RU2232989C1 - Method for preparing test-system for determination of helicobacter pylori cytotoxin-associated protein antigens in biological material in infected patients by co-agglutination reaction - Google Patents

Abstract

FIELD: medicine, immunology.

SUBSTANCE: invention can be used in diagnosis of diseases associated with H. pylori using co-agglutination reaction. As material for preparing anti-toxic rabbit helicobacter serum the homogenate from chicken embryos infected with the strain H. pylori NCTC 11637 (VAC-A⁺, CAG-A⁺) and died for two days is used. Prepared serum contains mainly antibodies raised to large-molecular proteins (above 90 kDa), among them, to cytotoxin-associated CAG-A protein exhibiting high immunogenicity. On the basis of this serum Hp-tox-diagnosticum (test-system) is made for carrying out the co-agglutination reaction on plates that shows high sensitivity and specificity. Diagnosticum allows detecting antigens of large-molecular proteins in H. pylori, among them, proteins VAC-A and CAG-A being in human biological material directly (feces, saliva, circulating immune complexes). The application of method provides simplifying and accelerating the preparing test-system for carrying out the co-agglutination reaction.

EFFECT: improved preparing test-system.

2 cl, 11 dwg, 2 tbl, 6 ex

Description

Appointment: biotechnology. It can be used in the field of medicine for the diagnosis and monitoring of Helicobacter pylori infection caused by toxigenic strains, to evaluate the effectiveness of antibiotic therapy, as well as in scientific studies of the pathogenesis and immunogenesis of this infection.

The inventive serum is obtained by immunizing rabbits with fetal fluid of chicken embryos infected with toxigenic (VAC-A ⁺ , CAG-A ⁺ ) culture of Helicobacter pylori (H. pylori) and died within 1-2 days as a result of reproduction and lethal action of these bacteria . Serums containing predominantly antibodies to large molecular weight proteins with a molecular weight of 90-140 kDa and taken in selected optimal dilutions sensitize the Cowan I staphylococcus cells treated with formalin and by heating.

The obtained antibody diagnosticum is used in the formulation of the coagglutination reaction on immunological tablets with a U-shaped bottom with various dilutions of biological material (coprofiltrate, saliva, circulating immune complexes, blood serum) - experimental series. In the second row of biomaterial diluted in the same way, instead of a diagnosticum, non-sensitized staphylococcus (control row) is introduced.

The results are taken into account by the difference in coagglutination titers in the experimental and control series, 4-fold or more difference in the titers in which indicates a positive reaction to the presence of a complex of large molecular proteins, which are a highly specific antigen marker for cytotoxin-producing H. pylori strains.

The method allows to diagnose and verify H. pylori infection in the acute period of the disease, to determine the ability of the pathogen to express toxin in the body in dynamics, to evaluate the effectiveness of eradication therapy.

The formulation of the coagglutination reaction on immunological tablets makes it highly sensitive and informative.

H. pylori is known to have several pathogenetically significant protein antigens, among which great importance is given to the vacuolizing VAC-A cytotoxin with a molecular weight (mM) of about 90 kDa and the associated highly immunogenic CAG-A protein with mM 120-140 kDa, which is specific marker of pathogenicity island (PAI), which contains about 40 genes that control the synthesis of pathogenetically significant proteins of these bacteria.

Strains of H. pylori, isolated from patients and healthy individuals, have different ability to express VAC-A and CAG-A proteins and are divided according to this characteristic into 2 main types: I - (VAC ⁺ , CAG ⁺ ), II - (VAC ^- , CAG ^- ).

To diagnose H. pylori infection, it is important not only to determine the fact of infection, the presence and quantity of bacteria in the body, but also to identify the ability of bacteria to express these proteins (VAC-A and CAG-A) in the body. Strains of H. pylori type I have increased virulence and are much more often found in peptic ulcer and gastric cancer.

Known methods for the diagnosis of H. pylori infection, based on the determination of the genome regions responsible for the synthesis of VAC-A and CAG-A proteins using polymerase chain reaction (PCR) in strains. In principle, biopsy samples of the gastric mucosa, gastric juice, plaque, and feces can theoretically serve as material for studies using PCR (1, 2). However, the use of PCR for the study of feces is very difficult due to the presence of inhibitory polysaccharide components, it requires preliminary complex multi-stage processing of the material, which makes this method practically unacceptable for use in conventional diagnostic laboratories.

A known method for the diagnosis of H. pylori infection by determining antibodies to VAC-A and CAG-A proteins in the blood serum of patients by ELISA and immunoblot methods (3). However, like other serological methods, these methods do not allow one to judge the current infection, since antibodies remain in the body for a long time after a clinically pronounced infection, for the same reason, the method cannot be used to assess the effectiveness of eradication therapy.

