RU2717671C1 - Method for multiplex immunological analysis of biological samples from air in automatic mode - Google Patents

Abstract

FIELD: medicine; biology.SUBSTANCE: invention relates to research in environmental protection, medicine and microbiology and can be used for analysing air for presence of biopathogens in it. Method for multiplex immunological analysis of biological samples from air in automatic mode includes a number of consecutive stages: collecting bioaerosol particles, converting them into a liquid state, sample preparation for analysis by incubating the sample with magnetic microspheres, biotinylated antibodies and fluorescent labels and analysing the sample on a multiplex immunoassay. Immunoassay is performed in an immunoassay reactor, which is a reservoir of chemically resistant material, volume of 1.5 ml in which automatic temperature is maintained at 37 °C. In order to retain magnetic particles during flushing to tank in an automatic mode by means of electromechanical drive annular permanent magnet is supplied. Immunoassay reactor is mounted on the shaft of an orbital shaker, which in automatic mode drives it at a speed of up to 1,500 rpm.EFFECT: method enables to automate the process of immunological examination of air samples, shorten the time of sample analysis, provide continuous monitoring of air composition, as well as increase accuracy of determining biopathogens in air.1 cl, 1 dwg

Description

FIELD OF THE INVENTION

The invention relates to the field of research in environmental protection, medicine, microbiology. This invention can be used to analyze air for the presence of biopathogens in it, to protect humans or animals from the harmful effects of bacteria, viruses, genetic vectors and nanotechnology objects.

State of the art

The prior art method of multiplex immunological analysis (MIA). The technique of multiplex immunoassay of biomarkers consists in the fact that at the first stage, they incubate fluorescent microspheres conjugated to specific analyte molecules (antibodies or antigens) with a sample (serum, plasma, cell culture supernatant) containing the desired analyte, after which washing of the unreached microspheres of components. Each microsphere contains within itself a certain amount of fluorescent dye, thereby spectral coding is carried out, where the spectral code of the microsphere is determined by the fluorescence intensity of the dye.

Two methods can be used for washing, depending on the magnetic properties of the microspheres used. If non-magnetic fluorescent polymer microspheres are used, then washing is performed by filtration using a special vacuum collector and filtrational 96-well flat-bottomed plates. When using magnetic fluorescent polymer microspheres, washing can be carried out in two ways: by filtration and by magnetic separation. Magnetic washing consists in using a special magnetic tripod, in which a flat-bottomed 96-well plate is installed, and magnetic microspheres are concentrated on the bottom of the tablet well, and unbound components are removed by turning the tablet over together with a magnetic tripod. In addition, it is possible to perform washing with the help of an automatic flushing station (wash) completely in automatic mode. The type of microspheres used (magnetic or non-magnetic) depends on the manufacturer of the specific reagent kits.

In the next step, analyte microspheres are incubated with biotinylated antibody antibodies. Unbound components are washed off again, after which a fluorescent dye - streptavidin-phycoerythrin conjugate (SAPE), which binds to the biotin label on detector antibodies, is added to the resulting sandwich. In this case, phycoerythrin plays the role of a fluorescent tag, which is read using a reporter laser. In some types of analysis, detection antibodies are used that are conjugated to phycoerythrin directly without the formation of the biotin-streptavidin conjugate.

At the final stage of the analysis, in order to detect the corresponding analytes in the sample, the signals of the fluorescent labels are read in a multiplex immunological analyzer, for example, a flow cytometer or a planar immunological analyzer. During the measurement, each microsphere is exposed to light with two different wavelengths (for example, red and green) from two radiation sources. The type of radiation source used depends on the type of analyzer, for example, solid-state lasers are used in a flow cytometer and LEDs in a planar immunological analyzer. Light from the first laser (e.g., red) serves to excite fluorescence from the microspheres to determine the microsphere code and their classification, and light from the second laser (e.g., green) serves to excite fluorescence from the fluorescent label in order to determine the fact of binding to the analyte. The signal emitted by the fluorophores is recorded by the analyzer's detectors, which can be used in a flow cytometer as photomultiplier tubes (PMTs) or avalanche photodiodes, and in a planar immunological analyzer, a CCD.

