RU2298798C1 - Method and analytical system for controlling biological sample in latex agglutination reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves incubating reacting mixture in transparent biochip cells treated with water-repellent agent. Results are checked in reacting mixture monolayer zone from changing light transmission or geometrical form of latex particles crystals. Analytical system for realizing latex agglutination reaction has tablet having transparent cells for storing reacting mixtures and their optical control unit. The tablet is manufactured as biochip, transparent cells are hydrophobic, and optical control unit has optical microscope or video camera.

EFFECT: increased sensitivity of method; small-sized tablet design.

3 cl, 5 dwg, 5 tbl

Description

The invention relates to analytical instrumentation and can be used in the manufacture and use of biochip microanalytical control systems for biological suspensions in the latex-agglutination reaction.

A known method of controlling a biological sample in a latex-agglutination reaction, comprising treating the test sample with complementary components immobilized on the surface of the latex particles, followed by incubating the reaction mixture on a glass slide or in transparent wells of the tablet and visually taking into account the results of the reaction based on the precipitation of conglomerates from the aggregated reaction mixture in case of binding of complementary components (see, for example: Chaika N.A. // Immunological diagnosis of viral infections.- M .: Medicine, 1985; US 4331649, G01N 33/50, 33/54, 33/68, 1982; RU 2015512, G01N 33/53, A61K 39/09, 1994; RU 2113172, A61B 10 / 00, A61K 39/05, G01N 33/536, 1998).

To increase the specificity and sensitivity of the method, the operation of immobilization of latex particles with complementary components is carried out under the influence of the field of ultrasonic vibrations (RU 2177617, G01N 33/567, 33/546, 2001).

Sample preparation and incubation operations can be optimized for pH using a buffer solution (RU 2188428, G01N 33/569, 33/50, 2002).

To detect several components of the analyzed sample, diagnostic test systems are used in which latex particles are sensitized by one or several complementary components giving color latex-agglutination reactions, while the content of the corresponding components in the sample is judged by the color of conglomerates of the aggregated reaction mixture (see, e.g. US 4745075, G01N 33/53, 33/545, 33/577, 1988).

The development trend of this type of technique is the microminiaturization of the agents used to conduct the latex-agglutination reaction, and to automate the accounting of its results. In this regard, a method for controlling a biological sample is known, which involves conducting a latex agglutination reaction on a biochip under the influence of various physical factors, such as electrophoresis or electromagnetic field, on the complementary components of biological structures immobilized on latex particles for their processing and analysis (WO 0230562, B01L 3 / 00, C12N 13/00, G01N 27/447, G01N 33/543, 2002).

The main disadvantage of conducting a latex-agglutination reaction on a biochip is the difficulty in filling the channels and wells of the biochip with a reaction medium due to the hydrophilicity of the surface layer formed on the latex carrier as a result of immobilization of its complementary components and the formation of aggregates, which is well known (see, for example: Muroi S ., Hosoi K., Ishikawa T. // J. Appl. Polym. Sci, 1967, v. 11, p. 1963; Vyzayendran BR // J. Appl. Polym. Sci, 1979, v.23, p. 893 ) In addition, hydrophilized sites screen the polymer chains of immunoreagents in the surface layer of latex particles, which reduces the effectiveness of the target reaction (Kondo A. // J. Immunol Methods, 1990, v. 135, p. 111). The latter drawback is also inherent in macro variants of the method.

To eliminate these disadvantages, a hydrophobic latex material is used as a carrier. This principle is implemented in the closest to the claimed method of controlling a biological sample in a latex-agglutination reaction, which involves treating the test sample with complementary components immobilized on the surface of substantially hydrophobic latex particles of natural or synthetic rubber, as well as styrene or a styrene butadiene copolymer, followed by incubation of the reaction mixture microtiter tablet and taking into account the results of the reaction visually on a glass slide located on a dark background, or about the change in turbidity using a turbidimeter (US 4429040, G01N 33/54, C12Q 1/56, 1984).

However, this method does not allow the study of microprobe, and taking into account the results is inconvenient to use.

The technical task of the method is to enable the reaction of latex agglutination with a microvolume of the biological sample under study. The technical task, a derivative of the indicated, is to automate the procedure for taking into account the reaction results, since obvious obstacles to visual control are created at the micro level.

