the first peak and some adjacent to it, demonstrating the best ratio of the signal of the OD of positive control samples (containing antibodies to antigens of the Rickettsia LP group) to the OD of negative control samples (not containing antibodies to the antigens of the Rickettsia LP group), are combined and used in further to obtain an immunosorbent enzyme immunoassay test system. The quantitative determination of protein is assessed spectrophotometrically according to one of the known methods (OFS.1.2.3.0012.15. Protein determination), the quantitative determination of bacterial lipopolysaccharides is assessed in the LAL test according to one of the regulated methods (OFS.1.2.4.0006.15. Bacterial endotoxins).

To prepare an immunosorbent, a working dilution of an inactivated soluble purified protein-lipopolysaccharide substance is sorbed onto a solid phase (a suitable model of a polystyrene 96-well plate for ELISA purposes). The selection of optimal dilutions is well understood by a specialist and does not require additional explanations.

The technical result is also achieved by the selection of conditions for ELISA and the fact that in order to increase the information content and sensitivity of the method for diagnosing rickettsioses of the LP group, the test sample (human serum or plasma) is simultaneously and differentially examined not only for the presence of specific antibodies of classes G and M, but also for the presence class A antibodies with separate use of three antibody conjugates (anti-IgA, anti-IgM, anti-human IgG, labeled with peroxidase).

The method for diagnosing rickettsioses of the LP group is that the serum / plasma of a patient with suspected rickettsiosis caused by the causative agent of the LP group is examined for the presence of specific antibodies by an indirect ELISA method. To simplify the method for diagnosing rickettsioses of the LP group, an enzyme immunoassay diagnostic test system has been developed, which is a multicomponent set of reagents and includes an immunosorbent based on an inactivated soluble purified protein-lipopolysaccharide antigenic substance, three conjugates (human anti-IgA, human anti-IgM and anti-IgG human labeled with horseradish peroxidase), auxiliary solutions for ELISA, positive and negative control samples (K + and K). Auxiliary solutions are sample dilution solution (PPO), conjugate dilution solution (PPK), chromogen (3 ', 5' - tetramethylbenzidine, hereinafter - TMB), stop reagent, concentrate of phosphate-saline solution with tween 20 (PSR-T ). Dilutions of the test and control samples (K + and K-) prepared using PPO are introduced into the wells of three parallel rows of immunosorbent (for example, row 1 is isolated to detect specific antibodies of class A, row 2 - to detect specific antibodies of class M, wells row 3 - to detect specific antibodies of class G). Initial dilutions of samples for the purpose of detecting specific class A antibodies to the antigens of rickettsiae of the LPL group (inactivated soluble purified protein-lipopolysaccharide antigenic substance) - 1: 10, for detecting specific antibodies of class M - 1: 20, for detecting specific antibodies of class G - 1: 40. Next, the dilutions of the test and control samples are incubated, for which the lid-protected immunosorbent is kept at a temperature of 37-38 ° C, preferably in a humid chamber, for 45-60 minutes. The wells of the immunosorbent are washed with the FSR-T working solution 5-6 times, adding at least 200 μl of the solution to the well. Working dilutions of three conjugates (human anti-IgA, human anti-IgM and human anti-IgG labeled with horseradish peroxidase) are prepared using PPK. Separately, 100 μl of the corresponding conjugates are added to the wells of the immunosorbent rows, incubation is carried out at a temperature of 37-38 ° C for 30-60 minutes. Then the plate (immunosorbent) is washed with FSR-T 5-6 times and 100 μl is added to each well of the TMB chromogen and incubated for 10-30 minutes at room temperature, protecting the plate from direct light. To stop the reaction, 50-100 μl of stop reagent is added to the wells of the immunosorbent and the optical density (OD) is measured using a suitable model of the spectrophotometer at a wavelength of 450 nm with the possible use of a reference wave of 620-680 nm. To evaluate the results of ELISA, the threshold value of OD - ODcrit is calculated separately for each class of antibodies according to the formula: ODcrit = average OD (K-) + 0.2, where OD (K-) is the average value of optical density in the wells of the corresponding rows with negative control sample (K-). The test sample contains specific antibodies of class A and / or M and / or G, if its OD value is greater than or equal to ODcrit.

Based on the results of ELISA using the created immunoassay diagnostic test system, it is concluded that there are specific antibodies of classes A, M, G to the antigens of rickettsia of the LP group in human serum / plasma.

The invention is illustrated by the following examples.

Example 1.

Obtaining inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from the whole-soluble antigen of Rickettsia sibirica.

To obtain an inactivated soluble purified protein-lipopolysaccharide antigenic substance, an inactivated lyophilized whole-soluble preparation of the Rickettsia sibirica antigen (strain “Netsvetaev”) produced at the Federal State Budgetary Institution “NITsEM named after V.I. N.F. Gamalea "of the Ministry of Health of Russia (the accumulation of rickettsia was carried out in the yolk sacs of developing chicken embryos).

