RU2451083C1 - Method for detecting bacterial antigens - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves preparing a biochip containing immobilised antibodies to various antigens, incubating the biochip with analysed cell suspension for 1-2 hours at room temperature, washing off the biochip from unconjugated cells and detecting an antigen-antibody reaction with using an UV light source. A bacterial cell suspension is taken in the concentration of min. 1x10⁵ microbial cell/ml inactivated in any method not destroying the analysed antigen and resuspended in a buffer solution containing Tris HCl 50 mM, 0.02 % Tween 20 and NaCl 50 mM and propidium iodide in the concentration of 1.0-2.0 mcg/ml and used to coat the biochip surface in an amount sufficient to coat all its segments.

EFFECT: invention provides more simple and cheap method to detect the presence of bacterial antigens.

3 tbl, 3 ex

Description

The invention relates to microbiology and veterinary medicine and can be used to determine the antigenic structure of bacteria.

Traditional methods for identifying pathogenic microorganisms in environmental objects include taking a sample from certain places in a sterile dish and sending it to a laboratory for analysis (Handbook edited by M.O. Birger. M., "Medicine", 1973, p. 387), obtaining a pure culture and its identification based on the study of antigenic and / or biochemical properties. However, microorganisms have an extremely diverse antigenic structure, for example, the S.enterica species includes about 2500 serotypes and the identification of each serotype requires the use of a large number of antigen-specific sera; conducting separate tests to identify each antigen requires a lot of labor and a large number of diagnostics.

When using this method to indicate pathogenic microorganisms in environmental objects, a number of difficulties arise due to the fact that various foreign microflora components, individual fragments of genetic material are present in the samples of the studied material, which reduces the reliability of the method. The method requires careful preparation of the test material. The analytical sensitivity of the method depending on the type of infection is 100-1000 MK./ml, therefore, at low concentrations of the microorganism, culture is required.

A known method of quantitative immunoassay of low molecular weight and high molecular weight compounds using a biological microchip (RF Patent No. 2363955, publ. 10.08.2009). The method provides for the incubation of a biological microchip (biochip) in a reaction medium, including the analyzed sample for the formation of complexes. A biochip is an array of three-dimensional hydrogel elements of a given volume, formed on a substrate by photo- or chemically induced polymerization and containing biological molecules (ligands) that are the same or different in nature.

The disadvantages of this method are low sensitivity and limited functionality, since the biochip used does not allow the use of whole bacterial cells to identify their antigens and provides for preliminary cleaning of the studied analytes. The use of antigens rather than whole cells makes it possible to quantify the method, but sharply reduces its sensitivity; therefore, the use of highly sensitive detectors to take into account the reaction (mass spectrometry, PCR, ELISA, the use of powerful radiation sources for fluorescence detection of the formed complexes) is required.

The closest to the claimed method, the prototype is a method for determining antigens of eukaryotic cells using immunological biochips, which consists in the fact that the biochip containing immobilized antibodies is placed in a flow chamber and incubated with a suspension of eukaryotic cells. Cells having corresponding antigens on their surface bind to antibodies immobilized in strictly defined regions of the substrate. Then the biochip is washed from cells that did not bind to the antibodies with a fluid flow at a given speed. The fluid flow rate can be selected such that only those cells that do not bind to antibodies are detached, and cells that even bind weakly to antibodies remain on the surface of the biochip. The presence of bound cells is established by viewing the biochip spots with a microscope (A.V. Shishkin, I.I. Shmyrev, S. A. Kuznetsova and others. "Immunological biochips for the study of human red blood cells." Biological membranes, 2008, v.25, No. 4, p. 267-276).

The disadvantages of this method are its limited functionality, since it is unsuitable for conducting a biochip study outside the flow chamber, it does not allow morphological studies and identification of cells bound in the area of the biochip spots (which in some cases leads to errors in interpreting the result). Also, this method is not intended for the study of bacterial cells. In addition, the known method does not allow storage of a biochip located in a fluid-filled flow chamber for more than several hours after analysis, because with longer storage, cell death and destruction occurs, which excludes the possibility of re-examination of cells in controversial cases.