The solution of the technical problem lies in the fact that the following changes are made to the method for controlling a biological sample in the latex-agglutination reaction, which involves treating the test sample with complementary components immobilized on the surface of latex particles with subsequent hydrophobic incubation of the reaction mixture and taking into account the results of the reaction:

1) the reaction mixture is incubated and analyzed in transparent hydrophobized wells of the biochip;

2) the volumes of the reaction components are determined based on the formation at the bottom of the cell of at least one monolayer portion of the reaction mixture in the zone of its optical control;

3) the positive result of the reaction is judged by the change in light transmission or the geometric shape of the crystals of latex particles in monolayer sections.

The causal relationship of the changes made to the method with the achieved technical result is as follows. To enable analysis of the microvolumes of the reaction mixture in the biochip, it is necessary to prevent the spreading of samples along its neighboring cells (especially when filling them) and to ensure reliable formation of monolayer sections in the zone of optical reaction control, which is achieved by hydrophobization of at least the working surface (bottom) of each cell, which more technologically advanced than the use of latex particles with a substantially hydrophobic surface, which took place in the prototype. Moreover, a monolayer coating of the bottom of the cell with the reaction mixture is also a necessary condition for the observability of the reaction. If this condition is not met, the result of a positive reaction is doubtful or not observed, which is illustrated by examples. In addition, in contrast to the traditional analysis of a biological sample in a latex-agglutination reaction on glass, in the proposed method the feature by which its result is judged depends on the composition of the reaction mixture. Therefore, it is necessary to first determine the nature of the change in the transparency or geometric shape of the crystals of latex particles in monolayer sections caused by the aggregation of related complementary components at their ratios in the region of the sensitivity threshold of the method.

In the technical implementation of the method, monolayer sections of the reaction mixture can be formed by optimizing the ratio of volumes and concentrations of the ingredients of the reaction mixture (sensitized latex particles and the sample) by titration of the sample under the control of a light or volume microscope. This procedure is performed once for each series of latex and sample. In the future, the reaction mixture is based on the found ratios. It is also possible to ensure a monolayer filling of the chamber by choosing a biochip with an optimal hole size in depth by calculation or experimentally. It is convenient to use the so-called negative sample as a sample in a dilution exceeding the reaction sensitivity threshold. a sample that does not contain a detectable component.

The specifics of the reaction and, especially, the consideration of its results determine the special design of the analytical system used for their implementation.

A known analytical system that can be used to carry out and control the latex agglutination reaction containing a polystyrene 96-well microplate, the wells of which are sensitized with the corresponding complementary antigen, and a scanning spectrophotometer for optical reaction control (RU 2196609, A61K 39/265, G01N 33 / 569, 33/535, 33/543, 2003).

An analytical system for the implementation and control of the latex-agglutination reaction is also known, which contains a biochip, on which are applied platforms with a binder for capturing the analyte component, connected to a scanning optical analyzer (EP 1365249, G01N 37/00, 33/53, 2003).

Closest to the claimed is an analytical system for the implementation and control of the latex-agglutination reaction, containing a tablet with transparent holes made for it in the reaction mixtures and a unit for determining the optical density of the reaction mixtures at a wavelength of 530 nm (WO 2004/077011, G01N, 2004).

However, this device fundamentally does not allow to study microprobe of reaction suspensions due to the significant volume of the wells of the tablet.

The technical task of the device is to ensure the study of microprobe of reaction suspensions.

The solution to the technical problem of the device is that the following changes are made to the analytical system for monitoring the reaction of latex-agglutination, containing a tablet with transparent wells for reaction mixtures made in it and a unit for their optical control:

1) the tablet is made in the form of a biochip, the transparent wells of which are made hydrophobic;

2) the optical control unit is based on an optical microscope or a video camera.

The hydrophobic surface of the wells prevents spreading of the components introduced into them (including along neighboring wells) and provides the possibility of forming monolayer sections at the place of optical reaction control.

To ensure hydrophobicity and optical transparency of the holes, it is advisable to produce a biochip from polymethyl methacrylate. It is also possible execution of wells with a hydrophobic coating mainly from paraffin or stearin.

It is advisable to make holes in the biochip with a diameter of 0.2 to 3 mm and a depth of 5 ÷ 60 μm with a distance between the centers of the holes from 0.3 to 2 mm.

Figure 1 shows a diagram of an analytical system; figure 2 ÷ 5 shows micrographs of the study of positive and negative samples in reactions with latex antigenic and antibody diagnostic diphtheria, as well as antibody diagnosticum of rheumatoid factor and Shigellosis Sonne to the examples. Table 1 shows the technical characteristics of biochips, the surface of the holes of which are hydrophobized with various materials; Table 2 ÷ 5 shows the results of titration of biological samples in the latex-agglutination reaction to the examples given.