The inactivated lyophilized whole-soluble Rickettsia sibirica antigen was resuspended in a buffer of 10 mM Tris-HCl and 0.5 M sodium chloride, pH 7.4-7.6, containing 10% isopropanol. Next, the antigen suspension was subjected to ultrasonic treatment at a frequency of 23 kHz using an ultrasonic disintegrator SoniPrep 150 Plus (MSE, UK), for which the stem was immersed in a glass of water, the test tube with the antigen suspension was placed in the same glass. Ultrasonic treatment was carried out in the mode of 5 min sonication, followed by cooling the test tube and changing the water in the glass. The sonication time was 30 min. The resulting suspension was centrifuged at 10000 g and the supernatant was collected. Size exclusion chromatography (gel filtration) of the supernatant was carried out using an AktaExplorer 10 chromatograph (GE, Sweden) at a constant temperature of + 4 ° C. The collected supernatant was applied to a 10 × 300 mm column packed with Superdex 200 sorbent (GE, Sweden). Used a mobile phase of the following composition: YutM Tris-HCl and 0.5 M sodium chloride, pH 7.5, containing 10% isopropanol. The resulting chromatogram is shown in FIG. 1. As seen in FIG. 1, three main peaks are clearly distinguished on the chromatogram. The fractions were evaluated for specific antigenic activity in ELISA using human serum / plasma, containing and not containing specific antibodies to the antigens of the rickettsia group of the KPP group (Table 1).

Table 1. Results of testing the fractions obtained as a result of the gel filtration for specific antigenic activity.

Note: * OD (+) / OD (-) is the ratio of the mean optical density values of positive human serum / plasma samples containing antibodies to rickettsia of the CPP group to the mean optical density values of negative control serum / plasma samples of human blood that do not contain antibodies to the rickettsia of the KPP group.

Fractions of the first peak (nos. 6-8), demonstrating the best ratio of the mean value of the optical density signals of five positive human blood serum samples containing antibodies to rickettsia antigens of the CAT group (OD +), to the mean optical density of five negative samples human blood sera that did not contain antibodies to the antigens of the rickettsia group of the LP (OP-) group were combined. Obtained by such a combination of fractions 6,7,8, the target fraction was an inactivated soluble purified protein-lipopolysaccharide antigenic substance. The rest of the fractions demonstrating a decrease in OD (+) / OD (-) by 30% or more in ELISA compared to the average OD (+) / OD (-) calculated for the fractions of the first peak (in this example, the average value is 8, 1) were excluded.

For a qualitative assessment of the obtained antigenic substance, electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate was carried out according to the known method (OFS 1.2.1.0023.15. Polyacrylamide gel electrophoresis). The gel was stained using the EZ Blue Gel Staining Reagent (Sigma-Aldrich, USA). Electrophoresis is shown in FIG. 2.

The quantitative determination of protein in the obtained antigenic preparation was assessed spectrophotometrically according to the Bradford method (OFS.1.2.3.0012.15. Protein determination). The quantitative determination of bacterial lipopolysaccharides was assessed using the turbidimetric kinetic LAL test (OFS.1.2.4.0006.15. Bacterial endotoxins). The protein content in the obtained antigenic substance, determined by the Bradford method, was 0.292 mg / ml; the content of bacterial lipopolysaccharides, determined using the kinetic turbidimetric LAL test, was 6654 EU / ml.

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

By a similar technique, an inactivated soluble purified protein-lipopolysaccharide antigenic substance can be obtained using corpuscular or whole-soluble or soluble antigens of other pathogenic rickettsia species included in the LPS group.

Example 2.

Obtaining an immunoassay diagnostic test system for implementing a method for diagnosing rickettsioses of the group of tick-borne spotted fever.

An enzyme immunoassay diagnostic test system for implementing a method for diagnosing rickettsial diseases of the tick-borne spotted fever group is a set of reagents (components) designed to detect in human serum / plasma specific antibodies of classes A, M, G to antigens of rickettsia of the LP group.

For the preparation of the main component of the test system - immunosorbent - used inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from a suspension of whole-soluble Rickettsia sibirica (strain Netsvetaev). A detailed description of the preparation of inactivated soluble purified protein-lipopolysaccharide is given in example 1. Inactivated soluble purified protein-lipopolysaccharide antigenic substance, diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.6, to a working concentration (for protein 10 μg / ml , the content of bacterial endotoxins is 225 EU / ml), was sorbed for 18 h at room temperature (22 ° C) in the wells of the Maxi Sorp Costar plate in a volume of 100 μl per well. Then the contents of the immunosorbent were discarded and added for 1 h at room temperature, 150 μL in each well with a solution to block nonspecific binding (0.05M carbonate-bicarbonate buffer pH-9.6, containing 0.5% sodium caseinate solution and preservatives), after the solution was removed, the plate was dried, placed in a foil bag with embedded silica gel and sealed under vacuum.

To prepare conjugate No. 1 (11-fold concentrate of human anti-IgA conjugate with peroxidase), a preparation of affine-purified goat antibodies against human immunoglobulins A labeled with horseradish peroxidase (OOO Imtek, Russia), diluted to a concentration of 7 μg / ml on a mixture of glycerin 30% and cattle serum 70%. To prepare conjugate No. 2 (11-fold concentrate of human anti-IgM conjugate labeled with peroxidase), a preparation of affine-purified goat antibodies against human immunoglobulins M labeled with horseradish peroxidase (OOO Imtek, Russia) was used, diluted to a concentration of 2 μg / ml on the mixture from glycerin 30% and cattle serum 70%. To prepare conjugate No. 3 (11-fold concentrate of human anti-IgG conjugate, labeled with peroxidase), a preparation of affine-purified goat antibodies against human immunoglobulins G labeled with horseradish peroxidase (OOO Imtek, Russia) was used, diluted to a concentration of 8 μg / ml on the mixture from glycerin 30% and cattle serum 70%.

