RU2712149C1 - Kit for differentiated detection of blood serum markers at all stages of infectious disease by multiparametric dot-immunoassay - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to sets for immunochemical analysis. Set of deposited on the substrate-immunochip capture reagents is presented by a species antigen of the Dengue virus or a mixture of Dengue antigen types I–IV, an anti-human IgM antibody and monoclonal antibodies against the Dengue NS1 protein for extracting from the sample and detecting early pathogen proteins; multicomponent conjugate is represented by a mixture of gold sols bound with anti-human IgG antibodies for IgG detection, a specific antigen of Dengue virus or a mixture of Dengue virus antigen types I–IV for detecting specific IgM and monoclonal antibodies against NS1 protein Dengue virus for extraction from the sample and detection of early proteins pathogen, and the inbuilt controls contain human IgG and Denge NS1 protein immobilized on separate segments of the substrate-immunochip and include a segment with human IgG applied to monitor the performance of antiviral detector antibodies to the detected antigens and protein-free segment to assess the developing system and background phenomena.EFFECT: proposed is a kit for differentiated detection of blood serum markers at all stages of infectious disease by multiparametric dot-immunoassay.1 cl, 2 dwg, 2 tbl, 1 ex

Description

The invention relates to kits for immunochemical analysis, aimed at improving test systems for identifying specific markers of infectious diseases in blood products and can be used in virology and medicine.

Infectious diseases are characterized by periods during which serological disease markers succeed one another. From the onset of the disease (from the onset of clinical symptoms), progenitor proteins (early pathogen proteins) are determined in the blood, indicating the reproduction of the pathogen in the body. Class 5 antibodies (IgM) appear in the blood 5-7 days after the onset of clinical signs, and the precursor proteins gradually disappear. In the third week, class G antibodies (IgG) begin to be produced, and the level of IgM progressively decreases. If the precursor proteins and IgM indicate an acute disease, then the presence of IgG indicates a late, chronic stage of the disease or a previous infection. For different infections, the definition of these markers has a different meaning. So for HIV infection, it is important to detect early p24 protein and total antibodies, since the presence of both IgM and IgG indicates the presence of infection. Whereas, for example, for dengue fever (LD) and Zika (LZ), it is important to differentially identify all three markers (NS1 protein, IgM and IgG), since the presence or predominance of a marker allows you to diagnose the stage of the disease and judge the probable time and source infection, which is important in the epidemiological plan.

Currently, a serological examination is carried out using test systems that identify 1-2 markers and to identify all markers requires a series of tests, which implies the simultaneous presence of all the necessary test kits and a considerable investment of time and money.

The present invention suggests the possibility of simultaneous (in one analysis) differential identification of specific immunological markers (early pathogen proteins, IgM antibodies and IgG antibodies) in the test samples from all stages of the infectious disease, in particular dengue fever markers. The essence of this identification is the formation of specific complexes between markers from the test sample and the known capture immunoreagents, in a certain order, discretely fixed on a dense substrate. The resulting complexes are then detected using detection immunoreagents associated with an easily identifiable label - catalytically active gold sols.

Known test systems for enzyme-linked immunosorbent assay in plates for immunological reactions, for example Anti-Dengue virus ELISA (IgM), Anti-Dengue virus ELISA (IgG) and Dengue virus ELISA (NS1), Euroimmun (Germany) [1], which allow to detect corresponding single LD markers. To determine all markers, it is necessary to perform analyzes in three test systems that require increased volumes of the test sample, cash and labor.

Dot immunoassay systems are known that allow one sample to simultaneously determine the presence of IgM and IgG for LD. For example, in the ImmunoComb® II Dengue IgM & IgG BiSpot kit (Orgenics, Israel) [2], anti-human IgM and IgG antibodies are used as capture reagents, which are individually stained on a plastic substrate and allow the entire pool of corresponding antibodies to be isolated from the sample. Identification of specific antibodies among them is carried out by binding antigens of dengue virus type I-IV. The detection of specific IgM in this approach can be effective, since antibodies to the current acute infection will prevail or make up a significant proportion in the total IgM spectrum in the sample. In contrast, specific IgGs make up an insignificant proportion of the total IgG acquired as a result of previous diseases or vaccinations, and a very sensitive detection system is needed to detect them. Therefore, the kit uses sequential long-term operations of treating the substrate with biotinylated anti-dengue antibodies, and then with an avidin conjugate with alkaline phosphatase, which increase the analysis time to 2 hours and negatively affect the cost of the kit. The described set allows you to simultaneously differentiate between primary and secondary dengue fever only from the second or third week of the disease.

