RU2464572C1 - Test system for immunochromatographic assay of cardiac protein binding fatty acids and troponin i in whole blood sample for instant diagnosing of myocardial infarction (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is a test system for the immunochromatographic assay of cardiac protein binding fatty acids and troponin I in whole blood for the purpose of instant diagnosing of myocardial infarction wherein it contains an immunological tray (1) with a window (2) for introducing an analysed blood sample and a window (3) for viewing the results which comprises a substrate (4) for porous material lamination whereon there are lengthwise arranged and overlapped a pad (5) with conjugates for applying the analysed blood sample, a filtration pad (6) for separating the analysed blood sample, a porous membrane (7) for immunochromatographic reaction, and an absorption pad (8) for fluid flow; and on the pad (5) with conjugates and across the latter there are formed a first strip (9) of a conjugate of colloidal gold particles and first monoclonal murine antibodies specific to cardiac protein binding fatty acids, and a second strip (10) of the conjugate of colloidal gold particles and first monoclonal murine antibodies specific to troponin I; the porous membrane (7) across the latter there are formed a competing strip (11) containing a layer of first or second adsorbed monoclonal murine antibodies specific to cardiac protein binding fatty acids, a first test strip (12) containing the second adsorbed monoclonal murine antibodies specific to cardiac protein binding fatty acids, a second test strip (13) containing the second adsorbed monoclonal murine antibodies specific to troponin I and a reference strip (14) containing the adsorbed monospecific polyclonal rabbit anti-mouse (RAM) antibodies. According to the second and third versions of implementing the test system, the filtration pad has an additionally formed a strip (16) of a conjugate of the second monoclonal troponin I antibodies bound to biotin. According to the third version of implementing the test system, the competing strip 11 is arranged on the filtration pad (6) and contains the second monoclonal murine antibodies specific to cardiac protein binding fatty acids.

EFFECT: invention provides creating more simple and reliable bed combined immunochromatic test system for simultaneous detection of cardiac protein binding fatty acids and troponin I.

9 cl, 3 tbl, 4 dwg

Description

The invention relates to devices for immunochromatographic simultaneous detection of a cardiac protein that binds fatty acids and troponin I protein in whole venous blood for the rapid diagnosis of myocardial infarction and can be used in biotechnology, immunology and healthcare.

Cardiac BSA is a cardiospecific cytoplasmic low molecular weight protein (15 kDa), contained in large quantities in cardiomyocytes, carries out the binding and transport of fatty acids inside the cell and quickly enters the bloodstream with myocardial necrosis. The advantage of sBSSF is its high cardiospecificity due to the maximum concentration of sBFSFA in myocardial tissue. Cardiac BSA is an early marker of myocardial necrosis, it has kinetics similar to myoglobin, but it has great specificity. Diagnostically significant increase in the level of sBSLC is observed after 1 hour from the onset of pain. The level of sBSLC in the blood reaches its maximum value after 6 hours after myocardial damage and returns to its normal value after 16-24 hours. The amount of sBSLC in the blood increases in proportion to the vastness and depth of the infarction zone and reaches a level of more than 200-300 ng / ml at the upper limit of normal 15 ng / ml Thus, the cardiomarker sBSLC is the earliest of all markers of myocardial necrosis, which is especially important given the swiftness of events developing in ACS and its high level of adverse outcomes precisely on its first day. The test system for the determination of sBCLC provides significant assistance in the diagnosis of ACS, especially in situations with an atypical clinical picture and the absence of significant diagnostic changes in the electrocardiogram. In addition, an increase in the level of sBCLC is a predictor of adverse events in patients with ACS (myocardial infarction and cardiovascular death) for 1 year. The determination of the content of sBSLC in the blood of patients with suspected ACS for the early detection of myocardial necrosis was recommended by the decision of the annual congress of the European Cardiology Society in 2000.

Troponin I is a protein that is part of the troponin complex, the difference in isoforms of which in the myocardium and skeletal muscle is about 90%. Due to this, cardiac troponin-I (cTn-I) is a highly sensitive and highly specific marker of myocardial damage. Normally, troponin I is not detected in the bloodstream. With ischemic myocardial damage, troponins enter the peripheral bloodstream, where they can be determined after 6-8 hours. The maximum concentration is noted after 14-28 hours and persists for 3-7 days, forming a long “diagnostic window”. The release of troponin I has a single-phase character, which is due to the high content of its cytoplasmic fraction. If sBSLC dissolved in the cytosol is relatively quickly washed out of the necrosis zone, the destruction of the contractile apparatus of cardiomyocytes is longer in time, therefore, an increase in the level of troponins is determined up to 3-7 days after the onset of MI. This long period of troponin release into the blood increases the likelihood that a positive result of its determination was correct, especially in the subacute phase of myocardial infarction. The specificity and sensitivity of the methods for determining troponins in the blood with MI is 92-95%.