The coagglutination reaction was first used in the present invention, which allows one to study directly in biological material the antigens of pathogenetically significant high molecular weight proteins of H. pylori (more than 90 kDa), including, obviously, vacuolating toxin VAC-A and cytotoxin-associated CAG-A protein.

It is known that pathogenic microorganisms grown on nutrient media do not fully show their potential metabolic potential (1).

Once in the body of a sensitive host (human or animal), bacteria rebuild their metabolism to withstand the body's defenses. At the same time, microorganisms can acquire the ability to form a capsule, express toxins, and change antigenic and immunogenic properties.

Helicobacter pylori, the genome of which has been studied in detail, is particularly pronounced instability. With its difficult cultivation on artificial nutrient media, we suggested that in a sensitive organism the production of pathogenetically significant antigens and especially toxins can be much more pronounced and complete. This applies not only to known, but possibly not yet known pathogenetically and immunologically important antigens and toxins. In this case, the quantitative ratio of antigens formed during H. pylori infection is also important.

Earlier, we found that 9-day-old chicken embryos are a sensitive model for the propagation of H. pylori (4).

Based on the position that in a sensitive organism, in contrast to the growth of pathogenic microorganisms in nutrient media, the expression of pathogenetically significant antigens and toxins is most pronounced, and chicken embryos are a sensitive model of the reproduction and lethal action of H. pylori, as a material for obtaining antitoxic serum was used homogenate of fluids and tissues of chicken embryos infected with VAC-A ⁺ , CAG-A ⁺ H.pylori strain NCTC 11637.

The serum obtained after hyperimmunization of rabbits contained antibodies to a complex of pathogenetically significant antigens and toxins of H. pylori, mainly to high molecular weight proteins with mM from 90 to 120-140 kDa, which include VAC-A and CAG-A proteins. The latter, as you know, has a particularly pronounced immunogenicity.

Designed on the basis of these sera and stabilized with formalin and heating staphylococcus strain Cowan I, the diagnosticum designated by us as the "HP-tox-diagnosticum", when setting up the coagglutination reaction on a plate, allows detecting the presence of antigens of large molecular proteins H.pylori (including, obviously, VAC- A and CAG-A proteins) in biological material (feces, saliva, blood serum, CEC) in infected people.

Helicobacter tox diagnostic for RCA was developed for the first time. It allows you to identify the complex of the most important pathogenetically significant antigens and toxins in H. pylori infected people during the period of exacerbation of the disease and the growth of bacteria in the body, thus verify the fact of the participation of H. pylori in the infectious process, and also evaluate the effectiveness of treatment. The method for determining high molecular weight H. pylori proteins directly in biological material is simple, non-invasive, safe (therefore, it can be used in children and the elderly), it is short in time (18-24 hours), and economical. The method can be used in scientific research, the study of pathogenesis and immunity.

The aim of this work is to develop a method for obtaining a test system for determining pathogenetically significant protein antigens of H. pylori with a high molecular weight, including the vacuolizing toxin VAC-A and its associated protein CAG-A, directly in biological material (feces, saliva, blood serum) in H. pylori infected people.

The technical result achieved using the developed method is to obtain a diagnosticum for detecting high molecular weight proteins of 90-120-140 kDa, including probably VAC-A and cytotoxin-associated protein CAG-A, in coprofiltrate, saliva in the coagglutination reaction (RCA) serum, which allows not only to diagnose H. pylori infection (VAC-A and CAG-A proteins are markers of H. pylori), but also to determine the possibility of their expression in the body. In addition, the detection of H. pylori toxins in circulating immune complexes in the serum of patients with peptic ulcer disease additionally indicates the formation of specific antibodies to these proteins.

The method for determination of high molecular weight H. pylori proteins does not require endoscopy; it is carried out in vitro by setting the coagglutination reaction on immunological tablets, it is simple to set up, it speeds up the analysis, and is economical. It is achieved by using the H. pylori I strain of group VAC-A ⁺ , CAG-A ⁺ (e.g., H. pylori NCTC 11637). As a material for immunization of rabbits, the embryonic fluid of 9-day-old chicken embryos infected with the intragastric agar culture of H. pylori and died 1-2 days after their reproduction is used. The embryonic fluid is treated with 0.11% glutaraldehyde and rabbits are immunized with the resulting “toxoid”. Blood is taken after the third cycle (8 injections) of hyperimmunization.

Based on the obtained serum, coagglutinating diagnosticum is prepared; the reaction is placed on immunological tablets with a U-shaped bottom of the wells in two rows with different dilutions of the test material from 1: 4 to 1: 4096 and above (in the analysis of serum).

