RU2345365C1 - Method for differential diagnostics of yersiniosis and respective diagnostic kit - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to diagnostic methods for infectious diseases, and refers to immune-enzyme assay (IEA) of intestinal yersiniosis, caused by different serum variants of Yersinia enterocolitica, as well as yersiniosis diagnostic kits. Ground of the method: as antigen, species-specific pore-forming protein (porin) of Y. enterocolitica external membrane is used, which ensures determination of specific antibodies to Y. enterocolitica porin in human blood serum, regardless of the pathogen serum variant. As non-specific binding prevention agent, 0.1% solution twin-20 or twin-80 is used. Diagnostic titer represents patient blood serum, diluted 1:800 to 1:1600. The diagnostic method and diagnostic kit proposed increase efficiency of intestinal yersiniosis diagnostics, as they enable determination of specific antibodies to any Y. enterocolitica variant in human blood serum, both in early (1^st week) and in later (2^nd to 4^th week) disease stages.

EFFECT: increased efficiency of intestinal yersiniosis diagnostics.

2 cl, 4 dwg, 1 ex

Description

The invention relates to medicine, in particular to methods of diagnosis and kits for the diagnosis of infectious diseases, namely yersiniosis.

The problem of diagnosing yersiniosis, acute intestinal infection caused by pathogenic for humans Yersinia enterocolitica, currently remains relevant for practical health care. The widespread and pronounced variety of clinical variants of yersiniosis determine the difficulty in recognizing this disease and the significant frequency of diagnostic errors. Yersiniosis has a similar clinical picture with acute hepatitis, rheumatism, appendicitis, Crohn's disease, acute gastroenterocolitis, erythema nodosa, etc. [1-3]. In addition, the etiological role of Yersinia in the development of secondary focal forms of diseases (immunopathologies) associated with damage to the cardiovascular and nervous systems, musculoskeletal system, as well as the gastrointestinal tract and urinary tract has been proven [4, 5]. In recent years, a steady increase in the incidence of yersiniosis infections of the population throughout the Russian Federation has been recorded. The most common cause of intestinal yersiniosis in humans is Y. enterocolitica serotypes 0: 3 and 0: 9. However, there is an increase in the disease caused by Y. enterocolitica serotypes 0: 5, 0: 6, 0: 7, 0: 8, etc. [3]. In addition, it was found that in the so-called “healthy” person, Yersinia markers are found that cause a prolonged, low-manifest disease, or asymptomatic carriage, which can lead to serious complications [3].

Establishing the nature of yersiniosis infection is possible only as a result of a comprehensive clinical and laboratory diagnosis using specific analysis methods.

Laboratory diagnosis of yersiniosis is carried out using bacteriological and serological methods [6-15]. In the diagnosis of infectious diseases, bacteriological confirmation plays an undeniable role [6]. However, given the ambiguity of the results, the complexity and duration of bacteriological analysis, accelerated methods for the detection of Y. enterocolitica, of which the serological identification of the disease plays a dominant role, are of particular importance in the diagnosis of yersiniosis.

Serological methods for the diagnosis of yersiniosis have been described quite a lot [7-15]. Existing methods for serodiagnosis of yersiniosis are based on the determination of a bacterial antigen (the first group of methods) in various biological fluids (blood, saliva, urine, coprofiltrate) and on the determination of antibodies in the patient's blood serum to pathogen antigens (the second group of methods).

The first group of serological methods for diagnosing yersiniosis includes the coagglutination reaction (RKoA) [7] and the polymerase chain reaction (PCR) [8]. The use of PKoA is effective in the early stages of the disease (up to 90% of positive results at the 1st week of the disease), but is ineffective when examining patients at 2-4 weeks of the disease, during which the percentage of visits to medical institutions remains quite high. PCR, being a high-tech method for the diagnosis of yersiniosis, has not found wide practical use in view of its high cost and low technical equipment of clinical laboratories.