The above disadvantages are associated with the fact that the separation of cells (all or part) from the surface of the biochip occurs when the capillary of the flow chamber is filled with air, necessary to extract the biochip. Detachment of cells occurs at the boundary between air and liquid. This makes it impossible to stain cells bound to the biochip by methods used for further morphological studies and requiring preliminary drying of the biochip and fixation (for example, according to Romanovsky-Giemsa). For the same reason, reading the result and performing microscopic examination of the bound cells can only be carried out when the biochip is in the flow chamber. Staining of cells bound to the biochip by other methods suitable only for their approximate study (for example, by incubation with a methylene blue solution) directly in the flow chamber leads to adsorption of the dye (or other substances used in the staining process), which violates their transparency. This eliminates or at least complicates their reuse.

The technical task of the claimed invention is to expand the functionality of the known method and its cost.

The stated technical problem is achieved by the proposed method, namely, that the object of the study is using a native suspension of inactivated bacterial cells stained with a fluorescent dye, the cells are applied to a biochip with immobilized antibodies in the form of dots, the biochip is incubated with a cell suspension and washing from unbound cells followed by detection of the reaction "antigen-antibody" using a source of ultraviolet radiation.

The proposed method is as follows.

Prepare a biochip. As a substrate for the biochip, a glass slide coated with a 3% albumin solution with the addition of 0.1% glycerol and 0.01% amide black dye 10B is used. Glasses are dried and incubated at a temperature not exceeding 80 ° C for 2-4 hours. Various antibodies to the antigens of microorganisms are applied on the prepared glasses in the form of dots at 0.1-0.5 μl per point. The biochip is dried for 3-5 hours, immersed in a 0.2% solution of glutaraldehyde and incubated at + 5 ° C for 16-18 hours. The biochip is rinsed with distilled water, immersed in a 1% solution of bovine serum albumin with the addition of 0.02% tween-20 in phosphate-buffered saline (50 mM, pH 7.2-7.4). Incubated for 1 hour, rinsed with distilled water and dried. Store at + 5 ° C in a dry airtight container.

The analysis is as follows. A suspension of bacterial cells at a concentration of at least 1 × 10 ⁵ microbial cells / ml, inactivated in any way that does not destroy the analyzed antigen, is resuspended in a buffer solution containing 50 mM Tris HCl, 0.02% tween-20, 50 mM NaCl and propidium iodide at a concentration of 1.0-2.0 μg / ml. The suspension is applied to the surface of the biochip in an amount sufficient to cover all areas of the biochip containing antibodies, covered with a plastic film and incubated for 1-2 hours at room temperature.

After completion of the incubation, the biochip is washed from unbound bacterial cells using a buffer solution containing 50 mM Tris HCl, 0.02% tween-20, 50 mM NaCl, then rinsed in distilled water. The detection of the reaction of formation of the complex "antigen-antibody" is carried out using a source of ultraviolet radiation, for example a transilluminator. Bacterial cells containing a fluorescent dye, when interacting with specific antibodies immobilized on the surface of the biochip, form an antigen-antibody complex in the form of a cluster of luminous cells visualized in ultraviolet rays in the form of luminous spots localized in places where antibodies specific for given antigen.

To determine the serotype of the microorganism, the reaction results are interpreted in accordance with existing diagnostic tables.

The determining significant differences of the proposed method from the prototype are:

- to determine the antigens of bacterial cells, a native suspension of inactivated bacterial cells stained with a fluorescent dye is used, which allows to expand the functionality of the method, as well as increase its sensitivity, because binding of the antigen to the antibody is accompanied by the immobilization on the biochip of whole bacterial cells containing a much larger number of DNA fluorescent dye molecules than a single molecule of purified and fluorophore-labeled antigen can contain (prototype);

- fluorescent labeling of bacteria is carried out with an intercalating DNA fluorescent dye - propidium iodide, which simplifies the process of labeling the antigen by eliminating secondary labeled antibodies from the reactions, chemical methods of covalently “sewing” various labels to the antigen, and also cheapening the method by using cheaper detection systems .

