RU2528099C2 - Biological microchip for detection and multiparameter analysis of anticholeraic antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a biological microchip for the detection and multiparameter analysis of anticholeraic antibodies in human blood serum containing a massive of V. cholerae O-antigens discretely applied and immobilised on a carrier surface, and a cholera toxin grouped separately.

EFFECT: parallel immunological analysis of several samples of human blood serum for anticholeraic antibodies to a wide spectrum of cholera agents with determining G and M immunoglobulins for a relatively short time.

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Description

The invention relates to the field of clinical laboratory diagnostics, biotechnology and medicine, in particular to the immunochemical analysis of human blood products for the presence of specific cholera antibodies, and can be used in laboratory diagnosis of cholera, namely, in serological examination of people for cholera.

At present, a detailed agglutination reaction of the studied human blood sera with a three-hour broth culture of Vibrio cholerae isolated in this focus or with test strains of cholera vibrios of serovars Ogawa, Inaba and O139 serogroup is used to detect anticholera antibodies in patients who have been ill, vibrio-carriers and vaccinated people. ]. In this case, the analysis time is 18 hours. The long analysis time and the subjectivity of taking into account the results make it urgent to create a new generation of test systems that provide high analysis performance, receive an answer in a shorter time, and reduce the volume of the test sample.

At present, the technology for the analysis of complex biological systems using biological microchips (biochips) has received significant development. In biological microarrays, in contrast to traditional serological methods, the possibility of simultaneous parallel analysis of the test sample by many parameters, that is, a multi-parameter analysis for the presence of antibodies of different specificity, is realized. This significantly increases the effectiveness of the analysis, reduces the required amount of the studied material, allows you to miniaturize the research, increase the reliability of the results and automate the stages of the analysis.

Separate detection of antibodies of different specificity is important when conducting an epidemiological investigation of cholera cases. Determination of antibodies to cholera toxin in the blood serum of people allows us to draw a conclusion about the epidemic significance of strains of cholera vibrios circulating in the focus of cholera.

A number of authors have developed biological microchips based on oligonucleotide probes — the so-called “DNA chips” [2-5], for identification and intraspecific typing of infectious disease agents. However, DNA chips, in the absence of pathogen isolation, including during antibiotic treatment, do not allow to assess the dynamics of the infection process, conduct a retrospective diagnosis of cholera, and determine the state of humoral immunity in vaccinated people.

For immunological analysis, protein microarrays or immunochips are used. Protein microarrays are divided into two-dimensional (proteins are immobilized on the surface of the carrier) and three-dimensional (proteins are immobilized inside the carrier) [6].

Known three-dimensional biological microchip based on polyacrylamide hydrogel for conducting multiple parallel immunological analysis of compounds formed by the polymerization of hydrogel elements of a given volume on the surface of the microchip [7], and a method for detecting biological toxins using a hydrogel microchip [8]. Hydrogel biological microarrays allow for multiple immunological analysis of a wide range of low molecular weight and high molecular weight compounds. A significant drawback of three-dimensional microarrays is the low diffusion rate of sample molecules within the hydrogel volume, requiring many hours of exposure, which practically eliminates the advantage obtained by increasing the spatial density of the sample. In addition, the described biological microchips are designed to analyze a single sample.

Two-dimensional microarrays are known for determining bacterial antigens [9], detecting antibodies to a number of infectious agents in human serum: hepatitis C virus [10, 11], HIV infection [12], syphilis pathogen [13], borreliosis [14], TORCH infections [15]. In two-dimensional microarrays, proteins are adsorbed or covalently bound to the substrate and directly contact with the test material, which significantly reduces the analysis time and simplifies the technology of manufacturing the microarray. For covalent binding of proteins, the surface of the carrier is subjected to chemical modification to introduce amino, aldehyde, or epoxy groups [6], which ensures strong binding of proteins to the surface of the substrate.

The closest in technical essence is a diagnostic test system in the format of a two-dimensional immunochip for serological diagnosis of syphilis [16]. The diagnostic test system in the format of an immunochip for serological differential diagnosis of syphilis consists of an immunosorbent with the antigens of the syphilis causative agent Treponema pallidum separately conjugated and the reagents necessary to detect the antigen-antibody complex immobilized on it. However, this test system, which allows simultaneously and differentially detecting antibodies to Treponema pallidum of different classes, is not intended to detect cholera antibodies.

An object of the present invention is the development of a biological microchip, allowing to detect in a short time antichole antibodies to antigens of the pathogen cholera O1 of the serogroup Ogawa and Inaba, O139 serogroup and cholera toxin.