From the second group of serological methods based on the determination of antibodies in the blood serum of patients, the first was the agglutination reaction (RA) with O-diagnostics (suspension of bacteria killed by boiling or autoclaving) and OH-diagnostics (suspension of live or formalin-treated bacteria) [ 9]. The disadvantage of this method is the type-specificity of the reaction: for the formulation of RA, reference strains of Y. enterocolitica of the most common serovariants (0: 3, 0: 9, 0: 4.3, 0: 5.27, etc.) are required, which significantly reduces the diagnostic capabilities of this method. In addition, these diagnostics give a positive reaction with heterologous serum against enterobacteria and brucellosis serum. The disadvantages of the method include the retrospectiveness of diagnostic results, since antibodies in diagnostic titers are detected, as a rule, at 2-4 weeks of the disease. An improved version of RA, a latex diagnosticum based on the use of polystyrene microspheres stained with various dyes corresponding to a specific specificity of Y. enterocolitica O-antigens of various serotypes immobilized on them, is described [10]. This method is also characterized by low specificity and effectiveness in the early stages of the disease (1st week), which reduces the possibility of serological diagnosis. In addition, the method is time-consuming to implement and requires a significant investment of time and money both for obtaining O-antigens of Y. enterocolitica of various serovariants, and their subsequent immobilization on the surface of polystyrene microspheres.

The reaction of indirect hemagglutination (RNGA), which is used in almost all clinical laboratories, is widespread. The method is based on the determination of antibody titer in the blood serum of patients using lipopolysaccharide (LPS) from Y. enterocolitica 0: 3 and 0: 9 serological variants as antigen [11]. The disadvantage of RNGA is low specificity, because the reaction is type-specific, which does not allow to diagnose a disease caused by other serovariants of Y. enterocolitica. In addition, antibody titers reach diagnostically significant values at a later stage of the disease, i.e. by 15-20 days, which reduces the effectiveness of the diagnosis of yersiniosis in the early stages of the disease.

In recent years, enzyme-linked immunosorbent assay (ELISA) using antigens of various nature is widely used for serodiagnosis of yersiniosis.

A known method for the diagnosis of yersiniosis, based on the use in ELISA of a type-specific antigen - lipopolysaccharide (LPS) from Y. enterocolitica 0: 3 serotype [12]. The disadvantage of this method is the low specificity and sensitivity in verifying yersiniosis due to other serotypes (0: 5, 0: 6, 0: 7, 0: 8, 0: 9) of Y. enterocolitica.

A method has been developed for the diagnosis of yersiniosis (yersiniosis and pseudotuberculosis), which uses a protein antigen that is freely secreted into the culture medium by pathogenic yersinia, followed by the detection of antibodies in the blood serum of patients using ELISA [13]. The method does not allow to reliably diagnose yersiniosis caused by Y. enterocolitica of serotype 0: 9 in view of the common antigenic determinant of LPS (4,6-deoxy-4-formamido-L-D-mannopyranosyl) inherent in this serotype Y. enterocolitica and some types of brucella.

Bogautdinov et al. Proposed a method of enzyme-linked immunosorbent diagnosis of yersiniosis, based on the use of inactivated corpuscular antigen from Y. enterocolitica 0: 3 serovariant [14]. This method includes the following steps:

1) planting antigen on a polystyrene plate; inactivated corpuscular antigen from Y. enterocolitica 0: 3 serovariant is used as antigen;

2) washing the tablets;

3) applying the test serum, working dilution of serum 1: 400;

4) washing the tablets;

5) application of the conjugate;

6) washing the tablets;

7) applying a substrate mixture;

8) registration of reaction results.

The kit for implementing the known method for the diagnosis of yersiniosis using inactivated corpuscular antigen from Y. enterocolitica 0: 3 serovariant is a set of reagents for detecting antibodies to the Y. enterocolitica antigen by ELISA. The kit contains the following reagents:

plate with immobilized antigen - LPS from Y. enterocolitica 0: 3 serotype;

a solution for washing the tablet and for diluting blood serum;

inactivating reagent (0.5% Tween-20 solution);

conjugate (second general antibody);

substratin indicator solution;

samples of positive and negative controls.

The disadvantages of this method and set include the limitation of the scope

Applications - the field of veterinary medicine. The use of this method in medical practice for the diagnosis of yersiniosis in humans requires additional research. In addition, the low value of the diagnostic titer (1: 400) in ELISA does not exclude cross-reactions with other pathogens of acute intestinal infections, and the method does not allow to detect yersiniosis caused by a pathogen that is not related to 0: 3, 0: 9, 0: 6, 0: 5, 0: 8 to serovariants. In this regard, this method is characterized by low specificity and sensitivity.