A device for chromatographic analysis for detecting analyte in a sample is known (RF patent No. 2144729, IPC G01N 33/53, publ. 10.01.1999). The device includes (1) at least two strictly flat opposed blocks, one of which contains a chromatographic medium on its surface; and (2) means for mounting opposing blocks against each other and applying enough pressure there to transfer fluid from one opposing block to another in a direction strictly perpendicular to the opposing block in order to deposit a sample on a chromatographic medium to detect and / or subsequently determine the analyte. The device may also include: (1) at least three strictly flat opposed blocks, characterized in that one of them contains on its surface a chromatographic medium having first and second ends; (2) means for mounting opposing blocks against each other in at least two different combinations and applying enough pressure there to transfer fluid from one opposing block to another in a direction strictly perpendicular to the opposing blocks, so that the sample is applied to the chromatographic medium and flows through it from the first end to the second end to detect and / or determine the analyte on a chromatographic medium; and (3) at least one applicator and one absorber located on one of the opposing blocks and arranged so that when an opposing block with an applicator and an absorber is mounted on it opposite the opposing block containing the chromatographic medium, a liquid is applied to the latter, which flows through it from the second end to the first, as a result of which the direction of flow through the chromatographic medium is reversed, in order to detect and / or determine the analyte, carried out after reverse flow through romatograficheskuyu Wednesday.

However, this device has a rather complex design, includes a source of high pressure to ensure the passage of a liquid sample along its porous elements, and also does not contain components to ensure the detection of sBCLC and troponin I in blood samples.

Known test system for immunochromatographic determination of cardiac troponin I for rapid diagnosis of myocardial infarction (Chinese patent No. 101059519, IPC G01N 33/558, publ. 24.10.2007), containing an immunological tablet with a window for entering the sample and a window for viewing the results inside which there is a substrate for laminating porous materials, on which a filter pad for applying a blood sample to be examined, a pillow for applying a conjugate, a porous membrane and an abs a pillow pad, the pillow for applying the conjugate is impregnated with a conjugate of colloidal gold nanoparticles (size 15-25 nm) with the first monoclonal mouse antibodies specific for cardiac troponin I, on the porous membrane, a test strip containing adsorbed second ones is formed across the latter and parallel to each other monoclonal murine antibodies, and a control strip containing adsorbed rabbit antibodies against mouse immunoglobulins. The invention has a simple preparation, action and high sensitivity (1.0 ng / ml) with a stable result. An increase in the concentration of troponin I with myocardial infarction is observed after 4-6 hours. The kinetics of its release into the blood may represent a two-phase curve with an initial peak after 15-20 hours and a lower second peak after 60-80 hours after the development of myocardial infarction.

However, this test system does not allow detecting an early marker of sBFLC, the level of which is significantly higher (from 15 ng / ml and higher) compared with healthy people in the first 1-6 hours after myocardial infarction. The level of sBSLC in the blood reaches its maximum value 6 hours after myocardial damage and returns to its normal value after 16-24 hours.

A known test system for immunochromatographic determination of a cardiac protein that binds fatty acids for the rapid diagnosis of myocardial infarction (Chinese patent No. 1704757, IPC G01N 33/558, published 07.12.2005), containing an immunological tablet with a window for entering the sample and a window for viewing the results, inside which there is a substrate for laminating porous materials, on which a filter pad for applying a test blood sample, a pillow for applying a conjugate, are arranged sequentially along the length and lap a porous membrane and an absorption cushion, wherein the cushion for applying the conjugate is impregnated with a conjugate of colloidal gold particles with the first monoclonal mouse antibodies specific for a heart protein that binds fatty acids on the porous membrane, across the latter and parallel to each other, a test strip containing adsorbed second monoclonal murine antibodies, and a control strip containing adsorbed rabbit antibodies against mouse immunoglobulins. The cardiomarker sBSLC is present in the blood of healthy people. The individual protein content ranges from 5 to 7 ng / ml. Diagnostically, the value (level of pathological values) of sBSSF varies from 10 to 15 ng / ml. As a result of these protein features, non-specific marker detection occurs.

