RU2717671C9 - 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

The technical field to which the invention relates

The invention relates to the field of research in environmental protection, medicine, microbiology. This invention can be applied 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

A multiplex immunoassay (MIA) method is known from the prior art. The technique of multiplex immunoassay of biomarkers consists in the fact that at the first stage, incubation of fluorescent microspheres conjugated with analyte-specific molecules (antibodies or antigens) with a sample (serum, plasma, cell culture supernatant) containing the desired analyte is carried out, after which the unbound microspheres of components. Each microsphere contains inside itself a certain amount of a fluorescent dye, thereby carrying out their spectral coding, where the spectral code of the microsphere is determined by the fluorescence intensity of the dye.

For washing, two methods can be used 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 manifold and 96-well flat bottom filtration plates. When using magnetic fluorescent polymer microspheres, washing can be carried out in two ways: filtration and by magnetic separation. Magnetic washing consists in using a special magnetic rack, into which a flat-bottomed 96-well plate is installed, and the magnetic microspheres are concentrated on the bottom of the plate well, and unbound components are removed by inverting the plate along with the magnetic rack. In addition, it is possible to perform cleaning using an automatic washing station (washer) in a fully automatic mode. The type of microspheres used (magnetic or non-magnetic) depends on the manufacturer of the specific reagent kits.

At the next stage, the microspheres with the analyte are incubated with detector biotinylated antibodies. Unbound components are washed off again, after which a fluorescent dye is added to the formed "sandwich" - streptavidin-phycoerythrin conjugate (SAPE), which binds to the biotin label on the detector antibodies. Phycoerythrin in this case acts as a fluorescent label, which is read using a reporter laser. Some assays use detector antibodies conjugated directly to phycoerythrin without forming a biotin-streptavidin conjugate.

At the final stage of the analysis, in order to carry out the detection of 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 irradiated with light of two different wavelengths (eg, 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 are used in a planar immunoassay analyzer. Light from the first laser (for example, red) serves to excite fluorescence from microspheres in order to determine the code of the microspheres and their classification, and light from the second laser (for example, green) serves to excite fluorescence from a fluorescent label in order to determine the fact of binding to the analyte. The signal emitted by fluorophores is recorded by the analyzer's detectors, which can be photomultiplier tubes (PMTs) or avalanche photodiodes in a flow cytometer, and a CCD matrix can be used in a planar immunological analyzer.

Registration of fluorescence from phycoerythrin indicates the presence of the desired analyte in the test sample. In this case, the intensity of phycoerythrin fluorescence is proportional to the concentration of the desired analyte. Signal readings are expressed in relative units of MFI (median fluorescence intensity). The analyzer registers about 100 microspheres with different spectral codes and goes on to the next well. The entire 96-well plate is read in 20 minutes to 1 hour, depending on the type of analysis. Thus, the spectral code of the microsphere and the presence (amount) of the desired analyte on the microsphere are analyzed simultaneously. Scheme of multiplex immunoassay on the Luminex platform (Rudik D.V., Gulidova O.V., Multiplex analysis on the Luminex platform, Drug Development and Registration, 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, from the prior art there is known a method for conducting a multiplex immunological analysis of air, disclosed in WO2007001458 23.08.2007, which is selected as a prototype. The known method includes detecting and quantifying a plurality of analytes in air. The specified process includes: drawing the air flow into the manifold and ensuring the passage of the specified flow through a non-linear passage within the specified manifold, while the analytes are held in the specified passage. Passage of the specified passage by a stream of air is necessary for binding with a liquid that carries multiplexed binding elements consisting of many subsets of binding elements, each subset being distinguished from all other subsets by a characteristic that is distinguishable by analysis, and each contains a distinctive binding member that selectively binds one an analyte from the specified set of analytes. Detection of the fact of binding of the analyte with the specified linking member, which distinguishes the specified subsets. However, the known method does not have a specific sample preparation for immunological analysis in the framework of the air study, which reduces the reliability of the results obtained.

Implementation of the invention

The claimed method for multiplex immunological analysis of biological samples from air in an automatic mode, including a number of successive known stages, in which the collection of particles of bioaerosol; transfer them to a liquid state; perform sample preparation for analysis; analysis of the sample is carried out on a multiplex immunological analyzer, according to the invention, the method also includes the following steps:

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

• while the 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, an annular permanent magnet is brought to this container in an automatic mode using an electromechanical drive, which holds the magnetic particles during flushing;

• in this case, the immunological analysis reactor is installed on the shaft of the orbital shaker, which automatically sets it in motion at a speed of up to 1500 rpm;

• in this case, all operating modes of the reactor 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 refrigerated platform, also installed on the shaft of an orbital mixer;

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

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

• manipulations with liquids during the preparation of multiplex immunological analysis are performed with a syringe pump and a multiport valve in automatic mode.