A common disadvantage of known methods for the diagnosis of yersiniosis is the use of type-specific antigens of Y. enterocolitica, which significantly reduces their diagnostic value.

The objective of the invention is to improve the diagnosis of yersiniosis, namely the development of a highly effective method for early and differential diagnosis of yersiniosis and the development of a kit for its implementation.

As a result of solving this problem, a method for differential diagnosis of yersiniosis has been developed, which includes the determination of specific antibodies to Yersinia enterocolitica in the patient's blood serum by enzyme-linked immunosorbent assay, in which according to the invention the species-specific porous protein Yersinia enterocolitica is used as an antigen, serum dilution is taken as a diagnostic titer 1: 800-1: 1600, and the result is considered positive at a value corresponding to the ratio of the optical density ora enzymatic reaction to a specific serum absorbance value of the reaction solution with normal serum, is greater than or equal to 2.1.

Pore-forming proteins of the outer membrane (HM) of gram-negative bacteria are immunological markers characteristic of the species or genus of microorganisms and are highly immunogenic components of the bacterial cell, and the determination of antibodies to them is used to diagnose infectious diseases [15].

The authors of the proposed method found that porin NM Y. enterocolitica is a species-specific antigen of the causative agent of yersiniosis, antibodies to which are detected regardless of the serological variant of the pathogen both in the antisera of laboratory animals immunized with a lysate of Y. enterocolitica cells and in the blood sera of patients with yersiniosis. These results were a prerequisite for the use of porin NM Y. enterocolitica as a species-specific antigen for serodiagnosis of yersiniosis using ELISA.

Porin was isolated by the standard method in the modification of the authors of the proposed method [16].

ELISA based on porin from Y. enterocolitica is carried out according to the standard method for detecting specific antibodies in human serum (indirect, non-competitive analysis of antibodies) [17].

The analysis consists of the following steps:

1) landing of antigen on a solid carrier (96 well polystyrene plate) (antigen concentration of 5.0-2.5 μg / ml in carbonate-bicarbonate buffer with a pH of 9.0);

2) washing the plate 3-5 times for 5 minutes from unbound components with buffer "B", consisting of phosphate-buffered saline with a pH of 7.4 (buffer "A") + 0.05% tween-20 (or tween-80) ;

3) inactivation of the plate (prevention of nonspecific binding of antigen to antibodies) with buffer "C", consisting of phosphate-saline buffer with pH 7.4 (buffer "A") + 0.1% tween-20;

4) applying the test serum (a dilution series of 1: 800-1: 1600 is prepared using buffer "B");

5) washing the tablet;

6) application of the conjugate (the second General antibody, labeled with horseradish peroxidase):

working dilution is prepared using buffer "C";

7) washing the tablet;

8) application of a substratin indicator solution: 0.04% o-phenylenediamine in citrate phosphate buffer (pH 5.0) + 10 μl 33% H ₂ O ₂ (calculated per 10 ml of buffer);

9) applying a stop reagent: 0.5 M sulfuric acid solution (the solution is prepared by diluting 1 ml of concentrated sulfuric acid with 15 ml of distilled water);

10) registration of reaction results.

To study the diagnostic qualities and specificity of the ELISA test system based on porin, we examined 657 blood serums of patients with characteristic clinical signs of yersiniosis (57 of them with bacteriologically confirmed diagnosis), of which 357 were children aged 1 to 14 years, and 200 blood serums of adult patients. In addition, taking into account the polymorphism of yersiniosis infection, 256 blood sera of patients with acute intestinal infections not caused by Y. enterocolitica (pseudotuberculosis - 134, salmonellosis - 54, acute viral hepatitis (A and B) - 68) were analyzed. Verification of these diseases was carried out taking into account clinical and epidemiological, bacteriological and / or specific serological studies. The control group consisted of 86 practically healthy people, comparable in age and gender with the examined patients.

Diagnostic titer by ELISA was determined by comparing the statistically significant values [18], an equal ratio of the average absorbance values of the reaction solution with sera of patients yersiniosis and healthy people (F _on / F _zd) at various dilutions, where F _on = F _a + F _f

F _c - the value of the optical density of the solution of a specific antigen / antibody reaction (antibodies are the patient’s blood serum with a confirmed diagnosis, or a positive control),

F _health - the value of the optical density of the reaction solution with the blood serum of a healthy donor, or a negative control,

F _f - the value of the optical density of the solution of a non-specific background reaction.