In this regard, the immunochromatographic test proposed in this analogue allows the detection of an early marker of sBSLC in the amount of 5 ng / ml, i.e. it has too high sensitivity, which leads to the appearance of a large number of false positive results and, as a result, to low specificity and low accuracy of the test, which allows them to be used only as an indicative test. In addition, this test system does not allow the detection of the late marker troponin I, which narrows its functionality.

Known test system for immunochromatographic determination of cardiac protein that binds fatty acids in whole blood for rapid diagnosis of myocardial infarction (RF patent for utility model No. 87262, IPC G01N 33/558, publ. 09/27/2009), containing an immunological tablet with a window for depositing a sample and a window for viewing the results, inside which there is a substrate for laminating porous materials, on which a filter pad for applying a test blood sample is arranged sequentially along the length and overlap a conjugate coating plate, a porous membrane and an absorption cushion, wherein the conjugate coating cushion is impregnated with a conjugate of colloidal gold particles with the first monoclonal murine antibodies specific for the cardiac fatty acid binding protein on the porous membrane, a test strip is formed across the latter and parallel to each other containing adsorbed second monoclonal murine antibodies, and a control strip containing adsorbed rabbit antibodies against mouse immunoglobulins. On the porous membrane, a competing band is formed across the latter, located along the edge of the pillow for applying the conjugate and containing a layer of adsorbed first monoclonal mouse antibodies specific for the heart protein that binds fatty acids. This test system is free from the disadvantages of the previous analogue.

However, the test system does not allow the detection of the late marker troponin I, which narrows its functionality.

The closest analogue (prototype) is a combined test microchip for the diagnosis of eight cardiovascular diseases (Chinese patent No. 2783324, IPC G01N 33/543, publ. 05.24.2006) and contains antibodies such as anti-crp, anti-Myoglobin, anti-cTnI (cardiac troponin I), anti-cTnT (cardiac troponin T), anti-nt-probnp, anti-ck-mb (creatine kinase isozymes-MB), anti-h-fabp (cardiac fatty acid binding protein) , anti-GPBB (glycogen phosphorylase isoenzyme BB).

However, such a test is difficult to manufacture and expensive, requires special equipment to read the results, which does not provide the requirements of a “bedside” rapid diagnosis.

The technical result of the invention is the creation of a simpler and more reliable "bedside" combined immunochromatographic express test system for the simultaneous rapid determination of sBSLC and troponin I.

The specified technical result is achieved by the fact that the test system (the first version of its implementation) for immunochromatographic determination of cardiac protein that binds fatty acids and troponin I in whole blood for rapid diagnosis of myocardial infarction, according to the invention contains an immunological tablet with a window for entering the sample and a window for viewing the results, inside of which there is a substrate for laminating porous materials, on which a pillow with conjugates is arranged sequentially in length and lap For applying a test blood sample, a filter pad for separating a test blood sample, a porous membrane for carrying out an immunochromatographic reaction and an absorption pad for creating a fluid flow, the first band of a conjugate of colloidal gold particles with the first monoclonal mouse antibodies formed on the pillow with conjugates and across the latter (F9 ) specific to the cardiac protein binding fatty acids and the second band of the conjugate of colloidal gold particles to the first murine monoclonal with antibodies specific to troponin I (1-60), a competing band is formed on the porous membrane and across the latter containing a layer of adsorbed first (F9) or second (F10) monoclonal murine antibodies specific for a cardiac fatty acid binding protein, the first test a lane containing adsorbed second monoclonal mouse antibodies (F10) specific for a heart protein binding fatty acids, a second test lane containing adsorbed second monoclonal antibodies to troponin I (IC-19), and control lane containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM).

The indicated technical result is also achieved by the fact that the test system (second embodiment) for immunochromatographic determination of cardiac protein binding fatty acids and troponin I in whole blood for rapid diagnosis of myocardial infarction, according to the invention, contains an immunological plate with a window for specimen entry and a window for viewing the results, inside of which there is a substrate for laminating porous materials, on which a conjugate pillow is arranged sequentially along the length and lap for applying a test blood sample, a filter pad for separating a test blood sample, a porous membrane for conducting an immunochromatographic reaction and an absorption pad for creating a fluid flow, the first band of a conjugate of colloidal gold particles with the first monoclonal mouse antibodies formed on the pillow with conjugates and across F9) specific for a cardiac fatty acid binding protein and a second band of conjugate of colloidal gold particles to the first murine monoclo On the filter pad and across the latter, a band of conjugate of second monoclonal antibodies to troponin I (IC-19) coupled with biotin is applied on the porous membrane and across the latter a competing band containing a layer adsorbed first (F9) or second (F10) monoclonal mouse antibodies specific for a cardiac fatty acid binding protein, a first test strip containing adsorbed second monoclonal mouse antibodies (F10) specific for cardiac a fatty acid binding protein, a second test strip containing adsorbed avidin or streptavidin specific for biotin, and a control strip containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM).