Prepared auxiliary solutions for ELISA:

- solution for dilution of samples (PPO) composition: 0.1 M phosphate-buffered saline, pH 7.3-7.5, containing 1% sodium caseinate solution, 0.25 M urea, 0.5 M sodium chloride, 0, 01% tween-20, 0.01% sodium merthiolate;

- solution for dilution of conjugates (RRK) composition: 0.1 M phosphate-buffered saline, pH 7.3-7.4, containing 10% bovine serum, 0.05% Tween-20;

- chromogen (one-component aqueous solution of 3 ', 5'-tetramethylbenzidine, hereinafter - TMB) produced by CJSC "NEYU IMMUNOTECH", Russia;

- stop reagent (0.75 normal sulfuric acid);

- concentrate of phosphate-saline solution with tween 20 (FSR-T) for washing the plates (composition of the working solution FSR-T after diluting the concentrate with distilled water: 0.1 M phosphate buffered saline, pH 7.3-7.4, containing 0 , 15 M sodium chloride, 0.05% tween-20).

Prepared positive and negative control samples (K + and K). A positive control sample (K +) is represented by human serum / plasma or a pool of human serum / plasma containing antibodies of classes A, M, G to Rickettsia antigens of the LP group, inactivated by heating in a water bath for 30 min at 56 ° C. A negative control sample (K-) is represented by human serum / plasma or a pool of human serum / plasma that does not contain antibodies of classes A, M, G to antigens of the rickettsia group of the LP, also inactivated by heating in a water bath for 30 min at 56 ° C ...

All components are properly packed and labeled. The test system (set of reagents) is packed in a cardboard box, supplied with instructions for use with a description of the purpose, composition, detailed sequence of the analysis with its help, indication of the manufacturer, production time and shelf life, and other additional necessary information.

The above recipes for reagents (components) in the diagnostic enzyme immunoassay test system are given for a specific example. Correction of formulations is in the competence of a specialist.

Thus, the immunoassay diagnostic test system is a multicomponent set of reagents designed for the differential detection of specific antibodies of classes A, M, G to antigens of the LP group in human serum / plasma.

Example 3.

Study of the parameters of the sensitivity and specificity of the enzyme-linked immunosorbent assay in the comparative analysis of immunosorbents obtained on the basis of the whole-soluble antigens of Rickettsia sibirica and inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from a suspension of the whole-soluble antigen of Rickettsia sibirica.

For a comparative assessment of the parameters of the sensitivity and specificity of the enzyme immunoassay, immunosorbents were prepared separately based on lyophilized inactivated whole-soluble antigen Rickettsia sibirica, strain "Netsvetaev" (FGBU "NF Gamaleya NITsEM" (Ministry of Health of the Russian Federation), "Diagnostic Sibiricum rickettsial NPO Microgen, Russia) and inactivated soluble purified protein-lipopolysaccharide antigenic substance obtained by the original method (see example 1). For this, sorption was carried out in the wells of polystyrene plates Maxi Sorp Corning, USA in a volume of 100 μl in each well of the working dilutions of the above antigens and inactivated soluble purified protein-lipopolysaccharide antigenic substance prepared using 0.05 M carbonate-bicarbonate buffer, pH-9, 6. The plates covered with lids were kept for 20 hours at a temperature of 18 ° C, after the contents of the wells were removed without washing the plate, and a solution was added to block nonspecific binding for 1 hour of the composition shown in example 2 at a temperature of 18 ° C. The contents of the wells were removed, the plates were dried and used for ELISA. Thus, immunosorbent 1 was obtained as a result of sorption of "Diagnosticum of rickettsial Siberia dry for RSK" at a concentration of 200 μg / ml. Immunosorbent 2 was obtained as a result of sorption of an inactivated whole-soluble antigen of Rickettsia sibirica, strain "Netsvetaev" (NF Gamaleya Research Center for Electrochemistry, Ministry of Health of the Russian Federation), diluted to a concentration of 200 μg / ml. To obtain immunosorbent 3, an inactivated soluble purified protein-lipopolysaccharide substance was used, isolated from an inactivated whole-soluble antigen Rickettsia sibirica, strain "Netsvetaev", sorbed at a concentration of 10 μg / ml for protein and a content of bacterial endotoxins of 225 EU / ml.

In addition, to assess the specificity of the enzyme-linked immunosorbent assay using immunosorbents 1, 2, 3 and to identify possible cross-over (false-positive reactions), immunosorbent 5 was prepared in a similar way based on a control antigen, which is a homogenizate of tissues not infected with rickettsiae from the tissues of the yolk sac of developing chicken embryos (FGBU NITsEM them. NF Gamalei "of the Ministry of Health of Russia), which was sorbed at a selected concentration of 10 μg / ml. Also prepared immunosorbent 4 based on egg lysozyme (Amresco, United States) at a concentration of 5 μg / ml.

PPO, RRK, FSR-T, TMB, stop reagent, control samples (K + and K-) for ELISA used the composition shown in example 2.