For the studied sera (with a bacteriologically confirmed diagnosis) in dilutions of 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 1: 3200 these values were respectively 3.6; 4.0; 4.2; 5.5; 5.0; 4.1 (figure 1). For the diagnostic titer took serum dilution for which the ratio F _on / F _bldg had the greatest. Thus, in an ELISA based on porin from Y. enterocolitica, a diagnostic titer of 1: 800-1: 1600 was taken. An analysis of the blood sera of patients with yersiniosis using this porin-based test system showed that the level of specific antibodies was recorded within 0.64-2.0 units of the optical density of the solution of the enzymatic reaction, which significantly differed from the control values (0.1-0, 3 units of optical density of the solution of the enzymatic reaction) (figure 2). When testing a test system based on porin from Y. enterocolitica in blood serum, 12% of healthy donors showed an increased level of antibodies to Y. enterocolitica. This result coincides with the literature data that in places with constant circulation of pathogens of yersiniosis in healthy people in the blood, an increased level of antibodies to these pathogens is determined [3].

To determine the effectiveness of the ELISA test system based on porin, a parallel formulation of RNGA with a commercial type-specific yersiniosis diagnosticum was used. A comparative analysis of statistically reliable results of a serological examination of patients with yersiniosis (with a bacteriologically confirmed diagnosis) using ELISA based on porin and RNGA revealed that on the 1st week of the disease, ELISA detects specific antibodies in 85.2% of cases (the average value of the optical density of the enzymatic solution the reaction was 1.64 relative units), and when setting RNGA in the same patients, diagnostic antibody levels were detected in 32.3% of cases (titer 1: 100-1: 200) (figure 3). At the 2nd week of the disease in the blood serum of patients using ELISA based on porin, antibodies to Y. enterocolitica were detected in the diagnostic titer in 95.0% of cases (the average optical density of the solution of the enzymatic reaction was 1.94 relative units), and RNGA - in 42.7% of cases (titer 1: 100-1: 400). On the 3rd and 4th weeks of the disease, positive reactions in ELISA based on porin were found in 90.7% of cases (the average optical density of the solution of the enzymatic reaction was 1.78 relative units), in RNGA - 37.7% (antibody titers 1: 100-1: 800) (Fig. 3). The data obtained indicate that ELISA based on porin from Y. enterocolitica is a highly effective method and detects specific antibodies to Y. enterocolitica in the blood serum of patients with yersiniosis, 2 times more effective than rnga, traditionally used by clinical laboratories.

To confirm the specificity and sensitivity of ELISA based on porin from Y. enterocolitica, the blood serum of patients with acute intestinal infections not caused by Y. enterocolitica was examined. As a result, it was found that the blood serum of patients with pseudotuberculosis and salmonellosis in the diagnostic titer (1: 800) interact with porin. However, the optical density of the solution of the enzymatic reaction of these sera is 2-3 times less than the optical density of the solution in the interaction of porin with the blood serum of patients with yersiniosis. When diluting these 1: 1600 sera, antibodies to porin from Y. enterocolitica were determined at the negative control level. When analyzing the blood sera of patients with hepatitis, antibodies to porin from Y. enterocolitica was not detected in any of the cases (figure 4). This suggests that the proposed test system allows you to effectively and reliably carry out differential diagnosis of yersiniosis from other acute intestinal diseases. Therefore, ELISA based on porin from Y. enterocolitica is a sensitive method for the diagnosis of yersiniosis, has high specificity and does not cross-react with antibodies to pathogens similar in clinic to yersiniosis.

Thus, the ELISA test system based on the species-specific protein-porin HM Y. enterocolitica provides the detection of specific antibodies to Y. enterocolitica in the blood serum of patients with yersiniosis in dilutions of 1: 800 and 1: 1600 and, compared with existing methods for the diagnosis of yersiniosis, is a more sensitive and more specific way to diagnose yersiniosis by identifying all serovariants in enterocolitica that cause yersiniosis. The inventive method is more efficient in the diagnosis of yersiniosis, since it allows you to diagnose the disease, both in the early (at the 1st week of the disease - in 85.2% of cases) and at a later time of the infection process (at the 2nd and 3-4th weeks - in 95% and 90.7% of cases, respectively).