The indicated technical result is also achieved by the fact that the test system (third embodiment) for immunochromatographic determination of cardiac protein binding fatty acids and troponin I in whole blood for the rapid diagnosis of myocardial infarction, according to the invention, contains an immunological plate with a window for specimen entry and a window for viewing the results, inside of which there is a substrate for laminating porous materials, on which a conjugate pillow is arranged sequentially along the length and lap for applying a test blood sample, a filter pad for separating a test blood sample, a porous membrane for conducting an immunochromatographic reaction and an absorption pad for creating a fluid flow, the first band of a conjugate of colloidal gold particles with the first monoclonal mouse antibodies formed on the pillow with conjugates and across F9) specific for a cardiac fatty acid binding protein and a second band of conjugate of colloidal gold particles to the first murine monoclo by competing antibodies specific for troponin I (I-60), a competing band of the second monoclonal mouse antibodies (F10) specific for the cardiac fatty acid binding protein and a band of conjugate of the second monoclonal antibodies to troponin I (IC -19) associated with biotin, a first test strip containing adsorbed second monoclonal murine antibodies (F10) specific for a cardiac fatty acid binding protein is formed on the porous membrane and across the latter a test strip containing adsorbed avidin or streptavidin specific for biotin, and a control strip containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM).

The bands on the pillow with conjugates, on the filter pad and on the porous membrane are formed in parallel and at a distance from each other by impregnation with appropriate reagents, followed by removal of moisture.

The pillow for applying the conjugate is made of cellulose, or glass microfibre, or polyester microfibre 15 mm long with a pore size of 20-25 μm, a thickness of 350 μm and an absorption capacity of at least 55 mg / cm ² .

The filtration pad is made of a porous polysulfone material with a length of 20 mm, a thickness of 600-650 μm and a pore size of 55-57 μl / cm ² . The porous membrane is made of nitrocellulose or polyethersulfone with a length of 20 mm, a thickness of 125-155 microns, an absorption capacity of at least 30 mg / cm ² , a flow rate of 90-110 sec / 40 mm. The absorption cushion is made of porous material of cotton fluff or fiberglass fiber with a length of 15 mm and a thickness of 400-450 microns. The lamination substrate is made of polystyrene or acrylic adhesive material 60 mm long, 230-270 microns thick.

The specified technical characteristics of the elements of the test system allow reliable operation, reduce the number of false-positive results by increasing the specificity and accuracy of the test and at the same time determine sBSLC and troponin I.

The invention is illustrated by the following drawings. Figure 1 shows a diagram of the appearance of the test system, made in the form of an immunological tablet. Figure 2 presents the layout of the internal elements of the test system in the immunological tablet (the first embodiment). Figure 3 presents the layout of the internal elements of the test system in the immunological tablet (second embodiment). Figure 4 presents the layout of the internal elements of the test system in the immunological tablet (third embodiment).

The Cardiotest test system for immunochromatographic analysis contains an immunological plate 1 with a receiving oval window 2 for adding a whole blood sample and a rectangular window 3 for viewing the results. Inside the immunological tablet 1, a substrate 4 for laminating porous materials is placed, on which a pillow 5 with conjugates for applying the test blood sample, a filter pad 6 for separating the test blood sample, a porous chromatographic membrane 7 for carrying out an immunochromatographic reaction, and an absorption pillow are arranged sequentially in length and lap 8 to create fluid flow.

In the first embodiment of the test system (Fig. 2), on the pillow 5 with conjugates and across the latter, a first band 9 of a conjugate of colloidal gold particles with monoclonal mouse antibodies (F9) specific for a cardiac fatty acid binding protein and a second band 10 of the conjugate are formed colloidal gold particles with murine monoclonal antibodies specific for troponin I (I-60). A competing strip 11 is formed on the porous membrane 7, across the last, containing a layer of adsorbed first (F9) or second (F10) monoclonal mouse antibodies specific for a heart protein that binds fatty acids, the first test strip 12 containing adsorbed second monoclonal mouse antibodies ( F10) specific for a cardiac fatty acid binding protein, a second test lane 13 containing adsorbed monoclonal antibodies to troponin I (IC-19), and a control lane 14 containing adsorbed monos specific polyclonal rabbit antibodies against mouse immunoglobulins (RAM). The bands 9 and 10 on the pillow 5 with conjugates and the bands 11, 12, 13 and 14 on the porous membrane 7 are formed in parallel and at a distance from each other by impregnation with the appropriate reagents, followed by removal of moisture. The competing strip 11 on the porous membrane 7 is located along and at the edge of the filtering pad 6 for the separation of the test blood sample.