The registration of fluorescence from phycoerythrin indicates the presence of the desired analyte in the test sample. In this case, the fluorescence intensity of phycoerythrin is proportional to the concentration of the desired analyte. Signal readout results are expressed in relative units of MFI (median fluorescence intensity). The analyzer registers approximately 100 microspheres with a different spectral code and proceeds to the next well. The reading of the entire 96-well plate takes from 20 minutes to 1 hour, depending on the type of analysis. Thus, the spectral code of the microsphere and the presence (quantity) of the desired analyte on the microsphere are simultaneously analyzed. The scheme of conducting multiplex immunoassay on the Luminex platform (Rudik D.V., Gulidova O.V., Multiplex analysis on the Luminex platform, Development and registration of medicines, 2014, No. 8, 98-104). However, this method does not allow automatic continuous multiplex immunological analysis of air for the presence of biopathogens in it to protect humans or animals from the harmful effects of bacteria, viruses, toxins, genetic vectors and nanotechnology objects.

In addition, the prior art method for conducting multiplex immunological analysis of air, disclosed in WO2007001458 08/23/2007, which is selected as a prototype. The known method includes the detection and quantification of many analytes in the air. The specified process includes: drawing the air flow into the collector and ensuring the passage of the specified flow through the nonlinear passage inside the specified collector, while the analytes are held in the specified passage. The passage of this passage through an air stream is necessary for binding to a fluid that carries multiplexed binding elements consisting of many subsets of binding elements, each subset being different from all other subsets in a characteristic that is distinguishable in the analysis, and each contains a distinctive binding member that selectively binds one an analyte from the specified set of analytes. The fact of binding of the analyte to the specified binding member that distinguishes the specified subsets is detected. However, the known method does not have a specific sample preparation for immunological analysis in the framework of the study of air, which reduces the reliability of the results.

The implementation of the invention

The claimed method of multiplex immunological analysis of biological samples from air in an automatic mode, including a series of successive known steps in which particles are collected bioaerosol; translate them into a liquid state; perform sample preparation of the sample for analysis; analyze the sample on a multiplex immunological analyzer, according to the invention, the method also includes the following steps:

• preparation of immunological analysis includes incubation of the sample with magnetic microspheres, biotinylated antibodies and fluorescent labels, as well as washing between incubations;

• in this case, an immunological analysis is performed in an immunological analysis reactor, which is a container of chemically resistant material, with a volume of 1.5 ml, in which the temperature is automatically maintained at 37 ° C;

• when using magnetic microspheres, a ring permanent magnet that holds magnetic particles during washing is fed to this tank in automatic mode using an electromechanical drive;

• at the same time, the immunological analysis reactor is installed on the shaft of the orbital shaker, which automatically drives it at a speed of up to 1500 rpm;

• at the same time, all reactor operating modes are set automatically by the control computer in accordance with the analysis protocol;

• reagents for sample preparation of immunological analysis are stored at 4 ° С on a cooled platform, also installed on the shaft of the orbital mixer;

• before using reagents, the platform is set in motion with a frequency of 800 rpm to obtain a homogeneous suspension of microspheres;

• after performing all sample preparation operations, the suspension from the reactor is sent to a multiplex immunological analyzer;

• manipulations with liquids during sample preparation of multiplex immunological analysis are performed using a syringe pump and a multi-port valve in automatic mode.