Determination of specific antibodies to porin from enterocolitica in healthy individuals using ELISA is an early method for the differential and etiological diagnosis of yersiniosis or bacterial carriage, which is necessary for the timely appointment of treatment and prevention of adverse outcomes.

The invention is illustrated by the following drawings:

Figure 1 presents the results of determining the average level of specific antibodies to porin from Y. enterocolitica in the blood serum of patients with yersiniosis with a confirmed diagnosis (row 1) and healthy people (row 2) at various dilutions (1: 100, 1: 200, 1 : 400, 1: 800, 1: 1600, 1: 3200 - points 1, 2, 3, 4, 5, 6, respectively) using an ELISA test system based on porin from Y enterocolitica.

Figure 2 presents the average level of specific antibodies to porin from Y. enterocolitica in the blood serum of patients with yersiniosis (1) and healthy people (2) with a diagnostic titer (1: 800 -1: 1600) using an ELISA test system based on porin from Y. enterocolitica.

Figure 3 shows the comparative efficacy of ELISA based on porin from Y. enterocolitica and RNGA based on a commercial yersiniosis diagnosticum at various stages of the disease (1, 2, 3 - 1st, 2nd and 3rd weeks, respectively)

Figure 4 presents the analysis of blood serum of patients with acute intestinal infections (salmonellosis, yersiniosis, pseudotuberculosis, hepatitis) and donors at a dilution of 1: 800 (rows 1, 2, 3, 4, 5) and 1: 1600 (rows 7, 8, 9, 10, 11) using an ELISA test system based on porin from Y. enteracolitica.

The inventive method is illustrated by the following example:

100 μl of antigen solution (concentration 5 μg / ml in carbonate-bicarbonate buffer with pH 9.0) is added to the wells of a tablet (96-well polystyrene tablet from Dynatech, Switzerland or Costar, USA) and the tablet is sensitized at 37 ° C in within 2 hours. Then the contents of the wells are removed by strong shaking, and the plate is washed 3 times with buffer "B", consisting of 0.02 M phosphate-saline buffer pH 7.4 (buffer "A") + 0.05% tween-20. Buffer “A” contains 5.68 g of Na ₂ HPO ₄ + 6.24 g of NaH ₂ PO ₄ + 11.2 g of NaCl + 2 L of H ₂ O. At each stage of washing the plate, it is necessary to carefully remove moisture from the wells by tapping the inverted plate about filter paper. Then, 300 μl of buffer "C" consisting of phosphate-buffered saline pH 7.4 (buffer "A") + 0.1% tween-20 is added to each well of the plate, and the plate is inactivated for 16-18 hours at 4 ° C. Then, 100 μl of the tested blood serum, positive and negative controls, two replicates for each dilution are added. Dilutions (1: 800 and 1: 1600) of the studied blood serum samples of the patient, positive and negative controls are prepared using buffer "B" (buffer "A" + 0.05% tween-20, see paragraph 2). Next, the plates are incubated for 2 hours at 37 ° C, then washed 3-5 times for 5 minutes from unbound components with buffer "B", carefully removing residual moisture from the wells. Then, 100 μl of conjugate (enzyme-labeled second general antibodies) in working dilution (1: 1000) in buffer “C” (buffer “A” + 0.1% tween-20) was added to each well of the plate and incubated for 1.5 hours at 37 ° C, then the tablets are washed as described above. Then, 70 μl of a sub-stratification solution containing 0.04% orthophenylenediamine in citrate-phosphate buffer (pH-5.0) and 10 μl of 33% H ₂ O ₂ (per 10 ml of buffer used) are added to the wells of the plate. Citrate-phosphate buffer is prepared by mixing 49.3 ml of 0.2 M sodium disodium phosphate (1.42 g per 50 ml of H ₂ O) and 50.7 ml of 0.1 M sodium citrate (2.1 g per 100 ml of H ₂ O ) The plates are kept for 15-20 minutes in the dark (for the development of a color reaction) at room temperature, then 30 μl of 0.5 M H ₂ SO ₄ solution (stop reagent) is added to each well to stop the reaction. The reaction results are taken into account using a spectrophotometer, measuring the optical density of the solution in the wells of the tablet at a wavelength of 492 nm. When setting ELISA on each tablet at the same time measure the optical density of the solution in the wells containing the following controls:

- positive control (control of specific binding) - 100 μl of blood serum of a patient with yersiniosis with a bacteriologically confirmed diagnosis + 100 μl of conjugate + 70 μl of substrate solution + 30 μl of H ₂ SO ₄ ;

- negative control - 100 μl of blood serum of a healthy donor + 100 μl of conjugate + 70 μl of substratin indicator solution + 30 μl of H ₂ SO ₄ ;

- control of non-specific binding - 100 μl of buffer solution + 100 μl of conjugate + 70 μl of substratin indicator solution + 30 μl of H ₂ SO ₄ .

The result is considered positive if the value corresponding to the ratio of the optical density of the solution in wells with specific serum to the optical density of the solution in wells with normal serum is greater than or equal to 2.1.

Currently, the industrial production of enzyme-linked immunosorbent diagnostics (ELISA test systems) for yersiniosis in our country is not carried out. Differential methods for the diagnosis of yersiniosis, proposed by different authors [12-14], are not equipped with the necessary sets of reagents for the immunodiagnosis of yersiniosis.

The proposed diagnostic kit for yersiniosis for carrying out the proposed method, namely an enzyme-linked immunosorbent assay using a species-specific antigen - porin HM Y. enterocolitica, is designed to detect specific antibodies to Y. enterocolitica in human serum and can be used in clinical and epidemiological studies, as well as blood service.

The kit contains all the reagents required for ELISA, except for distilled water.

The kit includes:

1) 96-well polystyrene plate (Dynatech, Switzerland or Costar, USA) with an immobilized antigen - NM Y. enterocolitica porin in a hermetically sealed bag - 1 pc.;

2) phosphate-buffered saline (concentrated buffer solution for inactivation and washing of the plate, for dilution of serum and conjugate) - 2 bottles.

3) conjugate (fraction of general antibodies against the total fraction of human immunoglobulins labeled with horseradish peroxidase) - 1 bottle;

4) substrate (ortho-phenylenediamine, 4 mg) - 1 bottle;

5) citrate - phosphate buffer - 1 bottle;

6) negative and positive controls (freeze-dried blood serum samples of a patient with yersiniosis with a confirmed diagnosis and a healthy donor) - 2 bottles;

7) inactivating reagent (tween-20 or tween-80) - 1 bottle;

8) hydrogen peroxide (H ₂ O ₂ , concentrated solution) - 1 bottle;

9) stop reagent (solution of 0.5 M sulfuric acid) - 1 bottle;

The set is designed for 96 definitions, including controls.

In the claimed diagnostic kit as an inactivating reagent use tween-20 or tween-80 instead of the traditionally used expensive normal blood serum or albumin, which reduces the cost of the proposed kit.

Preparation of reagents for analysis:

1. Buffer "A": contents of 1 vial of phosphate-buffered saline concentrate (pH 7.4) (mixture of salts: 0.02 M sodium phosphate disubstituted 12-aqueous, 0.02 M sodium phosphate monosubstituted 2-aqueous and 0.1 M sodium chloride) must be dissolved in 1 liter of distilled water (buffer "A").

2. Buffer "B" for washing the plate and diluting the test blood serum of patients, positive and negative controls: add 1 ml of tween-20 (or tween-80) to 1 liter of buffer "A" and receive buffer "B".

3. Buffer “C” for inactivation of the plate and dilution of the conjugate: add 200 ml of tween-20 (or tween-80) to 200 ml of buffer “A” and receive buffer “C”.

4. Dilution of serum: test blood serum, control, positive and negative serum diluted with buffer "B" to the working dilution.

5. The substrate mixture is prepared immediately before use. Dissolve the contents of the vial with the substrate (4 mg of ortho-phenylenediamine) in 10 ml of citrate-phosphate buffer (ready to use) and add 10 μl of 33% H ₂ O ₂ .

6. Stop reagent (solution of 0.5 M sulfuric acid) is ready for use.

For the reliability of the results of the analysis, it is recommended that the test and control blood serum samples be duplicated using two wells for each sample.