The analytical system for the implementation of the latex-agglutination reaction in the embodiment of FIG. 1 comprises a biochip plate including a transparent base 1 and a photoresistive layer 2 deposited thereon, perforated through holes to form holes 3. The surface portions of the base 1 at the location of the holes 3, serving the bottom of the holes are provided with a hydrophobic coating 4. The analytical system of FIG. 1 is also equipped with an optical control unit based on a video camera. In this embodiment, the optical control unit includes a lighting device 5 mounted to illuminate the wells 3, a video camera 6 connected to the image processing module 7, which is capable of recording changes in light transmission or geometric shape of crystals in the wells 3 of the latex particles 8 of the reaction suspension.

In an embodiment of the optical control unit based on an optical microscope, a lighting device 5 is used that is integrated into the microscope in the transmitted light mode, and instead of the video camera 6, the optical system of the microscope is used. In this case, the reaction is viewed visually through the eyepiece of the microscope or using a camera installed instead of element 7. As an illumination device 5, an LED is installed in Fig. 1, and the CCD / CMOS matrix serves as the sensitive element of the video camera.

The microchip for the proposed analytical system can be manufactured as follows. On a transparent base 1 made of polymethylmethacrylate in the form of a plate 1 mm thick, a photoresistive layer 2 is applied by the method of screening, which is a composition of a GAWN 1109 Resist resist with a GAWN 1110 Hardener hardener with a volume ratio of components 2: 1, respectively. Then, a layer 2 is perforated by photolithography through holes with a diameter of 1 mm at a distance between the centers of the holes of 1.4 mm to form holes 3. The wells are hydrophobized by treatment with a 0.5 wt.% Solution of stearin in cyclohexane or paraffin melt. In this example, not only the bottom of the wells is hydrophobized, but also the entire surface of the tablet for reasons of manufacturability of the manufacture of the target product.

After filling the wells with a monolayer of reaction suspensions, the biochip is incubated at the temperature required for the reaction for a period sufficient to undergo the reaction. Observe the nature of the change in light transmission or the geometric shape of the crystals of latex particles in the wells, before and after incubation, by which they judge the result of the reaction.

Table 1 shows the technical characteristics of biochips, the wells of which are hydrophobized by sorption of a 2-4-micron layer of polydimethylsiloxane, stearin, solidol and paraffin, as well as by chemical binding of hydroxytrimethylsilane. Control - non-hydrophobized polymethyl methacrylate (no coating). The wetting angle for water and latex suspension (average particle diameter 0.8 μm), as well as the transparency of the bottom of the well were taken into account. As can be seen from the table, the water contact angle in the control and when coating the bottom of the hole with a solidol is 77-80 degrees, and when applying polydimethylsiloxane, stearin, paraffin and trihydroxymethylsilane from 100 to 102 degrees. According to latex for these coating groups, the wetting angle is 82-88 degrees. and 105-108 degrees. respectively. The best transparency (++, +++) has stearin, paraffin and trihydroxymethylsilane coatings. In this regard, stearin and paraffin coatings should be preferred. The trihydroxymethylsilane coating also has the highest values of these indicators, but its deposition is less technologically advanced because it requires chemical bonding to the substrate.

The proposed technical solutions are illustrated by the following examples.

EXAMPLE 1. Working dilutions of latex diphtheria antibody test diagnosticum (LDATD) manufactured by Avicenna JSC (St. Petersburg), which is 1.2 wt.% Suspension of polystyrene carboxylated latex particles with a diameter of 0.8 μm, sensitized with purified diphtheria antitoxin, treated with equal volumes of diphtheria toxoid in dilutions from 1:10 to 1: 10000 in physiological buffer solution (FBI) and immediately introduced into the hydrophobized stearin biochip wells with a diameter of 1 mm and a depth of 30 μm of the analytical system phi d.1 under the control of a video camera in transmitted light mode, as well as an optical microscope in reflected light. The reaction is taken into account when monolayer filling of the wells, as well as with 2-3 and more than 5 layers of latex or the reaction mixture. In parallel, they put the following controls:

a) LDAD with an FBI that does not contain diphtheria toxoid (negative control No. 1);

b) LDATD with an FBI containing a nonspecific antigen (negative control No. 2 of the manufacturer of LDATD);

c) latex, not sensitized by specific antibodies (control No. 3 for the absence of spontaneous agglutination).

A positive reaction result is hereinafter recorded during enlightenment of the sample to "++" and more.

The results are shown in table.2. As can be seen from the table, the proposed method is specific and has a sensitivity characterized by the possibility of reliable determination of diphtheria toxoid in the dilution of the sample from 1:40, whereas in the known method, diphtheria toxoid is determined during the dilution of the sample from 1:10. Moreover, in the case of non-compliance with the conditions for the formulation of the reaction in a monolayer sensitivity decreases by about 1 order.