Known kits for immunochromatographic (lateral-flow immunoassay) analysis, designed to identify all three markers of LD. Thus, the Dengue NS1Ag + Ab Combo system kit (Standard Diagnostics Inc., Korea) [3] allows the simultaneous detection of three Dengue fever markers, however, the analysis is performed on two separate devices: IgM and IgG antibodies are detected in separate bands on one side, and on the other is the NS1 protein. The kit allows you to diagnose LD at all stages of the disease, however, in fact, it is two separate tests in one package.

A known method of multidisciplinary immunochemical detection of antigens in liquid samples described in the patent of the Russian Federation No. 2296995 [4]. The essence of this identification is the specific immobilization of the antigens of the test sample with a set of known antibodies (primary immunoreagents), in a certain order, discretely fixed on a dense substrate of the analytical device. Immobilized antigens are treated with a mixture of antibodies specific to the entire spectrum of the analyzed antigens (secondary immunoreagent), as a result of which a triple complex is formed: the primary immunoreagent - antigen - secondary immunoreagent. The resulting complexes are then detected using the conjugate - antibodies specific for the secondary immunoreagent and associated with an easily identifiable label.

The disadvantages of this approach are the selection of primary and secondary immunoreagents, not only by specificity, but also by the type of animal in which they were developed, as well as the multi-stage and duration of the analysis procedure.

The closest technical solution (prototype) is a kit for multidisciplinary immunological analysis of antibodies in blood products described in the patent of the Russian Federation No. 2495434 [5], which includes the following components. 1. A device in the form of a substrate with a set of pathogens of infectious diseases discretely applied to its surface by antigens and additionally blocked by non-specific natural or synthetic polymers. The substrate is a plate with teeth in the form of a comb, on the surface of each tooth (immunochip) which are discretely applied antigens of viral pathogens and control (human immunoglobulins) to control the quality of the anti-species conjugate and the developing system, and, as a negative control, one of the tooth segments the substrate is left free of specific proteins for assessing background phenomena. The area of deposition of antigens and controls on each immunochip is outlined by a contrasting frame for positioning while instrumental analysis results. 2. Capacity for analysis, made in the form of a multi-cell analytical bath with the possibility of introducing each tooth of the substrate into a separate cell. The bathtub is filled with ready-made working solutions: for washing and diluting the sample, conjugate and development system. Bath cells are sealed.

As the antigens of the causative agents of viral infections in the kit, the antigens of the causative agents of vaccine-controlled infections are used: rubella, mumps, measles and poliomyelitis, or their recombinant or synthetic analogues.

Secondary immunoreagents are used as a conjugate: antibodies to human immunoglobulins, or staphylococcal protein A bound to alkaline phosphatase (for example, Protein A, Alkaline Phosphatase Conjugate from Merck, Cat. No. 539251), and as a developing system, a substrate for detection of alkaline phosphatase (preferably BCIP / NBT Liquid Substrate System from SIGMA-ALDRICH Cat. No. B1911). In an alternative embodiment of the conjugate, secondary immunoreagents (antibodies to human immunoglobulins or staphylococcal protein A) are used, associated with catalytically active gold sols, and a solution of a “physical developer”, including: 0.2% metol; 0.2% silver nitrate; 0.5% - citric acid, double-distilled water - the rest is up to 100%.

A particular example of a prototype is the one described by A. Poltavchenko et all. “A Kit for Multiplexed Detection of Antibodies to Agents of Hemotransmissible Infections.” OnLine Journal of Biological Sciences 2016, 16 (3): 137.144 (DOI: 10.3844 / ojbsci.2016.137.144) [6] kit for detecting antibodies to blood-borne pathogens of infectious diseases. The immunochips of this kit contain antigens from six GTI pathogens: HIV, HCV, HBV, syphilis, cytomegalovirus infection and toxoplasmosis, as well as monitoring the performance of the detection system - IgG from human blood; Antibodies against human IgG bound to gold nanoparticles are used as the conjugate, and the physical developer described in the prototype is used as the developing system.

The prototype kit is intended only for the simultaneous detection of specific antibodies, and does not allow to differentiate specific antibodies of the IgM and IgG classes.

The technical result of the claimed invention is the possibility of diagnosing the disease at all stages of the infectious process due to the simultaneous differentiated detection of specific antigens and antibodies of the IgM and IgG classes in the samples.