The indicated actions are carried out by means of an automatic analyzer of biopathogens in the air, which is a complex of equipment located in a single metal frame structure. The layout of the device is made in the form of two compartments. In the upper compartment are all the nodes and subsystems that are in direct contact with biological samples and reagents - from an aerosol collection device to PCR and an immunological analyzer. In this compartment, a controlled temperature and humidity regime is maintained. In the lower compartment there is all the auxiliary equipment and control electronics: power sources and distributors, backup batteries, a refrigeration unit, a control computer and automation units. The refrigeration unit provides cooling of the circulating water to a temperature of +4 ° C; this water, in particular, is used to cool reagents in order to ensure the autonomy of the device for up to 7 days. The sample preparation system of an automatic analyzer of biopathogens in the air is built according to a closed scheme.

In this case, the preparation of immunological analysis includes incubation of the sample with magnetic or non-magnetic microspheres, biotinylated antibodies and fluorescent labels, such as streptavidin-phycoerythrin conjugates (SAPE), as well as washing between incubations. In this case, the operations are performed in the immunological analysis reactor, which is a container of chemically resistant material, for example, polypropylene, with a volume of 1.5 ml in which, the required temperature is automatically maintained at + 37 ° C. When using magnetic microspheres, a ring permanent magnet that holds magnetic particles during washing is fed to a given container in automatic mode using an electromechanical drive. The immunological analysis reactor is mounted on the shaft of an orbital shaker, capable of driving it in automatic mode at speeds of up to 1500 rpm. All reactor operating modes are set automatically by the control computer in accordance with the analysis protocol. Reagents for sample preparation for immunological analysis (microspheres, antibodies, SAPE) are stored at + 4 ° C on a refrigerated platform, also installed on the shaft of the orbital mixer. Before using reagents, the platform is set in motion at a frequency of 800 rpm to obtain a uniform suspension of microspheres. After performing all sample preparation operations, the suspension from the reactor is sent to a multiplex immunological analyzer. All manipulations with liquids during sample preparation of MIA are performed by a syringe pump and a multi-port valve in automatic mode.

The objective of the present invention is the implementation of the possibility of analyzing an air sample in automatic mode in real time, which will reduce the time of study of the sample due to the complete automation of the process, provide continuous monitoring of the composition of the air, and also improve the accuracy of determination of biopathogens in the air.

The technical result of the invention is the automation of the process of immunological research of an air sample, reducing the time of study of the sample, ensuring continuous monitoring of the composition of the air, as well as improving the accuracy of determining biopathogens in the air.

Disclosure of Invention

The specified technical result is realized due to the following techniques. The air analysis process begins with the collection of the aerosol sample and its transfer to the liquid phase. At the same time, an impactor may be responsible for collecting ambient air - a device that creates a flow of intake air up to 4 m ³ per minute and is able to concentrate aerosol particles in the range from 0.3 to 10 μm by about 10 times. The air enriched with such particles is supplied to the aerosol collector, constructed on the principle of a water cyclone. Sample collection takes place within 4-5 minutes, after which, the required volume (about 5 ml) of the liquid is transferred to the next processing stages. The next step is the immunological analysis of the sample, which can be carried out on the basis of microfluidic components, including: tubes for supplying fluid, syringe pumps, valves, vortex shakers, during which the manual manual sample preparation technique is fully reproduced in automatic mode. In this case, the components participating in the sample preparation of the sample for MIA in accordance with the protocol are added via microtubules to the test tube reactor located on a shaker equipped with a magnet and heated to + 37 ° C. In this case, sample preparation includes a number of sequential actions that are performed automatically:

1. The primary antibody is conjugated with microspheres (A), for which a container with microspheres at a concentration of 50,000 pcs / ml is shaken with a primary antibody on a shaker for 60 seconds. Then, 50 μl of solution A is automatically transferred through the fluid system to a polypropylene reactor. 50 μl of sample solution (B) are delivered there via the fluid system. As a result, 50 μl A + 50 μl B in the well .

2. Then, the first incubation of the obtained AB sample is performed on a shaker for 30 minutes at a temperature of 37 ° C and 800 rpm. As a result , 100 μl AB + B in the well .