For a comparative assessment of the sensitivity of ELISA used:

- 18 sera / blood plasma of patients diagnosed with Astrakhan spotted fever, established by a set of clinical and epidemiological data and confirmed serologically using a commercial enzyme immunoassay test system "Rickettsia conorii ELISA IgG / IgM" produced by Vircell, Spain;

- 2 blood serum of a person with a preliminary diagnosis of "African spotted fever", established on the basis of a set of clinical and epidemiological data and confirmed by PCR and serologically (RNIF);

- 6 blood serums / plasma of patients with an established clinical diagnosis of tick-borne typhus of North Asia (tick-borne rickettsiosis, in the text - KR) from the collection of the laboratory of ecology of rickettsia of the Federal State Budgetary Institution NITsEM named after N.F. Gamalei "of the Ministry of Health of the Russian Federation and confirmed serologically in RSK / RNIF.

For a comparative assessment of the specificity of ELISA used:

- 6 control human blood sera that do not contain antibodies according to the RNIF, ELISA, dot blotting, RSK to antigens of rickettsiae of the KPL group;

- 30 sera of conditionally healthy blood of people who were uninfected at the time of receiving blood sera and did not previously tolerate rickettsial etiology diseases.

ELISA was performed using the prepared immunosorbents under the same conditions. In a specific example, all human serum / plasma samples were tested against only specific antibodies of classes M and G. Antibodies of class A were not evaluated in this example, because the aim of the study was a comparative assessment of not only the specificity, but also the sensitivity of the assay (diagnostic method) for one variable parameter - the composition of the immunosorbent.

In parallel wells of each immunosorbent, the test samples were added in a volume of 100 μl at dilutions of 1: 20 in order to detect specific antibodies of class M and 1:40 in order to detect antibodies of class G prepared on PPO; immunosorbents were incubated for 45 min at 37 ° C. Then the contents of the wells were removed into a container for collecting the infected material, the wells of the immunosorbents were washed 6 times with the working solution of FSR-T. In the wells of one row, 100 μl of affine-purified goat antibodies against human immunoglobulins M labeled with horseradish peroxidase (Imtek LLC, Russia) at a final concentration of 0.2 μg / ml were added, and the conjugate of affine-purified goat antibodies against immunoglobulins G was added to the wells of a parallel row. human labeled with horseradish peroxidase (OOO "Imtek", Russia) at a final concentration of 0.7 μg / ml. Dilutions of conjugates were performed using PPK. The plates (immunosorbents) were incubated for 30 min at 37 ° C, after which the contents of the wells were removed into a container for collecting infected material, the wells were washed 6 times with the working solution of FSR-T, 100 μL of TMB solution was added and kept for 15 min protected from light place at a temperature of 18 ° C. The reaction was stopped by adding 50 μl of stop reagent to all wells of the plate. The results were recorded spectrophotometrically at a wavelength of 450 nm. Interpretation of the results of ELISA with immunosorbents 1, 2, 3 was carried out as follows. The ODcrit was calculated separately to determine the threshold values of the level of antibodies of classes M and G by the formula: ODcrit = average OD (K-) + 0.2, where OD (K-) is the average value of optical density in the wells with K- introduced in rows, marked for the detection of class M antibodies, and in the wells with K- introduced in the rows for the detection of class G antibodies. The sample was considered positive for the presence of antibodies of a certain class (M or G) to the antigens of the rickettsia group of the LP, if the optical density of the analyzed sample was the corresponding value of OPcrit.

To interpret the results of ELISA with immunosorbents 4 and 5, the mean OD was calculated for all sera included in the study, excluding those sera whose OD deviated from the mean OD by more than 3 standard deviations, calculated using the methods of mathematical statistics. Calculated ODcrit = average OD + 0.2. The sample was considered positive for the presence of antibodies of a certain class to lysozyme or control antigen if the optical density of the analyzed sample was higher and equal to the ODcrit value. The results are shown in Table 2.

Table 2. Comparative evaluation of the sensitivity and specificity of ELISA when using the whole-soluble antigens of Rickettsia sibirica and inactivated soluble purified protein-lipopolysaccharide substance.

false positive samples in the study of negative control blood sera (n = 6) The number of false positive samples in the study of blood sera of apparently healthy people (n = 30) 3/30 3/30 0/30 1/30 4/30 Sensitivity,% 88.5 88.5 one hundred - - Specificity,% 88.9 88.9 one hundred - -

Note:

1 - "Diagnosticum rickettsial Siberica dry for RSK",

2 - whole-soluble antigen of Rickettsia sibirica (strain "Netsvetaev"),

3 - inactivated soluble purified protein-lipopolysaccharide substance isolated from whole-soluble Rickettsia sibirica,

4 - lysozyme from egg yolk,

5 - control antigen.

The presented data convincingly demonstrate that false positive reactions when using immunosorbents 1 and 2 were caused by the presence of impurities of ovoculture tissues in them, which was proved in the analysis with a control antigen (immunosorbent 5), which has a complex composition and also includes determinants common with lysozyme (immunosorbent 4 ). The specificity of ELISA using immunosorbent 3 was 100% and exceeded the specificity achieved by using insufficiently purified antigen of Rickettsia sibirica and Diagnosticum of Rickettsia Sibirica for RSC (immunosorbents 1 and 2).

The sensitivity of ELISA using immunosorbent 3 also exceeded the sensitivity parameters of the assay using immunosorbents 1 and 2. This is explained by the fact that for the preparation of inactivated soluble purified protein-lipopolysaccharide substance, the primary suspension of antigens is subjected to ultrasonic treatment, which additionally contributes to the complete conversion of the antigen into a soluble form, when this achieves the greatest availability of the main antigenic determinants for efficient binding by specific antibodies.