The specified technical result is achieved by the fact that in the kit for the differentiated detection of markers in the blood serum at all stages of an infectious disease by the method of multiparametric dot immunoassay, which includes an immunochip substrate made in the form of a comb with teeth with capture reagents and controls discretely applied to the surface of each tooth, as well as a container for analysis containing isolated cells with solutions for washing and diluting the sample, conjugate, solutions for development and stabilization staining, according to the invention, the capture reagent kit is represented by a common pathogen antigen (for isolating specific IgG from a sample), anti-human IgM antibodies (for isolating an IgM pool from a sample) and monoclonal antibodies for detecting early pathogen antigens; the built-in controls are located on separate segments of the immuno-chip substrate and include a segment coated with human IgG (monitoring the performance of antivirus detection antibodies), segments coated with detectable antigens (monitoring the performance of detection antibodies to detectable antigens) and a segment free of specific proteins for assessing background phenomena, and the conjugate contains a mixture of gold sols associated with antibodies against human IgG (detection of IgG), with a common pathogen antigen (detection of specific IgM) and from a monoclone nymi antibodies for the detection of early antigens of the pathogen.

As monoclonal antibodies for the detection of early pathogen antigens, pairs of monoclonal antibodies to different epitopes of the determined protein are used.

For the diagnosis of Dengue fever, a specific Dengue virus antigen or a mixture of Dengue type I-IV antigens, anti-human IgM antibodies and monoclonal antibodies against Dengue virus NS1 protein are used as a capture reagent kit; as a conjugate, a mixture of gold sols associated with: antibodies against human IgG, a specific dengue antigen or a mixture of dengue type I-IV antigens and monoclonal antibodies against the Dengue virus NS1 protein is used as conjugate, and the built-in controls contain human IgG and Dengue virus NS1 protein immobilized on individual segments of the immunochip, as well as a segment free of specific proteins for assessing background phenomena.

Thus, the inventive kit is intended to detect several markers at different stages of an infectious disease caused by a single pathogen. The ratio of sols associated with certain types of detector antibodies in the conjugate is selected empirically.

The following graphic figures are presented to illustrate the invention. In FIG. Figure 1 shows the main elements of a kit for the differentiated detection of serological markers from all stages of Dengue fever and the layout of test and control zones (capture reagents) on a substrate (immunochip):

K- - zone for monitoring background phenomena (without capture reagents);

K1 - zone for monitoring the performance of anti-species detector antibodies (human IgG);

K2 - zone for monitoring the health of detector antibodies to the early protein of NS1 dengue virus (NS1);

T1 - test area for antibodies of the IgG class against Dengue virus (a mixture of species antigens of Dengue virus type I-IV);

T2 - test area for antibodies to the IgM class against dengue virus (monoclonal antibodies against human IgM);

T3 - test area for early Dengue virus NS1 protein (monoclonal antibodies against Dengue virus NS1 protein).

In FIG. Figure 2 shows typical examples of the results of a multiplex analysis of LD markers in the blood serum of patients at different stages of the disease obtained using the proposed kit (the numbers under the immunochips indicate the periods of infection (weeks from the moment of the onset of clinical symptoms).

The proposed kit includes the following basic elements:

- a flat substrate-immunochip 1 for immunological analysis, made in the form of a comb, each tooth (immunochip) 2 of which on its surface contains a set of zones with capture reagents applied in the form of spots 3 and positive controls and is an analytical device; as a negative control, the K-zone of each tooth 2 of the substrate 1 is left free of specific proteins for assessing background phenomena;

- analytical multi-cell capacity (bath) 4 with insulated cells 5 filled with ready-made working solutions and sealed with a film;

- a puncher (not shown in the drawings) for opening the cells 5 of the analytical bath 4;

Description of the specific claimed kit.

The substrate 1 is made of non-porous synthetic material (preferably synthetic paper) in the form of a flat comb, for example, with five teeth (immunochips) 2, on each of which a set (in the form of spots 3) of capture reagents (T1, T2, T3), including: a specific antigen of a detectable pathogen (T1), monoclonal antibodies against human IgM (T2) and monoclonal antibodies against an early pathogen protein (T3); as well as built-in controls (K1, K2) located on separate segments of the immunochip and including: the K1 segment coated with human IgG (monitoring the performance of anti-type detector antibodies), the K2 segment with the applied early protein of the pathogen (monitoring the performance of specific detection antibodies), and free from specific Protein segment (K-) for assessing background events. The species antigen on the substrate can be represented by native, synthetic, recombinant proteins, their combination, or chimeric constructs containing sets of antigens of the corresponding pathogen. Antibody capture can be represented by monoclonal antibodies and should not compete for the binding sites (epitopes) of antigens with detector antibodies, and also react with antispecies immunoglobulins in the conjugate.