In addition, there is a plot 15 for applying a sample of whole blood on a substrate 5, located in front of the bands 9 and 10 of the conjugates of particles of colloidal gold with monoclonal antibodies. Moreover, the oval window 2 of the tablet 1 is located above section 15 on the substrate 5.

In the second embodiment of the test system (Fig. 3), a first band 9 of a conjugate of colloidal gold particles with monoclonal murine antibodies (F9) specific for a cardiac fatty acid binding protein and a second band 10 of conjugate are formed on a pillow 5 with conjugates and across the latter colloidal gold particles with murine monoclonal antibodies specific for troponin I (I-60). A competing band 11 is formed on the porous chromatographic membrane 7 and across the latter, containing a layer of adsorbed first (F9) or second (F10) monoclonal mouse antibodies specific for a cardiac fatty acid binding protein, the first test strip 12 containing adsorbed second monoclonal mouse antibodies ( F10) specific for a fatty acid binding protein, a second test strip 13 containing adsorbed avidin or streptavidin specific for biotin and a control strip 14 containing th adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulin (RAM). A band 16 of a conjugate of monoclonal antibodies to troponin I (IC-19) associated with biotin is applied on the filter pad 6 and across the latter.

In the third embodiment of the test system (Fig. 4), a first band 9 of a conjugate of colloidal gold particles with monoclonal murine antibodies (F9) specific for a cardiac fatty acid binding protein and a second band 10 of conjugate are formed on a pillow 5 with conjugates and across the latter colloidal gold particles with murine monoclonal antibodies specific for troponin I (I-60). A competing band 11 is formed on the filter pad 6 and across the latter, containing a layer of adsorbed second monoclonal murine antibodies (F10) specific for the cardiac fatty acid binding protein and band 16 of the conjugate of monoclonal antibodies to troponin I (IC-19) associated with biotin . A first test strip 12 containing adsorbed second monoclonal mouse antibodies (F10) specific for a cardiac fatty acid binding protein, a second test strip 13 containing adsorbed avidin or streptavidin specific for biotin, and a control one were formed on the porous chromatographic membrane 7 and across the latter lane 14 containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM).

Table 1 shows the characteristics of the materials from which the elements of the test system are made.

Table 1 Characteristics of the materials of the test system Name of the element of the test system Material Composition Characteristics of materials Pillow 5 with conjugates for applying a blood sample cellulose or glass microfiber; or polyester microfiber; - pore size 20-25 microns;
- absorption capacity of 55 mg / cm ² ;
- release of the conjugate (after 90 seconds) 89%;
- 350 microns thick; Filter cushion 6 porous polysulfone material - a thickness of 600-650 microns;
- pore volume 5b, 8 μl / cm ² ; Porous Membrane 7 nitrocellulose, or polyethersulfone, or nylon, or polyvinylidene chloride on a polystyrene substrate membrane thickness 125-155 microns
- the thickness of the substrate is 100 μm;
- absorption capacity of at least 30 mg / cm ² ,
- sample flow rate 90-110 sec / 40 mm; Absorption pad 8 porous material from cotton fluff, or fiberglass - thickness 429 microns;
- dry weight of 12.4 mg / cm ² ; Lamination substrate 4 white polystyrene or acrylic adhesive material - the thickness of the substrate 254 μm +/- 10%;

In all versions of the test system, bands 9 and 10 on the pad 5 with conjugates, band 16 on the filter pad, bands 12, 13 and 14 on the porous membrane 7 and strip 11 on the membrane 7 (1 and 2 test system options) or filter cushion 6 (3rd embodiment of the test system) are formed in parallel and at a distance from each other by impregnation with appropriate reagents, followed by removal of moisture.

In the first (FIG. 2) and second (FIG. 3) embodiments of the test system, a competing strip 11 on the porous chromatographic membrane 7 is located along and at the edge of the filtering pad 6 for separation of the blood sample to be studied, and in the third embodiment of the test system ( 4) a competing strip 11 on the filter pad 6 is applied along and at the edge of the conjugate pad 5.