Enzyme-linked immunosorbent assay using the proposed diagnostic kit is illustrated by the following example:

A pre-prepared tablet (after sensitization of the antigen - porin HM Y. enterocolitica and washing the tablet) must be inactivated for the next stages of analysis.

1. Inactivation of the plate (prevention of non-specific binding of antigen to antibodies): 300 µl of buffer “C” is added to all wells and incubated overnight (16-18 h) at 6-8 ° С. Buffer "C", consisting of phosphate-buffered saline with a pH of 7.4 (buffer "A") + 0.1% tween-20 or tween-80, is prepared by dissolving the contents of the 1st vial (mixture of salts - 0.02 M sodium phosphate disubstituted 12 - aqueous; 0.02 M sodium phosphate monosubstituted 2-aqueous and 0.1 M sodium chloride) in 1 liter of distilled water (receive buffer "A"). Then, 0.2 ml of Tween-20 or Tween-80 is added to 200 ml of buffer “A” and buffer “C” is obtained to inactivate the plate and to dilute the conjugate. The solution is not stored.

2. Washing the tablet: after inactivation, remove the contents of the wells by decantation and wash them 3-5 times for 5 minutes from unbound components with buffer "B". To prepare buffer "B", 0.5 ml of tween-20 or tween-80 is added to 1 liter of buffer "A" to obtain a solution for washing the plate and diluting serum. At each wash, 300 μl of wash buffer “B” is added to all wells. With each decantation, the residual liquid is carefully removed from the wells by tapping the plate in an inverted position on filter paper.

3. Apply in two parallel 100 μl of the test serum, the positive and negative controls: serum is applied in dilutions of 1: 800-1: 1600 in buffer "B";

4. Incubation of the tablet at 37 ° C for 2 hours.

5. At the end of the incubation, the contents of the wells are removed and the plate is washed thoroughly 3 times with buffer "B";

6. Pipette 100 µl of the conjugate solution in the working dilution into all wells: a working dilution (1: 1000) is prepared by dissolving the contents of the vial (dry preparation) in buffer “C”.

7. Incubation of the tablet at 37 ° C for 1.5 hours.

8. At the end of the incubation, the contents are removed from the wells and the plate is washed.

9. Application of the substrate mixture. A substratin indicator solution is prepared immediately before use: 10 μl of 33% H ₂ O _{2 is} added to 10 ml of 0.05 M citrate-phosphate buffer (pH 5.0) containing 0.04% ortho-phenylenediamine. To prepare a 0.05 M solution of citrate-phosphate buffer, the contents of the vial (mixture of salts) are dissolved in 10 ml of distilled water. Before applying the substrate solution, the tablet is rinsed with distilled water and dried.

10. 70 μl of substrate mixture are added to all wells of the plate and incubated in the dark for 15-20 minutes at room temperature.

11. In all wells of the tablet in the same sequence as the substrate mixture, add 30 μl of stop-reagent to stop the enzymatic reaction.

12. Registration of the results of the reaction: measure the optical density of the solution of the enzymatic reaction in the wells at a wavelength of 492 nm.

The result is considered positive if the value corresponding to the ratio of the optical density of the solution in wells with specific serum to the optical density of the solution in wells with normal serum is greater than or equal to 2.1.

Literature

1. Kononova D.G., Sharapova T.A. Intestinal yersiniosis and its significance in human pathology. Information mail. Vladivostok. 1980. S. 1-15.

2. Popova O. V., Shepeleva G. K., Shestokova I. V., Andreev I. V., Popova T. I., Yushchuk N. D. Clinical and immunological characteristics of yersiniosis infection. // Infectious. illnesses. 2006. V. 4. No. 3. S.51-55.

3. Tseneva G.Ya. Yersinia and Yersiniosis. SPb. St. Petersburg. 2006.168 s.

4. Akbarov S.V., Sultanov Kh.K., Matkarimov B.D., Baltabaeva M.A. Clinical variants of yersiniosis reactive arthritis in children. // Pediatrics. 1993. No. 6. S.54-56.

5. Kamantsev V.N., Skripchenko N.V., Tikhomirova O.V., Ankylosing spondylitis M.K. Lesions of the peripheral nervous system with yersiniosis infection in children. // Russian honey. journal 2003. No2. S.27-29.