The technical result is achieved due to the fact that the whole cell can contain much more fluorescence tag molecules than a single antigen molecule, and therefore the luminosity of the antigen-antibody complex, which is part of the bacterial cell / antibody, will be higher than complex pure antigen / antibody, which allows you to record reactions without the use of expensive equipment.

The invention is illustrated by the following examples of specific performance of the method.

Example 1

Determination of antigens of bacterial cells of Streptococcus sp.

A slide coated with a 3% albumin solution with the addition of 0.1% glycerol and 0.01% amide black 10B was used as a biochip. The glasses were dried and incubated at a temperature not exceeding 80 ° C for 2 hours. Antibodies to somatic O antigens Streptococcus sp. serogroups A, B, C, D and antibodies to O antigens of Salmonella enterica at 0.2 μl per point. The biochips were dried for 2 hours and incubated in a 0.2% solution of glutaraldehyde at + 5 ° C for 18 hours.

The biochips were rinsed with distilled water, immersed in a 1% solution of bovine serum albumin with 0.02% tween-20 in phosphate-buffered saline (50 mM, pH 7.2-7.4), incubated for 1 hour, rinsed with distilled water and dried.

The analysis was carried out as follows. Suspension Streptococcus sp. at a concentration of 1 × 10 ⁶ microbial cells / ml, inactivated by boiling for 30 minutes, resuspended in a buffer solution containing 50 mm Tris HCl, 0.02% tween-20, 50 mm NaCl and propidium iodide at a concentration of 1.0 μg / ml The suspension was applied to the surface of the biochip in an amount of 200 μl, covered with a plastic film and incubated for 1 hour at room temperature.

After incubation, unbound cells were removed by rinsing in a buffer solution containing 50 mM Tris HCl, 0.02% tween-20, 50 mM NaCl, rinsed in distilled water, and bound bacteria were detected using a transilluminator containing an ultraviolet light source such as a mercury lamp, light filter and video documentation system of radiation. When the complex "antigen-antibody" is formed on the biochip, luminous spots are visible that are localized in places where antibodies specific for this antigen are applied. The results are presented in table 1.

Table 1 Biochip Antibodies Test bacteria cultures Streptococcus sp. serotype B Streptococcus sp. serotype D Salmonella enterica O-specific antigen of Streptococcus sp. serogroup A negatively negatively negatively O-specific antigen of Streptococcus sp. serogroup B positively negatively negatively O-specific antigen of Streptococcus sp. serogroup C negatively negatively negatively O-specific antigen of Streptococcus sp. serogroup D negatively positively negatively Salmonella enterica O Antigen Specific (ABCD) negatively negatively positively

As follows from table 1, the method is characterized by sufficient specificity and allows you to distinguish microorganisms of the genus Streptococcus of different serotypes.

Example 2

The antigens of bacterial cells of Salmonella sp. the proposed method. At the same time, the possibility of using commercial antibody preparations intended for the agglutination reaction as part of the biochip was determined. As a control, the antigens of bacterial cells of S.enterica were determined in the agglutination reaction. For this purpose, the bacterial culture of S.enterica and commercial preparations (BioRad) of antibodies used to determine Salmonella serotypes were used.

A slide coated with a 3% albumin solution with the addition of 0.1% glycerol and 0.01% amide black 10B was used as a biochip. The glasses were dried and incubated at a temperature not exceeding 80 ° C for 2 hours. The prepared glasses were coated with antibodies to somatic (O antigens) and flagellum (H antigens) Salmonella enterica in the form of dots at 0.1 μl per point. The biochips were dried for 3 hours and incubated in a 0.2% solution of glutaraldehyde at + 5 ° C for 20 hours.

The biochips were rinsed with distilled water, immersed in a 1% solution of bovine serum albumin with 0.02% tween-20 in phosphate-buffered saline (50 mM, pH 7.2-7.4), incubated for 2 hours, rinsed with distilled water and dried.

The analysis was carried out as follows. Suspension Salmonella sp. at a concentration of 1 × 10 ⁵ microbial cells / ml, inactivated by boiling for 30 minutes, resuspended in a buffer solution containing 50 mM Tris HCl, 0.02% tween-20, 50 mM NaCl and propidium iodide at a concentration of 2 μg / ml. The suspension was applied to the surface of the biochip in an amount of 100 μl, covered with a plastic film and incubated for 2 hours at room temperature.