The results of microscopic examination of the samples indicate a decrease in the reflected light flux with a positive reaction result (1:10 dilution — FIG. 2-a) compared with the negative control (FIG. 2-b). In transmitted light, an inverse picture is observed: with a positive reaction (Fig. 2-c), the light transmission is higher than in the negative control (Fig. 2-d). A change in the geometric shape of the observed structures is also noted, which is especially clearly seen from the comparison of Fig.2-a and 2-b.

EXAMPLE 2. Working dilutions of a prototype of latex diphtheria antigenic diagnosticum (LDagD) of the joint production of the IVS RAS and the IEM named after L. Pasteur (St. Petersburg), which is 1.5 wt.% Suspension of polystyrene carboxylated latex particles with a diameter of 0, 81 microns sensitized with purified diphtheria toxoid with an activity of 0.1 Lf / ml are treated with equal volumes of a solution of specific monoclonal anti-diphtheria immunoglobulins with an activity of 0.1 IU / ml in dilutions from 1:10 to 1: 10000 in the FBI and immediately added to l APIS biochip diameter of 1.2 mm and a depth of 30 microns analytical system 1 under the control of the camcorder in the transmitted light mode, and also an optical microscope in reflected light.

In parallel, they set the controls:

a) LDag with FBI that does not contain diphtheria toxoid (negative control No. 1);

b) LDag with serum containing non-specific antibodies (negative control No. 2);

c) latex, not sensitized by a specific antigen (control No. 3 for the absence of spontaneous agglutination).

The reaction is taken into account, as in example 1.

The results are shown in table.3. As can be seen from the table, the proposed method is specific and has a sensitivity characterized by the ability to reliably determine diphtheria antitoxin in a sample dilution of 1: 2000, whereas in the known method, diphtheria antitoxin is determined when a sample is diluted from 1:10. Moreover, in the case of non-compliance with the conditions for the formulation of the reaction in a monolayer sensitivity decreases by about 2 orders of magnitude.

The results of microscopic examination of the samples indicate a decrease in the reflected light flux with a positive reaction result (Fig.3-a, b, c) compared with the negative control (Fig.3-d). In transmitted light, an inverse picture is observed: with a positive reaction (Fig. 3-d, e, g), the light transmission is higher than in the negative control (Fig. 3-z). A change in the geometric shape of the observed structures is also noted, which is especially clearly seen from a comparison of images in reflected light.

EXAMPLE 3. Workers diluting latex diagnosticum to detect rheumatoid factor (DRF) produced by the Company for the production of bacterial preparations of the St. Petersburg Research Institute of Vaccines and Serums (St. Petersburg), which is 1.5 wt.% Suspension of polystyrene carboxylated latex particles with a diameter of 0 , 6-0.8 μm, sensitized with antibodies to human rheumatoid factor, treated with equal volumes of a positive human blood serum sample from the manufacturer’s DRF kit in dilutions from 1:10 to 1: 1000 in glycine buffer nom solution (GBR), pH 8.1 and immediately added to the wells of a biochip diameter of 1.2 mm and a depth of 30 microns analytical system 1 under the control of video camera and a microscope.

In parallel, they set the controls:

a) DRF with HBR not containing a rheumatoid factor (negative control No. 1 of the manufacturer);

b) DRF with dilutions of a negative serum sample (negative control No. 2 of the manufacturer).

The results are shown in table 4. As can be seen from the table, the proposed method is quite specific and has a sensitivity, characterized by the ability to reliably determine the rheumatoid factor in the dilution of the sample from 1: 200, while in the known method, the rheumatoid factor is determined by dilution of the sample from 1:40. In this case, in case of non-compliance with the conditions for setting the reaction in a monolayer, the sensitivity decreases by one order of magnitude. In control No. 2, non-specific agglutination was observed with small dilutions of negative serum.

In transmitted light with a positive reaction (Fig. 4-a), the light transmission is higher than in the negative control (Fig. 4-b).

EXAMPLE 4. The reaction of latex agglutination is carried out, as in example 1, using a latex antibody shigellosis diagnosticum Sonne production company SPbNIIVS. A suspension of the diagnosticum and the sample are mixed in a volume ratio of 1: 5. Dilutions of the sample are carried out in phosphate buffered saline (PBS) containing serum albumin. The concentration of microbial cells in the initial sample is 1 billion / ml.