The design of the immunological tablet 1 provides the contact of the pillows 5 and 6, the porous membrane 7 and the absorption pillow 8 with each other and allows the blood sample to freely pass all porous materials due to the action of capillary forces.

On the conjugate pad on lines 9 and 10, the content of conjugates of monoclonal antibodies with particles of colloidal gold is 8-10 units of optical density (OD units). On the filter pad, lane 16 contains a conjugate of monoclonal antibodies to troponin I with biotin in a ratio of 1: 8-1: 16.

On membrane 7, competing strip 11 contains 0.9-1.0 mg / ml monoclonal antibodies; test strip 12 contains 1.0-1.1 mg / ml monoclonal antibodies; test strip 13 in the third embodiment of the test system contains avidin (streptavidin) at a concentration of 0.8-1.5 mg / ml, and control strip 14 contains polyclonal antibodies in an amount of 1.3-1.5 mg / ml.

The following is a description of the antibodies used in the test system for immunochromatographic determination of cardiac fatty acid binding protein and troponin I in whole blood.

1. Mouse monoclonal antibodies specific for sBSLC protein (FABP)

1.1. Monoclone F9, specific for the whole molecule of cardiac FABP, type IgG1 - used in band 9 (conjugated to colloidal gold particles) and competing band 11.

1.2. Monoclone F10, specific for the whole molecule of cardiac FABP, type IgG1 (applied to the first test strip 12 and the competing strip 11).

2. Mouse monoclonal antibodies specific for the troponin I protein

2.1. Monoclone No. 1 (I-60) cTnI, type IgG1, obtained by immunization with the troponin I molecule, specific for individual antigenic determinants of the cTnI molecule.

2.2. Monoclone No. 2 (IC-19) cTnI, type IgG2b, obtained by immunization with the troponin I molecule, is specific to the central part of the molecule with a stable antigenic determinant, tested for proteolytic degradation of the antigen (applied to test strip 13 in the first embodiment) and is used in strip 16 ( associated with biotin)

3. Monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM). The affinity-purified fraction of rabbit antiserum immunoglobulins against mouse immunoglobulin Fc fragments. It is used to bind conjugates of murine monoclonal antibodies in the control band 14.

Avidin-biotin reaction. The reaction between avidin (streptavidin) and biotin allows the connection between the various components of a specific complex on the solid phase. It is characterized by a high binding constant (10 ¹⁵ M ^-1 ), ease of attachment of biotin to antibodies without a noticeable violation of their antibody activity, and high stability of avidin (streptavidin).

Biotin is one of the many water-soluble components of the vitamin B complex and is a coenzyme for carboxylation enzymes. Its molecular weight is 244.3 Da. Streptavidin is a protein produced by the bacterium Streptomycces avidinii.

Avidin obtained from eggs is a protein with a molecular weight of 62400 Da and an isoelectric point pI = 10.5. One mg of avidin binds up to 14 μg of biotin. The resulting complex is stable in the range of pH 2-13. Avidin has 4 biotin binding sites. However, due to steric stresses, it can interact with only two biotinylated protein molecules.