The technical result is to increase the efficiency and reduce the time of serodiagnosis of cholera in humans, increase in information content by determining the classes of immunoglobulins, which allows you to set the duration of the disease. An additional result is an increase in the biological safety of serological diagnosis of cholera through the use of pure soluble antigens.

The technical result is achieved in that the biological microchip contains an array of V. cholerae O antigens and cholera toxin, discretely applied and immobilized on the surface of the substrate, in at least two repeats grouped into separate zones for parallel immunological analysis of several samples. The surface of the substrate is activated by aldehyde, epoxy or amino groups. V. cholerae O1 serogroups of the Ogawa and Inaba serovars and O139 serogroups are used to detect cholera antibodies.

Description of the design of the biological microchip.

The biological microchip is a substrate, which can be used as a slide according to GOST 9284-75 [17] 25x75x1 mm in size, activated by aldehyde, epoxy or amino groups. The specific antigens used in the manufacture of the microchip are preparations of soluble O-antigens V. cholerae O1 serogroup Serovar Ogawa, O1 serogroup Serovar Inaba, 0139 serogroups obtained in accordance with RF patent No. 2143280 [18] and cholera toxin obtained in accordance with the patent RF №2456996 [19]. Antigens are applied to activated glass by contact printing using a mini-plotter. Antigens are diluted in printing buffer to a final concentration of 0.5-1 mg / ml and sorbed in triplicate on activated glasses. Each antigen corresponds to an individual spot with a diameter of 0.3 mm. When printing, the set of antigens is grouped in the form of 16 identical zones on the same glass with a distance of 9 mm between the centers of the zones (Figure 1) and a distance between spots of 0.8-1 mm, providing a high density of antigens per unit surface area of the substrate. Immobilization of antigens is carried out for 1 h at a temperature of 37 ° C. Free glass surface binding sites are blocked with a 0.5% solution of bovine serum albumin (BSA) in phosphate-buffered saline for 1 h at 25 ° C. Then the glass is dried by centrifugation for 1 min at a rotor speed of 1000 rpm. Glasses with immobilized antigens are stored at 4 ° C until used. The maximum shelf life of 6 months.

A brief description of the drawings.

Figure 1 shows the general arrangement of antigens on a microchip (A), consisting of 16 identical zones (B) and the arrangement of antigens in one zone. Each zone is designed to analyze one sample. The numbers indicate the types of immobilized antigens: 1 - V. cholerae O1 antigen of Serovar Ogawa, 2 - V. cholerae O1 antigen of Serovar Inaba, 3 - V. cholerae O139 antigen of serogroup, 4 - cholera toxin, 5 - negative control (print buffer).

Figure 2 presents a schematic illustration of a stencil. Numbers indicate cell numbers. Stencil dimensions: 75x25.4 mm, mesh sizes: 7x7x4 mm.

Figure 3 shows an example of a positive result in the presence of antibodies to V. cholerae O1 serogroup Serovar Ogawa (A), V. cholerae O1 serogroup Serovar Inaba (B), V. cholerae O139 serogroup (C), cholera toxin (D) in the test material )

Examples of the use of a biological microchip.

Example 1. The identification and multi-parameter analysis of cholera antibodies class G / M in human serum during the on-line diagnosis of cholera (without determining the titer of specific antibodies)

Before staging the analysis from above, a stencil is applied and tightly clamped in a special frame to the designed biological microchip. The stencil contains 16 square-shaped cells (Figure 2), corresponding to 16 zones on a biological microchip (Figure 1A). When applying a stencil to a biological microchip, each cell is combined with the corresponding zone. 98 μl of buffer solution for washing the microchip from unbound BSA was added to each well and incubated on an immunoplate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 2-3 min. After incubation, the contents from the cells are carefully selected using a vacuum aspirator, without damaging the central field of the cell, corresponding to the location of the array of biological molecules. Then, 90 μl of buffer solution for sample dilution is added to each well. From 1 to 14 cells of the biological microchip, 2 μl of the studied sera are added, 2 μl of the positive control (positive human serum) is added to the 15 cell, 2 μl of sample dilution buffer (negative control) to 16 cells. The biological microchips are covered and incubated on an immuno-plate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 30 minutes. The contents of the cells are carefully selected using a vacuum aspirator. The microchips are washed twice with wash buffer, 100 μl of buffer solution are added to each well and incubated on an immunoplate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 2-3 minutes. 100 μl of conjugate containing antibodies against human G / M class immunoglobulins labeled with a fluorescent dye having a maximum absorption at a wavelength of 532 nm or 635 nm was added to each well in working dilution. The microchips are covered and incubated on an immunoplate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 30 minutes. The contents of the cells are carefully selected using a vacuum aspirator. The microchips are washed twice with wash buffer, adding 100 μl of buffer solution to each well, and incubated on an immuno-plate shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. Remove the stencil from the microchip. The microchip is washed with distilled water and dried by centrifugation for 1 min at a speed of 1000 rpm. After analysis, the microarrays are scanned on a fluorescence scanner at a wavelength of 532 nm or 635 nm. For analysis using the average value of the fluorescence intensity within the print area for each point of the array. Determine the location of the points of negative control for each test sample. Determine the value of the fluorescence intensity for each point of the array. The result is considered positive if the fluorescence intensity of the array point is 2.5 times higher than the fluorescence intensity of the negative control points. The result of the analysis is considered valid if there is a positive result in the positive control cell, with a negative result in the negative control cell. The sample is considered positive for the presence of antibodies to V. cholerae O1 of the serogovar Ogawa serogroup with a positive result in at least two repetitions in the first column of the array (Fig. 3A). The sample is considered positive for the presence of antibodies to V. cholerae O1 serogroup Inaba serogroup with a positive result in at least two repetitions in the second column of the array (Fig.3B). The sample is considered positive for the presence of antibodies to V. cholerae O139 serogroup with a positive result in at least two repetitions in the third column of the array (Fig.3B). The sample is considered positive for the presence of antibodies to cholera toxin V. cholerae with a positive result in at least two repetitions in the fourth column of the array (Fig. 3G). Analysis time is 1.5 hours