6. Yushchuk N.D., Karetkina G.N. Yersiniosis. // Ross. journal gastroenterol., hepatol., coloproctol. 1997. No5. S.58-60.

7. Klimanova E.M., Belaya O.F., Lavrova K.V. et al. Use of the coagglutination reaction in the diagnosis of yersiniosis in children. // Pediatrics. 1993. No. 4. S.48-49.

8. Malysheva E.B., Moskovkina E.D., Volnova O.M., Oskerko E.F., Shelalueva M.V. The importance of the PCR method in the diagnosis of yersiniosis: new technologies in assessing the effectiveness of treatment and prognosis. // Genodiagnosis in modern medicine. Thes. Doc. 3rd All-Russian Scientific - prakt.konf. Moscow. 2000. S. 169.

9. Koroliuk A.M., Golovacheva S.N., Dulatova M.V. The choice of rational methods for determining antibodies to Yersinia enterocolitica. The agglutination reaction. // Zh. Mikrobiol. Epidemiol. Immunobiol. 1988. No. 9. P.45-47.

10. Alexander S.K., Lukin Yu.V., Fidler L.M. Preparation of Ig G diagnosticum based on colored polyacrolein latexes for use in latex agglutination reaction. // ZhMEI. 1990. No. 6. S.84-88.

11. Korolyuk A.M., Golovacheva S.N., Dulatova M.V. Serological diagnosis of intestinal yersiniosis. Construction of a stable red blood cell preparation for an indirect hemagglutination reaction. // ZhMEI. 1990. No. 3. S.30-34.

12. Granfors K., Ogasawara M., Hill J. L., Lahesmaa-Rantala R., Toivanen A., Yu DTY.

Analisis of IgA antiboddies to lipopolisaccharide in Yersiniatriggered reactive artritis. // J. Infect. Dis. 1989. V.159. P.1142-1147.

13. Malov I.V., Rubtsov I.V., Yushchenko G.V., Leonenko V.V. Method for enzyme immunoassay of yersiniosis. // Patent SU 1767435 A1 of 1992.10.07.

14. Bogautdinov Z.F., Vylegzhanina E.S., Kuzmina V.B., Astakhova T.S., Niyazov U.E., Shumilov K.V. A method for the diagnosis of yersiniosis. // Patent RU 2152037 C1 of 2000.06.27.

15. Delvig A.A., Semenov B.F. The mechanisms of the formation of the immune response to porin proteins of the outer membrane of meningococcal serogroup B. // Zh. microbiol., epidemiol. and immunology. 1997. No. 6. S.92-96.

16. Vostrikova O.P., Kim N.Yu., Likhatskaya G.N., Guzev K.V., Vakorina T.I., Khomenko V.A., Novikova O.D., Solovyova T.F. The structure and function of pore-forming proteins of bacteria of the genus YERSINIA. II Bioorgan, chemistry. 2006.V.32. Number 4. S.371-383.

17. Egorov A.M., Osipov A.P., Dzantiev B.B., Gavrilova E.M. Theory and practice of enzyme immunoassay. M .: Higher school, 1991.228 s.

18. Ashmarin I.P. Vorobyov A.A. Statistical methods in microbiological research. - M., 1962. P.30-66.

Claims (2)

1. A method for the differential diagnosis of yersiniosis, including the determination of specific antibodies to Yersinia enterocolitica in the patient's blood serum by enzyme-linked immunosorbent assay, characterized in that the species-specific porosity protein of the outer membrane of Yersinia enterocolitica is used as the antigen, serum dilutions of 1: 800- are taken for the diagnostic titer 1: 1600 and the result is considered positive at a value corresponding to the ratio of the optical density of the solution in the reaction with a specific serum to the optical oh density of the solution in the reaction with normal serum is larger than or equal to 2.1. 2. The diagnostic kit for implementing the method according to claim 1, comprising a tablet with immobilized antigen, samples of control (positive and negative) blood serum and conjugate, containers with inactivating and stop reagents, substrate and buffer systems ready for use, characterized in that as an antigen, a species-specific porin protein of the outer membrane of Yersinia enterocolitica is used, and a 0.1% solution of Tween-20 or Tween-80 is used as an inactivating reagent.

Images