It also follows from the data presented that the diagnosis of rickettsioses of the LP group is group-specific, because Rickettsia sibirica antigen and antigenic substances obtained on its basis made it possible to successfully serologically detect not only tick-borne typhus of North Asia (tick-borne rickettsiosis), but also APL and African spotted fever.

Example 4.

A method for diagnosing rickettsial diseases of the LP group using the developed enzyme-linked immunosorbent assay on the example of studying serum or plasma samples from patients Sh., S., K., V. with suspected diseases of rickettsial etiology caused by causative agents of rickettsial diseases of the LP group.

The developed enzyme-linked immunosorbent assay was used, which allows the determination of antibodies of classes A, M, and G to the antigens of the Rickettsia group of the KPL group in human serum or plasma (a description of the test system and the composition of the main components are presented in example 2).

In the wells of row 1 of the immunosorbent, marked for the detection of specific IgA (antibodies of class A) to the antigens of rickettsiae of the LP group, 180 μl of PPO and 20 μl of blood sera of the studied patients Sh., S., K., V. and control samples K + and K were added. - (final dilution of samples - 1:10). In the wells of the 2nd row of the immunosorbent, marked by us for the detection of specific IgM (class M antibodies) to the antigens of the rickettsiae of the LP group, 100 μl of PPO and 100 μl of sera from the studied patients Sh., S., K., V. and control samples K + were added and K-, diluted preliminarily 1:10 in the wells of row 1. In parallel wells of 3 rows of immunosorbent, marked for the detection of specific IgG (antibodies of class G) to the antigens of the Rickettsia group of the LPO group, 100 μl of PPO and 100 μl of sera / plasma of the studied patients Sh., S., K., V. and control samples K + and K-, diluted previously 1:20 in the wells of row 2 (the final dilution of the samples was 1: 40), then 100 μl of diluted samples were removed from each well as follows so that the residual volume is 100 μl. The immunosorbent was incubated, protected with a lid, at 37 ° C in a humid chamber for 45 min. Next, the immunosorbent was washed 6 times with the FSR-T working solution. In the wells of row 1 of the immunosorbent, 100 μl of the working dilution of conjugate 1, diluted with PPK, was added. In the wells of row 2 of the immunosorbent, 100 μl of the working dilution of conjugate 2 diluted with PPK was added. In the wells of row 3 of the immunosorbent, 100 μl of the working dilution of conjugate 3 diluted with PPK was added. The plate was incubated at 37 ° C for 30 min. After that, the plate (immunosorbent) was washed with FSR-T 6 times and added with 100 μl of an aqueous single-component chromogen TMB, kept for 15 min at room temperature, protecting the plate from direct light. To stop the reaction, 50 μL of stop reagent was added to the wells of the immunosorbent. The OD was measured on a spectrophotometer at a wavelength of 450 nm. The ODcrit was calculated for each class of antibodies according to the formula: ODcrit = average OD (K-) + 0.2, where OD (K-) is the average optical density in the wells with K- added.

In a specific example, the OPcrit values are as follows:

ODcrit (IgA) = 0.2 + 0.074 = 0.274, where 0.074 is the average value for two holes OD (K-) in row 1,

ODcrit (IgM) = 0.2 + 0.061 = 0.261, where 0.061 is the average value for two holes OD (K-) in row 2,

ODcrit (IgG) = 0.2 + 0.068 = 0.268, where 0.068 is the average value for two holes OD (K-) in row 3.

OD (K +) in the holes of rows 1, 2, 3, respectively, were equal to the values of 0.564; 0.671; 0.988. The test system is considered valid because positive OD values were obtained in the wells with a positive control (K +) introduced.

Based on the ELISA results, it was concluded that antibodies A, M, G were present in the blood serum of patients Sh., S., K., V. to the antigens of rickettsiae of the LP group.

Excerpts from the medical history of patients Sh., S., K., V. and the results of specific laboratory tests are given below.

Patient Sh., 6 years old, was admitted to the regional infectious diseases hospital in Astrakhan with suspected clinical and epidemiological data for an acute APL disease (characteristic maculopapular rash, fever). There was no primary affect.

Conducted specific laboratory tests:

PCR for the presence of Rickettsia conorii in leukocyte blood sediment using the AmpliSens® Rickettsia conorii-FL reagent kit with hybridization-fluorescent detection of results (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor) - a negative result was obtained,

ELISA results obtained using the commercial immunoassay diagnostic test system "Rickettsia conorii ELISA IgG / IgM" (Vircell, Spain) - no specific antibodies of classes G and M were detected in the blood serum,

- ELISA results using the developed test system based on inactivated soluble purified protein-lipopolysaccharide substance isolated from Rickettsia sibirica - antibodies of classes M and G were not detected in blood serum, because The OD of the patient's blood serum in rows 2 and 3 corresponded to the values of 0.078 and 0.095 and did not exceed the ODcrit values calculated for specific IgM and IgG. However, antibodies of class A were detected, since Serum OD in row 1 was 1.1204 and exceeded OD crit (IgA) = 0.274.

Thus, in patient Sh. Suspected of having APL according to clinical and epidemiological data, the diagnosis was confirmed by the detection of specific class A antibodies to antigens of rickettsiae of the LP group in the blood serum.

Patient S., 75 years old, was admitted to the regional infectious diseases hospital in Astrakhan with suspicion of acute APL disease due to clinical and epidemiological data.