Example 2. The identification and multi-parameter analysis of cholera antibodies in human serum during a retrospective diagnosis of cholera (with the definition of the titer and class of specific antibodies)

Before staging the analysis on top of the biological microchip impose and tightly clamped in a special frame stencil. 100 μl of buffer solution for washing the microchip are added to each well and incubated on an immunoplate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 2-3 minutes. After incubation, the contents from the cells are carefully selected using a vacuum aspirator, without damaging the central field of the cell, corresponding to the location of the array of biological molecules. The test serum is diluted in the wells of a 96-well plate in a buffer solution for dilution of samples in a dilution of 1:20 to 1: 1280 in two replicates. To do this, 190 μl of sample dilution buffer is added to the first well of a 96-well plate, 100 μl of sample dilution buffer is added from the second to the seventh well. 10 μl of test serum (final dilution 1:20) was added to the first well and titrated twice 100 μl to the seventh well (final dilution 1: 1280). The procedure is repeated in the other 7 wells of a 96-well plate. The contents of each well in a volume of 100 μl are transferred to the cells of the biological microchip. The remaining two free holes contribute positive and negative control. The biological microchips are covered and incubated on an immuno-plate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 30 minutes. The contents of the cells are carefully selected using a vacuum aspirator. The microchips are washed twice with a buffer solution for washing the microchip, adding 100 μl of solution to each well and incubated on a shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 2-3 minutes. From 1 to 8 microchip cells are added in working dilution of 100 μl of conjugate containing antibodies against human G class immunoglobulins labeled with fluorescent dye, from 9 to 16 microchip cells are added in working dilution of 100 μl of conjugate containing antibodies against human class M immunoglobulins, labeled with fluorescent dye. The microchips are covered and incubated on an immunoplate shaker with a platform rotation of 500 rpm at a temperature of 37 ° C for 30 minutes. The contents of the cells are carefully selected using a vacuum aspirator. The microchips are washed twice with wash buffer, adding 100 μl of buffer solution to each well, and incubated on an immuno-plate shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. The further analysis procedure and the calculation of the results are carried out analogously to example 1. The titer and class of immunoglobulins are determined on the basis of positive results with the corresponding anti-species conjugates. For the titer of specific activity take the maximum dilution of the test sample, in which a positive result is observed. The analysis time is 2 hours.

Thus, the use of biological microarrays can increase the efficiency of serological testing and reduce its time due to the simultaneous parallel analysis of several samples of human blood serum for the presence of cholera antibodies to a wide range of cholera pathogen antigens of various specificity with the determination of class G and M immunoglobulins. reliability of the analysis results, standardization and automation of research. When conducting research using the inventive biochip requires a minimum amount of test material.

Information sources

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Claims (3)

1. A biological microchip for the detection and multi-parameter analysis of anticholera antibodies in human blood serum, characterized in that it contains an array of V. cholerae O-antigens and cholera toxin discretely applied and immobilized on the surface of the substrate, grouped into separate zones for parallel immunological analysis of several samples . 2. The biological microchip according to claim 1, characterized in that the substrate is activated by aldehyde, epoxy or amino groups. 3. The biological microchip according to claim 1, characterized in that O-antigens of V. cholerae O1 of the serogroup Ogawa and Inaba and O139 serogroups are applied to the substrate.

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