After incubation, unbound cells were removed by rinsing in a buffer solution containing 50 mM Tris HCl, 0.02% tween-20, 50 mM NaCl, rinsed in distilled water, and bound bacteria were detected using a transilluminator. When the complex "antigen-antibody" is formed on the biochip, glowing spots are visible that are localized in places where antibodies specific for this antigen are applied.

The agglutination reaction on glass was carried out in accordance with the recommendations of the manufacturer of antibody preparations.

The results are shown in table 2.

table 2 Commercial serum preparations of BioRad The inventive method Agglutination reaction results NMA Positively Positively NMV Negatively Negatively H 2 Negatively Negatively Hg p Positively Positively Hg, m Positively Positively Hr Positively Positively Vi Negatively Negatively O 12 Negatively Negatively OMB Negatively Negatively OMA Positively Positively O 9 Positively Positively O 4, 5 Positively Positively O 6, 14, 24 Positively Positively Note: NMA, NMV - group-specific serum on Salmonella H antigens, OMA, OMV - group-specific serum on Salmonella O antigens. H - flagellar antigen, O - somatic antigen, Vi - capsular antigen

From the results shown in table 2, it follows that the same antibodies used in the agglutination reaction on glass and in the biochip, allow to obtain comparable results, which allows the use of existing commercial antibody preparations. Also, the data in table 2 indicate the reproducibility of the reactions of formation of antigen-antibody during microagglutination and using the proposed method.

Example 3

To determine the serotype of the Salmonella enterica microorganism, 4 Salmonella enterica cultures of the following serotypes were used: typhimurium, enteritidis, dublin, paratyphi A, as well as BioRad serum from S.enterica O, H and Vi antigens. The method was carried out analogously to example 1. The results are presented in table 3.

Table 3 Serotype A complex of interaction with antibodies to antigens was detected typhimurium NMA, O 4, 5 enteritidis OMA, NMB, O 9, dublin OMA, O 9, Hg paratyphi a OMA (group A and B), H 2, O 4, 5

From table 3 it is seen that the detected antigens correspond to the antigenic structure of serotypes Salmonella enterica, serotypes typhimurium, enteritidis, dublin, paratyphi A.

Thus, a simple and sensitive method has been proposed that allows one to determine the antigenic structure of bacteria and to detect reaction results simultaneously without microscopy using cheap visualization systems.

Using the proposed method will allow the following.

1. To expand the functionality of the method due to the possibility of determining the antigenic structure of prokaryotic bacteria (microorganisms).

2. To simplify and reduce the cost of the method due to:

- exceptions from the stages of sample preparation stage of obtaining purified antigens;

- the use of standard commercial antibody preparations used to study the antigenic structure of bacteria;

- Exclusions of expensive equipment: fluorophore glow detection systems using laser technology, mass spectrometry systems, PCR equipment;

- providing the ability to visualize the reaction results simultaneously using a transilluminator, which eliminates the stage of microscopy and / or sequential scanning of each section of the biochip with antibodies deposited on its surface.

3. Provide the ability to store the biochip after the reaction at room temperature.

Claims (2)

1. The method of determining bacterial antigens, including the preparation of a biochip containing immobilized antibodies to various antigens, incubation of the biochip with an analyzed cell suspension, washing the biochip from unbound cells with subsequent detection of the antigen-antibody reaction, characterized in that the bacterial suspension is used as an object of study concentrations of at least 1 · 10 ⁵ microbial cells / ml, inactivated by any non-destructive bacterial cells, the latter is resuspended in a buffer solution containing We use 50 mM Tris HCl, 0.02% tween-20 and 50 mM NaCl and a fluorescent dye - propidium iodide at a concentration of 1.0-2.0 μg / ml, applied to the surface of the biochip in an amount sufficient to cover all areas of the biochip, and incubated for 1-2 hours at room temperature, followed by detection of the antigen-antibody reaction using a source of ultraviolet radiation. 2. The method according to claim 1, characterized in that a transilluminator is used as a source of ultraviolet radiation.