In the first (experimental) row, a diagnosticum is introduced, in the second row (control) - a suspension of non-sensitized staphylococcus and the result is taken into account by the appearance of an "umbrella" at the bottom of the hole and the difference in coagglutination titers in the I and II rows. A difference in titers of 4 or more times in the experimental and control series is considered positive for the presence of cytotoxin-associated proteins of H. pylori.

The test system is a kit that includes two containers: 1 filled with a specific H. pylori "tox-diagnosticum"; 2 - not sensitized staphylococcus Cowan I.

A method of obtaining a diagnosticum for the detection of high molecular weight proteins, including toxins, in biological fluids and its possible use are presented in the examples.

Example 1. The preparation of a full range of pathogenetically significant antigens and toxins Helicobacter pylori.

Chicken embryos of 9 days of age are infected inside the yolk sac with a culture of H. pylori strain NCTC No. 11637 grown on Columbia agar supplemented with 5% horse blood. Flushing 3-5 daily culture is introduced in an amount of 30 million MT in embryos that are incubated in a humid chamber at 37 ° C for two days.

The lysed embryo with vitelline fluid and protein is homogenized, freed from large particles by centrifugation at 2000 rpm for 30 minutes, poured into sterile ampoules and stored at -20 ° C.

This preparation - embryonic homogenate (EG) contains a full complex of protein antigens and toxins of H. pylori during the reproduction of bacteria in developing embryos, which seem to be optimal conditions for life (a rich set of nutrients, the presence of red blood cells and, therefore, hemin sufficient microaerophilic and temperature conditions).

Using polyacrylamide gel electrophoresis, we found that the composition of the embryonic homogenate contains at least 14-16 protein antigens that differ in molecular weight (mm) and the severity (intensity) of the lines formed by them (Fig. 1).

The entire electrophoretic field of the developed antigens was conditionally divided into three zones: I — zone of high molecular weight proteins with mM 90 and more kDa; II - zone of medium molecular proteins with mm 67-43 kDa; III - zone of low molecular weight proteins with mm 33-14 kDa. In the I zone, 3 major proteins were detected (possibly CAG-A with mM 120-140 kDa and VAC-A with mM about 90 kDa), in the II zone several minor proteins were detected, among which flagellum proteins (mM 57 kDa) and Ure A, and in III - low molecular weight proteins, among which a major protein is determined, possibly Ure B, mM of which is about 30 kDa.

In the lyophilized culture of H. pylori 11637, we found about 7 proteins: one in the first zone; 2-3 - in the second zone and 3-6 - in the third zone of the electrophoregram.

In the second zone, the protein (albumin) of the serum of newborn calves, which was used in the freeze drying of the strain, was also determined.

In the embryonic homogenate, a specific lipopolysaccharide (LPS-O antigen) of H. pylori was not detected by immunoelectrophoresis and precipitation in a gel with sera against a heated culture of H. pylori (O-antiserum); in a coagglutination reaction on glass with a diagnosticum prepared on the basis of this serum, it was found in a dilution of 1: 42-1: 84.

Thus, the embryonic homogenate of chicken embryos infected with H. pylori 11637 culture, after bacteria multiplication and embryo death, contained mainly a complex of pathogenetically significant major components of large molecular weight proteins with mM more than 90 kDa, including, obviously, VAC-A and CAG- toxin A protein.

Liquid extract of uninfected embryos and eggs did not contain these large-molecular proteins.

Example 2. Obtaining serum against the full complex of antigens and toxins Helicobacter pylori.

For immunization of rabbits, embryonic fluid (homogenate) of infected and dead embryos treated with 0.11% glutaraldehyde was used.

0.5 ml embryonic homogenate (toxoid) mixed with 0.5 ml of Freund's complete adjuvant was injected intradermally into the rabbits at several points of the back twice with an interval of two weeks, then after two weeks - 0.5 ml of the toxoid subcutaneously, after a week - 1.0 ml of toxoid intravenously. The second and third immunization cycles consisted of two intravenous injections of a toxoid of 1.0 ml. Blood was taken 7-10 days after each immunization cycle.

The study of sera against embryonic homogenate by agar precipitation, immunoelectrophoresis in agar, polyacrylamide gel electrophoresis and immunoblot showed that the obtained sera contained antibodies mainly against large molecular weight H. pylori proteins with mM more than 90 kDa, had a small amount of antibodies to medium and non-molecular proteins contained antibodies to low molecular weight proteins and H. pylori O-antigen. (figure 2). The latter is consistent with published data on the weak immunogenicity of the O antigen. No antibodies to human antigens of I (0) blood group were also detected.