3. Then perform the first flushing: raise the magnet for 60 seconds. A fluid sample is taken from the AB reactor. Lower the magnet. 200 μl phosphate-saline wash buffer (PBS-TBN) is added to the reactor. Shake the wash buffer on a shaker for 10 seconds. Then they again raise the magnet for 60 seconds. Liquid is taken from the reactor through the fluid system. Lower the magnet. 200 μl of PBS-TBN wash buffer is added to the reactor. Shake it on a shaker for 10 seconds. Lift the magnet for 60 seconds. Liquid is taken from the reactor through the fluid system. Lower the magnet. Transfer 50 μl of PBS-TBN Wash Buffer through the fluid system to the well. Shake it on a shaker for 10 seconds. As a result of this step , 50 μl AB in the well.

Then detecting antibodies conjugated with biotin are added (C), for which they are shaken in a container at a temperature of +4 ° C on a shaker for 20 seconds. Then, 50 μl (C) is transferred via the fluid system to PBS-TBN at a concentration of 8 μg / ml each into the reactor. As a result, in a well, 50 μl AB + 50 μl C.

4. Perform a second incubation on a shaker for 30 minutes at a temperature of 37 ° C and 800 rpm. According to the results of the stage in the well, 100 μl ABC + C.

5. Perform a second flush, similar to the first (see step 3). As a result of the step in the well, 50 μl ABC.

After that, the container with a conjugated solution of phycoerythrin (PE) with streptavidin (SA) (D) in PBS-TBN at a concentration of 8 μg / ml is shaken at a temperature of +4 ° C on a shaker for 20 seconds. After that, 50 μl of solution D is transferred through the fluid system to the reactor. At the end of this step , 50 μl ABC + 50 μl D.

6. Perform a third incubation on a shaker for 30 minutes at a temperature of 37 ° C and 800 rpm. At the end of this step, 100 μl ABCD + D in the well.

7. Perform the third wash, similarly to the first and second (see step 3). Then, 100 μl of PBS-TBN wash buffer is transferred through the fluid system to the reactor and it is shaken on a shaker for 10 seconds. As a result , 100 μl ABCD in the well.

8. Then, the indicated 100 μl ABCD sample is automatically transferred via the fluid system to a multiplex immunological analyzer and analysis is carried out.

The scheme of immunocomplexes resulting from sample preparation is shown in FIG. 1, where A is the primary antibody conjugated with microspheres, B is the antigen / target / sample, C is the detecting antibody conjugated with biotin (Bi), D is phycoerythrin conjugated with streptavidin (SAPE).

These actions are carried out by means of an automatic analyzer of biopathogens in the air, which includes an impactor, an aerosol collector, an ultrasonic homogenizer, a first rotary valve, a nucleic acid (NK) separation unit, containing: a tank with reagents for NK extraction, a third rotary valve, an NK recovery reactor, a unit for sample preparation of multiplex immunological analysis (MIA), including: a reservoir with microspheres, antibodies and dyes, a second rotary valve and a MIA reactor; a reservoir containing washing liquids such as: buffer, or 70% alcohol, or hypochlorite, or alkali; immunological analyzer; PCR reagent reservoir; PCR analyzer; sample archive; reservoir for cooled circulating water; reservoir of liquid intended for discharge.