The inventive test system "Cardiotest" (first option) works as follows. Blood in a volume of 100-150 μl (3-5 drops) is applied through the intake window 2 to the pillow 5 with conjugates. The blood sample, passing through pillow 5, impregnates bands 9 and 10 under the action of capillary forces, where the marker proteins present in the blood (sBSLC and / or troponin I) react with the corresponding protein with monoclonal antibodies labeled with colloidal gold, forming a colored immune complex antigen antibody. Next, the immune complex is absorbed by a filter pad 6, where blood separation occurs (erythrocyte mass separation). When determining troponin I, a high sensitivity of the test system is required - 1 ng / ml and the identification of both the whole molecule and its various fragments. The ultimate ability of a colloidal gold marker to detect a substance ranges from 0.5 to 1 ng / ml of a detectable agent. In the proposed design, antigen + antibody complexes labeled with colloidal gold particles are formed immediately on the conjugate pad 5 and, thereby, sufficient contact time is provided and the likelihood of hydrolysis of the labile molecules of the troponin I protein is reduced. Passing through the conjugate pad 5 and the filter pad 6, bound the complex is concentrated to some extent, does not decompose and does not settle on them together with red blood cells, freely passes with the plasma flow to the porous chromatographic membrane 7. The resulting the colored immune complex (consisting of one immune complex or two complexes) moves under the action of capillary forces along the porous membrane 7, crosses the competing band 11 and interacts with the adsorbed first or second monoclonal murine antibodies F9 or F10 specific for sBSLC. The function of lane 11 is to reduce the analytical sensitivity of the test to 15 ng / ml due to the competitive binding of sBSLC with F9 or F10 monoclonal antibodies immobilized in this lane 11. This strip is not stained because an increase in blood sBSLC from 15 ng / ml is considered to be diagnostically significant. A decrease in the analytical sensitivity of the test to 15 ng / ml leads to an increase in specificity while maintaining the necessary level of diagnostic sensitivity. Further, the immune complex, moving under the action of capillary forces, crosses the first test strip 12 and interacts with the second monoclonal mouse antibodies F10 to BSAI immobilized on it. As a result, in the presence of sBSLC in the blood sample above the threshold value, the first test strip 12 is colored purple. If the blood sample does not contain sBSLC in a concentration above the threshold, the first test strip 12 remains unstained. Further, the complex not bound in the first test strip 12 moves further along the porous membrane 7 to the second test strip 13 with the applied antibodies to troponin I No. 2. If the sample contains troponin I at a concentration of 1.1 ng / ml or higher, then the second test strip 13 is colored in purple (second colored strip). If troponin I in the sample is below the threshold value (below 1.1 ng / ml), then test strip 13 is not stained. Further, unbound conjugates reach control band 14 and interact with immobilized rabbit polyclonal antibodies to mouse immunoglobulins. As a result, the control strip 14 is also colored purple (third colored strip). Control strip 14 is the internal control of the test and, if the analysis is carried out correctly, should always occur, regardless of the presence of sBSLC and troponin I in the blood sample.

Results are determined visually within 5-30 minutes. With a positive test result, sBSLC and troponin I appear three colored bands - two test and control. If the sample contains only an early marker sBLC, then the first line is colored. This occurs in the time interval from 1 to 24 hours from the onset of the disease. If a later marker, troponin I, is present in the sample, the second test line is colored. The time interval from 6 to 48 hours from the onset of pain. If the sample is taken in the range from 6 to 24 hours from the onset of the disease, then all three bands appear. The color intensity of the control and test stripes may be different. With a negative result, only one band appears - the control. If the control strip does not appear, the test results are considered invalid.

The inventive test system "Cardiotest" (second and third option) works as follows.

Blood in a volume of 100-150 μl (3-5 drops) is applied through the intake window 2 to the pillow 5 with conjugates, which is absorbed by the porous material. The blood sample passes due to capillary forces along the conjugate pad 5. If the sample contains sBCLC, it interacts with the conjugate of colloidal gold and F9 antibody in band 9. As a result, a colored antigen-antibody immune complex is formed. The resulting colored immune complex moves under the action of capillary forces along the filter pad 6 and membrane 7, crosses the competing strip 11 (in the second embodiment of the test system on the membrane 7, and in the third version of the test system on the filter pad 6) and interacts with the applied on it adsorbed monoclonal murine antibodies F9, or F10 specific for sBSLC. The function of this band 11 is to reduce the analytical sensitivity of the test not lower than 15 ng / ml due to the competitive binding of sBSLC with F9 or F10 monoclonal antibodies immobilized on line 11 without decreasing the color intensity of this test strip. Since an increase in the level of sBSLC in the blood to 15 ng / ml is considered to be diagnostically significant, a decrease in the analytical sensitivity of the test to 15 ng / ml leads to an increase in specificity while maintaining the necessary level of diagnostic sensitivity.

Application of a competing band 11 with F10 monoclonal antibodies (filter 3 of the test system) to the filter pad 6 allows them to be used in a lower concentration, which saves reagents. For example, the concentration of F10 antibodies on the competing line 11 located on the membrane 7 is 1 mg / ml, and on the filter pad 6, the concentration of F10 antibodies on the band 11 is about 0.3 mg / ml. This property is determined by the location of the strip 11 on the tablet 1, the response time of the components, the washout of antibodies from the material.

Further, the immune complex moves under the action of capillary forces along the nitrocellulose membrane 7, crosses the first test strip 12 and interacts with monoclonal mouse antibodies F10 to BSCH immobilized on line. As a result, in the presence of sBSLC in the blood sample above the threshold value, the first test strip 12 is colored purple. If the blood sample does not contain sBSLC in a concentration above the threshold, the first test strip 12 remains unstained.