Conducted specific laboratory tests:

- PCR for the presence of Rickettsia conorii in leukocyte blood sediment using a commercial kit of reagents "AmpliSens® Rickettsia conorii-FL" - a positive result,

- ELISA using the developed test system based on inactivated soluble purified protein-lipopolysaccharide substance isolated from Rickettsia sibirica - antibodies of classes A (OD of serum = 1.141) and G (OD = 0.603) were detected in the patient's blood serum and no antibodies of class M (OD = 0.09).

Thus, the serological picture obtained using the developed test system additionally confirmed the picture of acute rickettsiosis, in particular, APL.

Patient K., was admitted to the clinical infectious diseases hospital No. 1 in Moscow with suspicion of rickettsiosis of the LP group (African spotted fever?) According to clinical and epidemiological data. Infection appears to have occurred as a result of tick sucking (primary affect noted) during a safari tour in Tanzania.

Conducted specific laboratory tests:

- when using a test system in the PCR format "RealBest DNA Rickettsia species" (CJSC "Vector-Best", Russia) and using a washout from a biopsy of primary affect as a biomaterial, a positive result was obtained,

- ELISA using a test system based on an inactivated soluble purified protein-lipopolysaccharide substance isolated from Rickettsia sibirica - specific antibodies of all three classes were detected in patient K.'s blood plasma: A (OD = 1.077), class M (OD = 0.456), class G (OD = 0.507), which, along with the PCR results, additionally confirmed the primary diagnosis of acute rickettsiosis of the LP group (African spotted fever).

Patient V., 45 years old, was admitted to the regional infectious diseases hospital in Astrakhan with suspicion of adenovirus infection on the 14th day of the disease from the moment of registration of the first symptoms of infection (there was a high temperature, conjunctivitis, catarrhal phenomena, at the time of hospitalization - the absence of a rash characteristic of APL). During the hospitalization of patient V., no laboratory studies were carried out to verify the diagnosis of APL. Retrospectively, only class A antibodies (OD = 1.198) were detected in the blood plasma of patient B. using the developed ELISA test system. Rickettsia conorii DNA was also retrospectively isolated from a blood plasma sample using the AmpliSens® Rickettsia conorii-FL reagent kit. Consequently, the rickettsial nature of the infectious disease (APL) was retrospectively proven, including serologically using the developed enzyme-linked immunosorbent assay.

Thus, the method for diagnosing rickettsioses of the LP group, which provides for the separate detection of specific antibodies not only of classes M and G, but also of class A in human serum or plasma, is the most informative and sensitive, which is especially important in the early period of infection.

Example 5.

The possibility of using the enzyme-linked immunosorbent assay not only for the diagnosis of rickettsiosis caused by Rickettsia sibirica. Since serological diagnostics of LP rickettsia is group-specific, it was of interest to study the sera or blood plasma of patients with tick-borne rickettsia caused by pathogens (rickettsia) of the LP group in relation to the efficiency of detecting specific antibodies in them using the proposed enzyme-linked immunosorbent assay. For the preparation of immunosorbents, an inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from a suspension of the whole-dissolving antigen of R. sibirica subsp. sibirica (Netsvetaev strain). The substance was obtained according to the original method described earlier (example 1). We used a test system, the description of which is described in example 2. The setting of ELISA was carried out according to the scheme given in example 4.

This study included sera or blood plasma of patients (86 samples in total) obtained on days 7-14 from the day of registration of clinical symptoms of acute infection of rickettsial etiology, which was confirmed by positive results of PCR testing of leukocyte blood fractions and / or washings from primary affects patients with the use of a commercial kit of reagents "RealBest DNA Rickettsia species" (CJSC "Vector-Best", Russia). The set of reagents "RealBest DNA Rickettsia species" is intended for the detection of genetic material containing a conservative region of the gltA (citrate synthase) gene, which is present in all species of rickettsia of the KPL group (E.I.Bondarenko, L.D.Schuchinova, D.I. Timofeev and other Identification of genetic markers of pathogens of tick-borne rickettsiosis in PCR using the kits of reagents "RealBest DNA Rickettsia species" and "RealBest DNA Rickettsia sibirica / Rickettsia heilongjiangensis". NEWS "Vector-Best", 2018, No. 1 (87)). Table 3 presents the data of testing the sera or plasma of patients' blood in ELISA.

Table 3. Detection of specific antibodies to Rickettsia antigens of the LPL group in ELISA using a test system based on inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from suspensions of Rickettsia sibirica whole-soluble antigens.

Spectra of detectable antibodies in the test samples The number of samples with the corresponding spectrum of antibodies to the antigens of the rickettsia group of the LP IgG nineteen IgA 21 IgM ten IgG + IgM 15 IgM + IgA 17 IgG + IgM + IgA 4 Total 86

In patients infected with human pathogenic rickettsia species of the LPL group, specific antibodies were detected in the ELISA based on inactivated soluble purified protein-lipopolysaccharide antigenic substance isolated from Rickettsia sibirica in samples (serum or blood plasma).