Thus, the serum we obtained contained mainly antibodies to large-molecular proteins with mM 90 and more kDa, including, obviously, VAC-A and CAG-A proteins.

In contrast, sera against warmed H. pylori cultures contained only antibodies to the thermostable O antigen and had virtually no antibodies to protein antigens.

Example 3. Construction of Helicobacter pylori tox-diagnosticum and formulation of the coagglutination reaction (RCA) on the tablet.

The selection of the dilution of serum for RCA on the tablet is as follows.

Based on a 5% suspension of formalinized (0.5% formaldehyde, 37 ° C, 3 hours) and warmed (80 ° C, 1 hour) staphylococci and sera against embryonic homogenate taken in different dilutions (1: 5, 1:10, 1:15, 1:20, etc.), prepare test diagnostics from the calculation:

0.1 ml of serum in different dilutions;

0.9 ml of a 5% suspension of stabilized staphylococci;

9.0 ml of phosphate-buffered saline (PBS), pH 7.2.

The suspension is gently mixed for several minutes and the obtained diagnostics are checked in RCA on the tablets.

To do this, in each row of holes with a U-shaped bottom of the immunological tablet make 0,075 ml of FSB. The test antigen (unheated embryonic homogenate) is instilled into the first 5 vertical wells of 0.025 ml and then titrated in each row four times in succession, transferring to the next wells of 0.025 ml from the previous dilution. From every seventh well of the horizontal row, 0.025 ml of liquid is discarded. Every eighth hole with FSB remains the control (1st control). After titration, the antigen test is added to all wells of the first row tox-diagnosticum, made on the basis of serum at a dilution of 1: 500, in the wells of the second row - a diagnosticum with a dilution of serum 1: 1000, in the wells of the third row - a diagnosticum with a dilution of 1: 1500, in the fourth row - a diagnosticum with a dilution of serum 1: 2000, etc. Instead of a diagnosticum, 0.025 ml of 5% suspension of non-sensitized staphylococcus is added to the last row (2nd control).

The tablets are shaken by gently tapping the edge of the tablet in different directions and left in a thermostat in a humid chamber (in plastic bags) at 37 ° C for 2 hours, after which they are transferred to room temperature until morning. Accounting for the reaction is carried out after 1-2 days upon the appearance of a uniform agglutinate - an “umbrella” at the bottom of the hole, in the inclined position of the tablet, not flowing to the lower edge of the hole (figure 3).

The optimal dilution of serum upon receipt of a diagnosticum for RCA on a plate is that dilution that gives the maximum titer with a test antigen and does not give a reaction in two control wells (1 — control of a diagnosticum with FSB, 2 — control of an antigen test with a suspension of non-sensitized staphylococcus).

From figure 3 it is seen that the optimal dilution of serum to obtain a diagnosticum is 1: 2000. This diagnosticum reveals large molecular H. pylori proteins in a titer of 1:64 in the test material.

Example 4. The manufacture of diagnostic kits for determination of high molecular weight proteins of Helicobacter pylori in the coagglutination reaction on a tablet (final stage).

Ready-made diagnostics in the required amount are obtained on the basis of sera selected in the study of test diagnosticum using optimal dilutions of sera.

The production technology is described in example 3. Briefly, it is as follows.

If when testing test diagnostics, the optimal final dilution of serum, for example, is 1: 2000, then the initial serum is diluted 1:20, taken in the amount of 0.1 ml, mixed with 0.9 ml of 5% suspension stabilized with formalin (0.5% formalin, 3 hours) and heating (80 ° C, 1 hour) of Cowan I staphylococcus, 9 ml of PBS (pH 7.2) are diluted. If more diagnosticum is needed, these volumes are increased by the required number of times.

The sensitivity of the diagnosticum is examined using the original embryonic lysate in various dilutions (Table 1).

The specificity of the HP-diagnosticum was tested with biological material (saliva, feces) in patients with chronic gastritis and a duodenal ulcer during exacerbation and remission.

With a clinically expressed disease in the feces and / or saliva of these patients, with a positive coagglutination reaction on glass to H. pylori O-antigen, positive RCA was detected on the tablets with tox diagnosticum in the same samples. During remission of H. pylori in RCA, glass was not detected. In the absence of propagation of H. pylori, there was no RCA with tox diagnosticum.

Fecal samples from patients with shigellosis, salmonellosis, yersiniosis, campylobacteriosis, the diagnosis of which was made on the basis of clinical data and positive RCA for thermostable O-antigen of the corresponding pathogens, were also investigated.