Multiplex immunoassay was carried out using xMAP microspheres manufactured by Luminex company of the capture-sandwich type. Primary antibodies in the amount of 10 μg / 10 ⁶ microspheres with three spectral codes: SY-25 (anti-adenovirus anti-hexone) was conjugated to the code No. 72 - ST1 (anti-LPS of S. typhimurium) and code No. 78 - CT8 (anti-beta subunit of cholera toxin). Microspheres were sensitized in accordance with the protocol given in the official Luminex publication xMAP Cookbook, 3rd. edition ”, by carbodiimide chemistry. Microspheres (1 × 10 ⁶ pcs.) Activated 10 μl 50 mg / ml (in distilled H2O) 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Sigma, # 22980) and 10 μl 50 mg / ml (in dist. H2O) N-hydroxysulfosuccinimide sodium salt (s-NHS) (Sigma, # 24520) in 80 μl of activating buffer solution (0.1 M NaH2PO4, pH 6.2) (Amresco, # Am-O823) for 20 min at 25 ° C with a rotation of 20 rpm on a vortex. After that, the activated microspheres were washed twice and resuspended in 500 μl of binding buffer solution (50 mM MES, pH 5.0) (Sigma, # M2933) with the addition of each antibody to its region. After incubation for 2 hours in the dark at 25 ° C with rotation at 20 rpm and three washing steps, the blocking buffer solution of the microspheres was resuspended in 1 ml of storage buffer solution and used for analysis after at least 16 hours. As a buffer for blocking and storage (microsphere blocking / storage buffer), PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% NaN3) for CT8, PBS-BN (PBS, 1% BSA, 0.05% NaN3) for SY-25 and PierceTM Protein-Free (PBS) Blocking Buffer (Pierce, # 37584) for ST1.

The following reagents were used to prepare PBS-TBN and PBS-BN: BSA (Sigma, # B-4287); Tween 20 (Amresco, # Am-O777), NaN3 (Amresco, # Am-O639). The microspheres remaining after the sensitization procedure were counted using an automatic TC-20 cell counter [Bio-RAD, USA].

Thus, due to the fact that the method of multiplex immunological analysis of air is performed in automatic mode and in the following sequence: collecting aerosol particles, transferring them to a liquid state, subsequent analysis of the selected sample using multiplex immunological analysis in automatic mode, with the following successive stages of sample preparation : agitation and connection of the sample with microspheres; the first incubation of the sample, the first washing, shaking and combining the sample with antibodies, the second incubation of the sample, the second washing, shaking and combining the sample with SAPE, the third incubation, the third washing, obtaining a sample for analysis, which is performed in a multiplex immunological analyzer, which allows automatic analysis of the air sample, while reducing the time of the study of the sample, to ensure continuous monitoring of the composition of the air, as well as to increase the accuracy of determining biopathogens in the air.

The claimed method can be implemented by a specialist in practice and, when implemented, ensures the implementation of the claimed purpose. The possibility of implementation in practice follows from the fact that for each feature included in the claims on the basis of the description, the material equivalent is known, which allows us to conclude that the criterion of "industrial applicability" for the invention and the criterion of "completeness of disclosure" for the invention are met.

Claims (16)

1. The method of multiplex immunological analysis of biological samples from air in automatic mode, including a series of sequential steps: collect bioaerosol particles; translate them into a liquid state; perform sample preparation of the sample for analysis; analyze the sample on a multiplex immunological analyzer, characterized in that Sample preparation for immunological analysis includes incubation of the sample with magnetic microspheres, biotinylated antibodies and fluorescent labels, as well as washing between incubations; wherein immunological analysis is performed in an immunological analysis reactor, which is a container of chemically resistant material, a volume of 1.5 ml, in which the temperature is automatically maintained at 37 ° C; when using magnetic microspheres, a ring permanent magnet that holds magnetic particles during washing is fed to a given container in automatic mode using an electromechanical drive; wherein the immunological analysis reactor is mounted on the shaft of the orbital shaker, which automatically drives it at a speed of up to 1500 rpm; in this case, all reactor operating modes are set automatically by the control computer in accordance with the analysis protocol; reagents for sample preparation of immunological analysis are stored at 4 ° C on a cooled platform, also installed on the shaft of the orbital mixer; before using reagents, the platform is set in motion at a frequency of 800 rpm to obtain a uniform suspension of microspheres; after all sample preparation operations have been completed, the suspension from the reactor is sent to a multiplex immunological analyzer; manipulations with liquids during sample preparation of multiplex immunological analysis are performed with a syringe pump and a multi-port valve in automatic mode. 2. The method according to claim 1, characterized in that conjugates of streptovidin-phycoerythrin are used as fluorescent labels.

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