Thus, the enzyme-linked immunosorbent system can be used not only for the diagnosis of tick-borne rickettsiosis (tick-borne typhus of North Asia) caused by Rickettsia sibirica, but also for the diagnosis of other rickettsioses of the LP group, the causative agents of which may be other pathogenic types of rickettsia, united in the group LP (Rickettsia conorii with isolated subspecies and the individual nosological forms of rickettsioses caused by them, among which Rickettsia conorii subsp.conorii - the causative agent of Marseilles fever, Rickettsia conorii subsp.caspensis - the causative agent of Astrakhan spotted fever fever ; Rickettsia africae - the causative agent of African tick fever and other pathogenic species).

It should also be specially noted that single specific class A antibodies to antigens of the LP group were detected in the blood serum of 21 patients (24.4%) with clinical signs of acute infection of rickettsial etiology. Consequently, additional detection of class A antibodies increases the sensitivity and information content of the method for diagnosing rickettsial diseases of the LP group.

It has been shown that additional detection of specific class A antibodies significantly reduces the number of seronegative results in the early period of infection. Detailed confirmation of this is given in the examples.

All the examples given prove that the problem posed by the invention has been solved.

INDUSTRIAL APPLICABILITY

A method for diagnosing rickettsial diseases of the tick-borne spotted fever group, based on separate differential detection by the method of indirect enzyme-linked immunosorbent assay in serum or plasma of human blood of specific antibodies of classes M, G and A to the antigens of rickettsia of the LPF group, using an immunosorbent obtained on the basis of inactivated soluble purified protein lipopolysaccharide antigenic substance isolated from a suspension of Rickettsia sibirica antigens is sensitive, specific and informative.

Due to the presence of a soluble (group-specific antigen) in the composition of rickettsia of the LP group, which is heterogeneous and includes a lipopolysaccharide with a high homology of antigenic structures in rickettsia within the group and other antigens of a protein nature, serological diagnosis of rickettsial diseases of the tick-borne spotted fever group using such antigens is a group specific makes it possible to detect specific antibodies to most types of human pathogenic rickettsiae within the LPL group, although the titer of specific antibodies to a homologous antigen (pathogen) is usually higher (K. Amano, M. Fujita, T. Suto. Chemical properties of lipopolysaccharides from spotted fever group rickettsiae and their common antigenicity with lipopolysaccharides from Proteus species Infection of Immunology, 1993, v. 61, no. 10, pp. 4350-4355;

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

D. Jones, B. Anderson, J. Olson, C. Greene. Enzyme-Linked Immunosorbent Assay for Detection of Human Immunoglobulin G to Lipopolysaccharide of Spotted Fever Group Rickettsiae. Journal of Clinical Microbiology, 1993, v. 31, no. 1, p. 138-141).

The original method of obtaining inactivated soluble purified protein-lipopolysaccharide antigenic substance from a suspension of primary antigens of Rickettsia sibirica allows the isolation of an antigenic substance with similar properties and characteristics from a suspension of corpuscular / whole-soluble / soluble antigens of other pathogenic rickettsia species included in the Rickettsia group.

The developed immunoassay diagnostic system can be used to diagnose rickettsial diseases of the LP group and to verify the diagnoses of infectious diseases of rickettsial etiology from other infections with similar clinical manifestations. The test system, in addition to detecting specific antibodies of classes M and G to antigens of rickettsia of the LP group, provides additional information on the presence / absence of specific antibodies of class A in serum or plasma of a person, which increases the reliability, information content and sensitivity of the enzyme immunoassay and a method for diagnosing rickettsial diseases of the group KPL with her help.

List of references

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

2. OFS.1.2.4.0006.15. Bacterial endotoxins.

3. OFS.1.2.1.0023.15. Polyacrylamide gel electrophoresis.

4. Rudakov N.V., Abramova N.V., Penievskaya N.A., Samoilenko I.E., Shpynov S.N., Reshetnikova T.A. Method for laboratory diagnosis of tick-borne rickettsiosis using enzyme-linked immunosorbent assay to determine antibodies to Rickettsia sibirica antigen. Patent RU 2477860, priority from 13.08.2010

5. K. Amano, M. Fujita, T. Suto. Chemical properties of lipopolysaccharides from spotted fever group rickettsiae and their common antigenicity with lipopolysaccharides from Proteus species. Infection of Immunology, 1993, v. 61, no. 10, p. 4350-4355.

6.http: //www.fullerlabs.com.

7. D. Jones, B. Anderson, J. Olson, C. Greene. Enzyme-Linked Immunosorbent Assay for Detection of Human Immunoglobulin G to Lipopolysaccharide of Spotted Fever Group Rickettsiae. Journal of clinical microbiology, 1993, v. 31, no. 1, p. 138-141.

8. Teysseire N, Raoult D. Comparison of western immunoblotting and microimmunofluorescence for diagnosis of Mediterranean spotted fever. Journal of Clinical Microbiology, 1992, v. 30, no. 2, p. 455-460.

9.E.I. Bondarenko, L. D. Shchuchinova, D.I. Timofeev et al. Identification of genetic markers of tick-borne rickettsiosis pathogens in PCR using reagent kits "RealBest DNA Rickettsia species" and "RealBest DNA Rickettsia sibirica / Rickettsia heilongjiangensis". NEWS "Vector-Best", 2018, №1 (87).