In the yolk fluid of some chicken eggs, H. pylori O-antigen was detected. However, in the yolk of these eggs, the reaction to the toxin was negative. This can probably be explained by the lack of conditions for the reproduction of these bacteria and the expression of toxins.

The data presented in Table 2 indicate the specificity of the HP-tox diagnosticum.

As can be seen from Table 1, the Hp-tox diagnosticum, prepared on the basis of anti-tox rabbit serum taken at a dilution of 1: 2000, gives a positive RCA on tablets with tox antigen before a dilution of 1: 800, which indicates that it is sufficient high sensitivity.

Example 5. The use of tox-diagnosticum in exacerbation of non-ulcerative dyspepsia associated with Helicobacter pylori.

Systematic long-term prospective studies were performed in two patients: with non-ulcer dyspepsia and duodenal ulcer, confirmed by the detection of O-antigen, which is a marker of N.R., in the coagglutination reaction on glass. The first patient was observed for 1.5 years. During this period, 200 samples of saliva and 274 samples of feces were examined.

It was found that during the period of exacerbation, a high rise in the titers of high molecular weight proteins (BMB) of H. pylori was observed, during the period of convalescence, the level of H. pylori BMB decreased and was practically not detected during the period of remission (Fig. 4).

In the second patient, operated on for a stomach ulcer 35 years ago and suffering from periodic exacerbations of non-ulcer gastritis, 73 samples of saliva and 42 samples of feces were examined for 1.5 years. An increased level of H. pylori BMD was observed during the exacerbation period and decreased during the period of remission (Fig. 5).

Example 6. The use of tox-diagnosticum to detect high molecular weight proteins of Helicobacter pylori in patients with duodenal ulcer before and after treatment.

We studied patients with duodenal ulcer (duodenal ulcer) in the period of exacerbation: before treatment, during treatment and at various times after it (Fig.6, 7, 8).

It was shown that during exacerbation (before treatment), all patients had a high level of H. pylori BMD in saliva and feces, which correlated with pathogen reproduction, as evidenced by positive RCA on glass on H. pylori O-antigen.

During the treatment period and in the next 1.5 months after it, the level of H. pylori BMD gradually decreased, but remained at a fairly high level. And only in the long term 1-2 years after treatment with successful eradication therapy, the level of H. pylori BMD decreased to low or negative indicators. With ineffective therapy continued to remain at a high level.

Thus, RCA with a tox-diagnosticum for detecting Helicobacter pylori BMD allows one to detect the rise of these pathogenetically significant antigens during the exacerbation of UBD, to verify the infection and the ability of the H. pylori strain to express H. pylori BMB, including, probably, VAC-A and CAG- A protein, makes it possible to evaluate the effectiveness of eradication therapy.

Literature

1. Kishkun A.A. Modern methods of diagnosis and evaluation of the effectiveness of the treatment of Helicobacter pylori infection. - Laboratory medicine, 2000, No. 3, p. 37-44.

2. Makristathis A., Paschiuq E., Schutze K. et al. Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. - J. Cl. Microb., 1998, v. 36, No. 9, p. 2772-74.

3. Salama S.M., Wefuan J.N., Shiro-Koulla S. et al. Value of whole-cell antigen extracts for serological detection of Helicobacter pylori. - J. Cl. Microb., 1993, v.31, No. 12, p. 3331-3332.

4. Belaya Yu.A., Belaya O.F. Coagglutination reaction. The signal method for the diagnosis of intestinal zooanthroponoses, control of food, feed, environmental objects. - M., 2001, 50 p.

Claims (2)

1. A method of obtaining a test system for the determination of antigens of high molecular weight Helicobacter pylori proteins in the biological material of infected individuals by a coagglutination reaction, including immunization of rabbits with a complex of Helicobacter pylori proteins followed by blood sampling, serum isolation, sensitization with obtained sera of Staphylococcus aureus Cowan I cells, from preservation, characterized in that the animals are immunized with a homogenate of fluids of chicken embryos after infection with their culture of Helicobacter pylori (vas-A ⁺ , cag-A ⁺ ) and death whether they are in 1-2 days. 2. The method according to p. 1, characterized in that the coagglutination reaction is placed on immunological tablets with a U-shaped bottom of the wells, in 2 rows of which unheated test material (coprofiltrate, saliva, blood serum) is diluted four times to 1: 4096, and then a diagnosticum is introduced into the first row, a suspension of non-sensitized staphylococcus is added (control), and the reaction is taken into account by the appearance of uniform agglutinate (“umbrella”) at the bottom of the well and is considered positive when the agglutination titer is four or more times higher in the first row compared with a titer of agglutination in the second.

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