These actions are carried out by means of an automatic analyzer of biopathogens in the air, which is a set of equipment housed in a single metal case of a frame structure. The layout of the device is made in the form of two compartments. The upper compartment contains all the units and subsystems that are in direct contact with biological samples and reagents - from an aerosol collection device to PCR and an immunological analyzer. This compartment maintains a controlled temperature and humidity regime. The lower compartment contains all auxiliary equipment and control electronics: power supplies and distributors, backup batteries, refrigeration unit, 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 to ensure the autonomy of the device for up to 7 days. The sample preparation system for an automatic analyzer of biopathogens in air is built in a closed circuit.

In this case, sample preparation for immunological analysis includes incubation of the sample with magnetic or nonmagnetic microspheres, biotinylated antibodies and fluorescent labels, such as streptavidin-phycoerythrin (SAPE) conjugates, as well as washing between incubations. In this case, operations are performed in an immunological analysis reactor, which is a container made of chemically resistant material, for example, polypropylene, with a volume of 1.5 ml, in which the required temperature of + 37 ° C is automatically maintained. When using magnetic microspheres, an annular permanent magnet is supplied to this container in an automatic mode using an electromechanical drive, which holds the magnetic particles during washing. The immunological analysis reactor is installed on the shaft of an orbital shaker capable of driving it in automatic mode at speeds up to 1500 rpm. All operating modes of the reactor 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 mounted on the shaft of an orbital stirrer. Before using the reagents, the platform is driven at 800 rpm to obtain a homogeneous suspension of microspheres. After completing all the sample preparation operations, the suspension from the reactor is sent to the multiplex immunological analyzer. All manipulations with liquids during MIA sample preparation are performed with a syringe pump and a multiport valve in automatic mode.

The objective of the present invention is to realize the possibility of analyzing an air sample in an automatic mode in real time, which will reduce the time for examining the sample by fully automating the process, ensure continuous monitoring of the air composition, and also improve the accuracy of determining biopathogens in the air.

The technical result of the invention is to automate the process of immunological examination of an air sample, reduce the time for examining the sample, ensure continuous monitoring of the air composition, and increase the accuracy of determining biopathogens in the air.

Disclosure of invention

The specified technical result is realized through the following techniques. Air analysis begins with collecting an aerosol sample and converting it to a liquid phase. At the same time, an impactor can be responsible for collecting ambient air - a device that creates an intake air flow of up to 4 m ³ per minute and is capable of concentrating aerosol particles in the range from 0.3 to 10 microns approximately 10 times. The air enriched with such particles is supplied to an aerosol collector, built on the principle of a water cyclone. The sample is collected within 4-5 minutes, after which the required volume (about 5 ml) of the liquid is transferred to the next processing stages. The next stage is the immunological analysis of the sample, which can be carried out on the basis of microfluidic components, including: tubes for fluid supply, syringe pumps, valves, vortex shakers, during which the manual procedure of sample preparation is fully reproduced in an automatic mode. In this case, the components participating in the sample preparation for MIA, in accordance with the protocol, are added via microtubules into a 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 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 the primary antibody on a shaker for 60 seconds. Then, 50 μl of solution A is automatically transferred through the fluid system into the polypropylene reactor. There, 50 μl of the sample solution (B) is delivered via the fluid system. As a result, in well 50 μl A + 50 μl B.

2. Then the first incubation of the obtained AB sample is carried out 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 carry out the first rinsing: raise the magnet for 60 sec. A sample is taken from the AB reactor through the fluid system. Lower the magnet. Add to the reactor 200 μl of phosphate buffered saline wash (PBS-TBN). Shake the wash buffer on a shaker for 10 seconds. Then the magnet is raised again for 60 seconds. Take liquid from the reactor through the fluid system. Lower the magnet. Add 200 µl of PBS-TBN wash buffer to the reactor. Shake it on a shaker for 10 seconds. Raise the magnet for 60 sec. Take liquid from the reactor through the fluid system. Lower the magnet. Transfer 50 μl of PBS-TBN Wash Buffer by fluid system to a well. Shake it on a shaker for 10 seconds. As a result of this step, there is 50 μl of 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) in PBS-TBN at a concentration of 8 μg / ml each is transferred through a fluid system into a reactor. As a result , 50 μl AB + 50 μl C.

4. Perform a second incubation on a shaker for 30 minutes at 37 ° C and 800 rpm. At the end of the stage, 100 μL ABC + C in the well.