The capture reagents and controls on the surface of the substrate are applied in a concentration of 5-20 μg / ml (preferably 10 ng / ml) in aliquots of 1.5-3 μl (preferably 2 μl) in the form of separate points (spots) 3 with a diameter arranged in a certain order from 2 to 5 mm (preferably 3 mm), which allows direct visual recording of analysis results.

The analytical bath 4 is made of inert plastic and has 12 rows, each of which consists of five insulated cells 5. Each row of cells 5 is filled with a specific ready-to-use working solution and sealed with a film. Each bath is designed to perform five analyzes.

As a conjugate, the kit uses a mixture of gold sols associated with detector molecules: antibodies to human class G immunoglobulins, a specific antigen of the pathogen, and monoclonal antibodies to the early protein of the pathogen. The ratio of sols associated with certain types of detector antibodies in the conjugate is selected empirically.

The list of contents of the bath cells is shown in table 1.

As a developing system, the composition of the “physical developer” is used, including: 0.2% metol; 0.2% silver nitrate; 0.5% citric acid in double-distilled water.

The principle of development is the catalytic deposition of silver from a developer on the surface of gold particles from a conjugate bound on a surface of a substrate. The developer solution is unstable, therefore, in the kit, the developer is used in the form of two components: a dry - tablet mixture of citric acid and metol in a ratio of 5: 2 and liquid - 0.4% silver nitrate in double-distilled water. Dry mix tablets are placed in cells 5 of the ninth row of bath 4. During the analysis, 200 μl of double-distilled water are added to these cells to dissolve the tablets, and immediately before development, 200 μl of silver nitrate solution. The solution is mixed by repeatedly immersing the matrix in the cell. The manifestation time is 8 minutes. To enhance and stabilize the color of the developed spots in the cell of row 11, a solution containing 1% thiourea and 1% sodium hydroxide in distilled water is used. The principle of amplification and stabilization is the conversion of precipitated silver into a chemically stable silver sulfide having an intense black color.

Additionally, the kit is equipped with:

- punch,

- a bottle with bidistilled water to dissolve the tablets of the developer,

- an opaque bottle with a liquid component of the developer - 0.4% solution of AgNO ₃ in bidistilled water,

- a sheet of black paper used as a dark background to position the immunochip when accounting and documenting the results.

Description of the process of using the claimed kit.

Before analysis, cells 5 are opened with a perforator. Test blood serum samples (15 μl) are introduced into the first row of cells and the teeth 2 of substrate 1 are immersed. Then, at certain intervals, substrate 1 is moved and immersed in cells 5 of the next row of bath 4 and, after being removed from the last row of cells 5, the result is visually taken into account by the presence or absence of dark spots at the sites of application of antigens. For single analyzes, separate teeth 2 are used, which are separated by scissors from the comb of the substrate 1. The analysis is carried out at a temperature above 20 ° C.

The following is an example 1 of a specific application of a kit.

Example 1. Comparison of the effectiveness of differential identification of markers of dengue fever in serum using kits for immunochromatographic and dot immunoassay.

In trial experiments using:

Blood serum of patients at different stages of the disease: 1st, 2nd, 3rd, 4th weeks after the onset of clinical symptoms, and serum of the patient (> 5 weeks).

Immunochips 2 with the scheme shown in FIG. 1, capture reagents and controls:

T1 - a mixture of species antigens of dengue viruses of types I-IV;

T2 - monoclonal antibodies against human IgM;

T3 - monoclonal antibodies against early dengue virus NS1 protein;

K1 - IgG from human serum - monitoring the performance of detector antibodies against human IgG;

K2 - early dengue virus NS1 protein - monitoring the performance of detector antibodies against NS1 protein;

One of the segments of (K-) immunochip 2 was left free of specific proteins for assessing background events.

Analytical bath 4 with 12 rows of cells 5 filled with solutions:

- row 1 - sample dilution solution - TSB-T (0.1 M Tris-HCl with 0.15 M NaCl and 0.1% tween-20, pH 7.2-7.4) with 0.05% ( w / v) casein, pH 8.0;

- rows 2, 3, 5 and 6 - solution for washing - TSB-T;

- row 4 - conjugate - a mixture of gold sols (20 nm) associated with detector molecules: antibodies to human G class immunoglobulins, a mixture of specific antigens of Dengue virus types I-TV and monoclonal antibodies to the early protein Dengue virus NP1.

- rows 7, 8, 10 and 12 - bidistilled water.

- row 11 - a solution of a color enhancer and stabilizer - 1% thiourea and 1% NaOH in distilled water.