LIST OF ABBREVIATIONS

AVI - adenovirus infection

APL - Astrakhan spotted fever

ELISA - enzyme immunoassay

K + - positive control sample in the test system

K - - negative control sample as part of the test system

CAT - tick-borne spotted fever

KR - tick-borne rickettsiosis

LAL (English LAL - abbreviation Limulus amebocyte lysate) - test for the content of bacterial endotoxins

LPS - lipopolysaccharide

OD - optical density

OPcrit - critical (threshold) value of optical density

PCR - polymerase chain reaction

RIF - reaction of immunofluorescence

RNIF - reaction of indirect immunofluorescence

RRK - dilution solution for conjugates

PPO - sample dilution solution

RSK - complement binding reaction

TMB - 3 ', 5'-tetramethylbenzidine (chromogen)

FSR-T - phosphate-saline solution with tween 20.

Claims (6)

1. A method of obtaining inactivated soluble purified protein-lipopolysaccharide antigenic substanceRickettsia sibirica for the preparation of the main component of the test system - an immunosorbent, which consists in the fact that the inactivated lyophilized whole-soluble antigenRickettsia sibirica resuspended in 10mM composition buffer tris-HCl and 0.5 M sodium chloride, pH-7.4-7.6, containing 8 M urea, then the antigen suspension is subjected to ultrasonic treatment at a frequency of 15-23 kHz for 15-30 minutes, followed by centrifugation at 10,000 - 12000g and the supernatant is taken, then size exclusion chromatography of the supernatant is carried out on a column with a chromatographic sorbent, after which the fractions are assessed for specific antigenic activity in ELISA using human serum or plasma, containing and not containing specific antibodies to the antigens of the rickettsia group of tick-borne spotted fever LPF, and carry out further combining of the fractions of the first peak and those adjacent to it, which demonstrated in ELISA the best ratio of the mean values of the optical density of the OD of positive control samples of human serum or plasma containing antibodies to the antigens of rickettsia tick-borne spotted fever LPF, to the mean values of the optical density of OD of negative control samples not containing antibodies to the antigens of rickettsiae of tick-borne fever LP, while the remaining fractions showing in ELISA a decrease in the optical density of OP (+) relative to the optical density of OP (-) by 30% or more compared to the average value of OP (+) to OP ( -) calculated for the fractions of the first peak are excluded. 2. Immunoassay diagnostic test system for the analysis of human serum or plasma and the diagnosis of rickettsioses of the group of tick-borne spotted fever, including: a plate for enzyme-linked immunosorbent assay with an immunosorbent adsorbed in the wells, obtained on the basis of inactivated soluble purified protein-lipopolysaccharide antigen spotty substance, Rickettsia sibirica according to claim 1, with marked parallel rows of wells: for detecting specific IgA - the first row, for detecting specific IgM - second row, for detecting specific IgG - third row, three conjugates: human anti-IgA, human anti-IgM and anti-human IgG labeled with horseradish peroxidase, in addition, auxiliary solutions: solution for diluting samples, solution for diluting conjugates, chromogen 3 ', 5'-tetramethylbenzidine TMB, stop reagent, concentrate of phosphate-saline solution with tween 20 - PSR- T, positive and negative control samples: K + and K, g de as positive and negative control samples K + and K use serum or plasma of human blood or a pool of serum or plasma of human blood containing and, accordingly, not containing specific antibodies of classes A, M, G to the antigens of rickettsia group of tick-borne spotted fever LP. 3. Immunoassay diagnostic test system according to claim 2, in which the antigens of the rickettsia group of tick-borne spotted fever LPF are at least Rickettsia conorii , Rickettsia sibirica, Rickettsia heilongjangensis. 4. A method for diagnosing rickettsioses of the group of tick-borne spotted fever LP, in which the serum or blood plasma of a patient with suspected rickettsiosis caused by the causative agent of the LP group is examined for the presence of specific antibodies by ELISA, characterized in that separate differential detection of antibodies of classes M is carried out, G and A in serum or plasma of human blood using an immunoassay diagnostic test system according to claim 2, in which an immunosorbent obtained on the basis of an inactivated soluble purified protein-lipopolysaccharide antigenic substance Rickettsia sibirica according to claim 1 is used, while in the wells of parallel rows with the adsorbed immunosorbent, 100 μl are added to the dilution well of the test samples of human serum or plasma and control samples K + and K- prepared using a solution for diluting samples: in initial dilutions of 1:10 to detect specific antibodies of class A - the first a row of holes; 1:20 for the detection of specific antibodies of class M - the second row of wells; 1:40 for the detection of specific antibodies of class G - the third row of wells, then the diluted test and control samples are incubated at 37 - 38 ° C for 45 - 60 min, the immunosorbent is washed with the FSR-T working solution 5-6 times, added to the appropriate rows of wells with immunosorbent and samples, 100 μl per well, working dilutions of human anti-IgA, human anti-IgM and human anti-IgG conjugates labeled with horseradish peroxidase, incubated at 37-38 ° C for 30-60 minutes, the immunosorbent is washed FSR-T 5-6 times, 100 μl of chromogen 3 ′, 5′-tetramethylbenzidine TMB is added to each well of the immunosorbent and kept for 10 - 30 minutes at room temperature, protecting the plate from direct light, then the enzymatic reaction is stopped by adding 50-100 μl stop reagent and carry out spectrometric measurement of optical density of OP at a wavelength of 450 nm, separately calculate the threshold value of optical density OPcrit for each class of antibodies M, G and A according to the formula: OPcrit = average value of OP K- + 0.2, where OD K- is the average value of the optical density in the wells of the corresponding rows with a negative control sample K-, while the test sample contains specific antibodies M, or G, or A, if its OD value exceeds or is equal to OD crit.

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