5. Perform a second wash in the same way as the first (see step 3). At the end of the stage, 50 μl ABC in the well.

Then 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 + 4 ° C on a shaker for 20 seconds. Thereafter, 50 μl of solution D is transferred through the fluid system to the reactor. By the end of this stage , 50 μl ABC + 50 μl D.

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

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

8. Then the specified 100 μl of ABCD sample is automatically transferred through the fluid system to the multiplex immunoassay analyzer and analyzed.

A diagram of the immunocomplexes formed as a result of sample preparation is shown in Fig. 1, where A is a primary antibody conjugated to microspheres, B is an antigen / target / sample, C is a detection antibody conjugated to biotin (Bi), D is phycoerythrin conjugated to 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) isolation unit, containing: a reservoir with reagents for NK isolation, a third rotary valve, an NK isolation reactor, a sample preparation unit for multiplex immunological analysis (MIA), including: a reservoir with microspheres, antibodies and dyes, a second rotary valve and a MIA reactor; a reservoir containing flushing fluids such as: buffer, or 70% alcohol, or hypochlorite, or alkali; immunological analyzer; reservoir with PCR reagents; PCR analyzer; archive of samples; tank for cooled circulating water; reservoir of liquid intended for draining.

Multiplexed immunoassay performed using the xMAP microspheres manufactured by Luminex type «capture - sandwich» .. Primary antibodies at 10 ug / ¹⁰ June microspheres with three spectral codes: a code №45 conjugated SY-25 (anti-adenovirus hexon) to the code No. 72 - ST1 (anti-LPS S. typhimurium) and to code No. 78 - CT8 (anti-cholera toxin beta subunit). The microspheres were sensitized according to the protocol given in the Luminex xMAP Cookbook, 3rd. edition ”, by the method of carbodiimide chemistry. Microspheres (1 × 10 ⁶ pcs.) Were activated with 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 distilled 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 ° С with rotation of 20 rpm on a vortex. Thereafter, the activated microspheres were washed twice and resuspended in 500 μl of binding buffer (50 mM MES, pH 5.0) (Sigma, # M2933) with the addition of each of the antibodies to their region. After incubation for 2 h in the dark at 25 ° С with rotation at 20 rpm and three stages of washing with blocking buffer, the microspheres were resuspended in 1 ml of buffer solution for storage and used for analysis after at least 16 h. As a buffer for blocking and storage (microsphere blocking / storage buffer), PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% NaN3) were used for CT8, PBS-BN (PBS, 1% BSA, 0.05% NaN3) for SY-25 and PierceTM Protein-Free (PBS) Blocking Buffer (Pierce, # 37584) for ST1.

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

Thus, due to the fact that the method of multiplex immunological analysis of air is performed in an automatic mode and in the following sequence: collection of aerosol particles, transferring them to a liquid state, subsequent analysis of the selected sample using multiplex immunological analysis in an automatic mode, with the following successive stages of sample preparation shaking and combining the sample with microspheres; first incubation of the sample, first washing, shaking and combining the sample with antibodies, second incubating the sample, second washing, shaking and combining the sample with SAPE, third incubation, third washing, obtaining a sample for analysis, which is performed in a multiplex immunoassay analyzer, which allows automatic analysis of an air sample, while reducing the time for examining the sample, ensuring continuous monitoring of the air composition, and also increasing the accuracy of determining biopathogens in the air

The claimed method can be carried out by a specialist in practice and, when implemented, ensures the implementation of the declared 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, a material equivalent is known, which allows us to conclude that the criterion "industrial applicability" for the invention and the criterion "completeness of disclosure" for the invention are met.

Claims (16)

1. Method for multiplex immunological analysis of biological samples from air in automatic mode, including a number of sequential stages: collecting bioaerosol particles; transfer them to a liquid state; perform sample preparation 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; when this immunological analysis is performed in a reactor for immunological analysis, 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, an annular permanent magnet is supplied to this container in an automatic mode using an electromechanical drive, which holds the magnetic particles during flushing; while the immunological analysis reactor is installed on the shaft of the orbital shaker, which automatically sets it in motion at a speed of up to 1500 rpm; in this case, all modes of operation of the reactor 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 refrigerated platform, also mounted on the shaft of the orbital mixer; before using the reagents, the platform is set in motion at 800 rpm to obtain a homogeneous suspension of microspheres; after completing all operations of sample preparation, the suspension from the reactor is sent to the multiplex immunological analyzer; manipulations with liquids during the preparation of multiplex immunological analysis are performed with a syringe pump and a multiport valve in automatic mode. 2. The method according to claim 1, characterized in that streptovidin-phycoerythrin conjugates are used as fluorescent labels.

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