- in the cells of row 9, 1 tablet (4 mg) of a dry developer-citric acid and metol mixture is added in a ratio of 5: 2, respectively.

Filled baths are sealed with a film. Before analysis, the sealing coating is opened with a perforator. Run analysis:

Multiplex dot-immunoassay of blood serum samples to identify dengue fever markers is carried out using the proposed kit. All operations are performed at a temperature not lower than 20 ° C. 15 μl of test sera are pipetted into the first row cells, mixed, mixed, immunochips are placed in them and incubated for 20 minutes. In the ninth row cells add 200 μl bidistilled water from the bottle. Then the immunochips are transferred from the cell to the cell of the following rows with incubations: 4th row - 20 min; 2, 3 and 5-8 rows - 2 min, 9 row - 8 min, 10-12 rows - 1 min. Before introducing immunochips into the cells of the ninth row, 200 μl of a 0.4% solution of silver nitrate from the bottle are added to them. After removal from the last cell, the analysis results are visually taken into account. The result is considered positive if a clearly visible dark spot is visually determined at the site of application of the appropriate capture reagent, all system health controls have an intense dark color, and zone K is colorless or slightly colored.

Immunochromatographic analysis was performed using the Dengue NS1Ag + Ab Combo system kit (Standard Diagnostics Inc., Korea). The immunoassay and the results are carried out in accordance with the manufacturer's instructions. The results obtained using the proposed kit, in comparison with the data of immunochromatographic analysis are shown in table 2.

Notes:

+ clearly defined spot at the site of application of antigen (positive result). The number of crosses indicates the color intensity of the test area.

- a faint spot at the site of antigen deposition or a clean background of the substrate (negative result).

The type of matrices after analysis of 5 sera from patients with different periods of Dengue fever disease is shown in FIG. 2.

The proposed kit contains a complete set of components, is autonomous and can be used not only in clinical laboratories, but also in off-laboratory conditions for performing both serial and individual analyzes. The sensitivity of the multiplex analysis using the proposed set is not inferior to the sensitivity of commercially available commercial diagnostic kits (see example 1). The determination of all markers allows diagnosis at all stages of the disease, including the early ones (before the development of specific antibodies). The proposed set allows, due to the determination of 3 markers in one analysis, to significantly reduce the time and cost of examining the patient, as well as the number of samples taken from the patient.

Thus, the achievement of the technical result of the claimed invention, namely: the possibility of diagnosing the disease at all stages of the infectious process due to the simultaneous differentiated detection of specific antigens and antibodies of the IgM and IgG classes in the samples, is confirmed by the description of the invention and example 1 of the specific application of this kit.

Sources of patent and scientific and technical information

1. https://www.euroimmun.com/startseite.html

2.http: //www.biograd.ru/content/immunocomb®-dengue-igm-igg- bispot

3. https://www.alere.com/en/home/product-details/sd-bioline-dengue-duo-nsl-ag---ab-combo.html

4. RF patent No. 2296995, IPC G01N 33/53.

5. RF patent No. 2495434, IPC G01N 33/53.

6. Poltavchenko et all. “A Kit for Multiplexed Detection of Antibodies to Agents of Hemotransmissible Infections.” OnLine Journal of Biological Sciences 2016, 16 (3): 137.144 (DOI: 10.3844 / ojbsci.2016.137.144).

Claims (1)

A kit for the differentiated detection of serum markers at all stages of the dengue fever infectious disease using a multiparametric dot-immunoassay method, including an immunochip substrate made in the form of a comb with teeth, the surface of each of which is blocked by non-specific natural or synthetic polymers and contains a set of discretely located capture reagents infectious disease markers and controls; analysis tank containing isolated cells with solutions for diluting the sample and washing, a multicomponent conjugate and solutions for developing and stabilizing the optical signal, characterized in that the set of capture reagents applied to the immunochip substrate is represented by a specific Dengue virus antigen or a mixture of Dengue virus antigens of types I-IV, anti-human IgM antibody and monoclonal antibodies against Dengue virus NS1 protein for isolating and detecting early pathogen proteins from a sample; the multicomponent conjugate is represented by a mixture of gold sols bound to anti-human IgG antibodies for detecting IgG, a specific dengue antigen or a mixture of dengue type I-IV antigens for the detection of specific IgM and monoclonal antibodies against the dengue virus NS1 protein for isolation from the sample and detection of early proteins pathogen, and the built-in controls contain human IgG and Dengue virus NS1 protein immobilized on separate segments of the immunochip substrate and include a segment coated with human IgG to control performance These are antispecific detector antibodies to identified antigens and a segment free of specific proteins for evaluating the developing system and background phenomena.

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