In parallel, they set the controls:

a) diagnosticum with FSB that does not contain Sonne antigen (negative control No. 1 of the manufacturer);

b) latex, not sensitized by specific antibodies (negative control No. 2 of the manufacturer).

The results are shown in table.5. As can be seen from the table, the proposed method is specific and has a sensitivity, characterized by the possibility of reliable determination of Shigella Sonnet in breeding microbial culture from 1: 1280 against 1: 640 in the known method.

The results of microscopy of the samples indicate a decrease in the reflected light flux with a positive reaction result (Fig. 5a, b) compared with the negative control (Fig. 5c) as the dilution of the microbial culture decreases. In transmitted light, an inverse picture is observed: with a positive reaction (Fig. 5d, e), the light transmission is higher than in the negative control (Fig. 5th). A change in the geometric shape of the observed structures is also noted, which is especially clearly seen from a comparison of images in reflected light.

As explained above, the use of the proposed technical solutions makes it possible to conduct a latex-agglutination reaction with a microvolume of the biological sample under investigation up to 0.01 ÷ 0.1 μl. Moreover, in the variants of examples No. 1 and 2, an increase in sensitivity of determining the corresponding complementary components by 1-2 orders of magnitude was achieved. The technical result, derived from the achieved, consists in miniaturization of the used, transported and stored carriers of reaction suspensions (biochips) and automation of the reaction accounting procedure by using a digital camera or microscope photo output. The miniaturization of the design results in a decrease in the material consumption of the manufacture of the tablet (based on the biochip).

Table 1. The wetting angle of the wells, water-repellent various materials Hydrophobic material The method of hydrophobization Wetting angle, deg. Transparency,
rel. on water latex Polydimethylsiloxane chemical one hundred 106 + Stearin adsorption 101 107 ++ Solidol adsorption 80 88 + Paraffin* adsorption 102 108 +++ Trihydroxymethylsilane chemical one hundred 105 +++ Polymethyl methacrylate - 77 82 +++ * Parafilm "M" (American National Can) Table 2. The results of the agglutination reaction with LDatD for example 1. Reverse titer Accounting for the reaction with the number of layers of latex Macrometer Controls monolayer 2 ÷ 3 > 5 No. 1 Number 2 Number 3 10 +++ ++ + ++ - - - twenty +++ + - + - - - 40 ++ - - - - - - one hundred + - - - - - - 1000 - - - - - - - 2000 - - - - - - - 5000 - - - - - - - 10,000 - - - - - - -

Table 3. The results of the agglutination reaction LDAGD for example 2. Reverse titer Accounting for the reaction with the number of layers of latex Macrometer Controls monolayer 2 ÷ 3 > 5 No. 1 Number 2 Number 3 10 +++ +++ ++ ++ - - + twenty +++ ++ + + - - - one hundred +++ + - - - - - 1000 ++ + - - - - - 2000 ++ - - - - - - 5000 + - - - - - - 10,000 - - - - - - - Table 4. The results of the agglutination reaction with DRF for example 3 Reverse titer Accounting for the reaction with the number of layers of latex Macrometer Controls monolayer 2 ÷ 3 > 5 No. 1 Number 2 10 +++ +++ ++ +++ - + twenty +++ ++ + +++ - + 40 ++ + + ++ - - 200 ++ + + - - - 400 + + + - - - 800 + - - - - - 1600 + - - - - - 3200 - - - - - -

Table 5. The results of the agglutination reaction with the latex antibody diagnosticum Sonne for example 4. Reverse titer Accounting for the reaction on the biochip Macrometer Cocktails No. 1 Number 2 twenty ++++ ++++ - - 40 +++ +++ - - 80 +++ +++ - - 160 +++ +++ - - 320 +++ +++ - - 640 +++ +++ - - 1280 +++ + - - 2560 + + - - 5120 + - - - 10240 - - - -

Claims (3)

1. A method for studying a biological sample in a latex-agglutination reaction under hydrophobic conditions, characterized in that the reaction mixture is incubated in transparent hydrophobized wells of the biochip, and the results are taken into account in a monolayer section of the reaction mixture by changing the light transmission or geometric shape of the crystals of latex particles. 2. The analytical system for the latex-agglutination reaction described in claim 1, containing a tablet made in the form of a biochip, including a transparent base with a photoresistive layer deposited on it, perforated through holes with the formation of holes, the bottom of which is provided with a hydrophobic coating, and a block optical control, including an optical microscope or video camera connected to the image processing module. 3. The analytical system according to claim 2, characterized in that the transparent wells of the biochip are hydrophobized with polydimethylsiloxane or stearin.

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