If troponin I is present in the sample, then this marker protein under the action of capillary forces is absorbed by the porous material of conjugate pad 5, during the passage of which troponin I interacts in band 10 with a conjugate of colloidal gold and monoclonal antibodies (I-60). As a result, a colored antigen-antibody complex is formed. The resulting colored immune complex moves under the action of capillary forces along the porous materials of pillows 5 and 6, crosses band 16 on the filter pad 6 with the conjugate of monoclonal antibodies to troponin I (IC-19) bound to biotin. The triple complex antibody (I-60) -antigen troponin I-antibody (IC-19) is formed.

Next, the bound complex moves along the nitrocellulose membrane 7 to the second test line 13 coated with avidin (or streptavidin). The complex binds through the affinity of biotin from the complex and avidin (streptavidin) applied to test strip 13. If the sample contains troponin I at a concentration of 0.9-1 ng / ml, then the second test line is colored in purple (second colored strip 13) . If troponin I in the sample is below the threshold value, then test strip 13 is not stained.

Then, unbound reagent conjugates reach control band 14 and interact with immobilized rabbit antibodies to mouse immunoglobulins. As a result, control line 14 is also colored purple (third colored strip 14). Control strip 14 is the internal control of the test and, if the analysis is carried out correctly, should always occur, regardless of the presence of sBSLC or troponin I in the blood sample.

Results are determined visually within 10-30 minutes. In the presence of sBSLC and troponin I in the sample, three colored bands appear - two test and control. If the sample contains only an early marker sBLC, then the first test line is colored. This occurs if blood sampling was carried out in a period of 1 to 24 hours from the onset of the disease. If only one later marker, troponin I, is present in the sample, only the second test line is stained. The time period for identifying this marker is from 6 to 48 hours from the onset of pain. If the sample is taken in the range from 6 to 24 hours from the onset of the disease, then all three bands appear. The color intensity of the control and test stripes may be different.

With a negative result, only one band appears - the control. If the control strip does not appear, the test results are considered invalid.

Research data confirming the technical result achieved by the claimed test system. The cardiomarker sBSLC is present in the blood of healthy people. The individual protein content ranges from 5 to 7 ng / ml. The diagnostic level (level of pathological values) of sBFLC varies from 12 to 15 ng / ml. As a result of these protein features, non-specific marker identification occurs. When using the test for express diagnostics, high sensitivity is not always necessary, which allows them to be used only as an indicative test. The competing lane 11 provides a decrease in the analytical sensitivity of the test to 15 ng / ml due to the competitive binding of BSAs with F9 monoclonal antibodies immobilized in this band 11. Since the level of sBSAI in the blood is considered to be diagnostic, from 15 ng / ml, a decrease in the analytical sensitivity of the test to 15 ng / ml leads to an increase in the specificity of the test while maintaining the necessary level of diagnostic sensitivity. Indeed, the use of competing strip 11 can significantly reduce the number of false-positive results, while not increasing the number of false-negative and, thus, while maintaining the same level of diagnostic sensitivity, the specificity and, as a result, the accuracy of the test are increased. Table 2 (first option) and table 3 (second and third options) show the diagnostic data of the claimed test system, which confirm the claimed technical result.

The test system for the determination of Troponin I requires high sensitivity up to 1 ng / ml. The ultimate ability of a colloidal gold marker to detect a substance is from 1 ng / ml of detectable agent.

In the second and third embodiments of the test system, increasing the sensitivity of the test is ensured by the interaction of biotin and avidin (streptavidin). Biotin bound to the antibody is washed off from the filter pad 6, binds to Avidin (alternatively streptavidin) on the test strip 13. The visualization of this complex provides a conjugate of colloidal gold.

The inventive test system allows you to increase the sensitivity in the range of 0.9-1 ng / ml.

table 2 Data diagnostic indicators of the inventive test system (first option) Time watch 1-3 3-6 6-12 12-24 24-48 Sensitivity SBSJK 88 93 99 98 Not determined Troponin I Not determined fifty 70 92 95 Specificity SBSJK 94 97 one hundred 98 Not determined Troponin I Not determined 70 91 93 95 Accuracy SBSJK 83 91 97 94 Not determined Troponin I Not determined 54 87 95 97

Diagnostic indicators were calculated by the formulas:

Sensitivity -

TPR = TP / (TP + FN);

Specificity -

SPC = TN / (FP + TN);

Accuracy -

ACC = (TP + TN) / N,

where TP is a reliably positive result,

FN-significantly negative result,

FP false positive

FN is a false negative result

N is the number of all patients.

The use of the triple complex antibody (I-60) colloidal gold antigen troponin I-antibody (IC-19) biotin, which then binds to avidin on the test line, can significantly reduce the number of false-negative results, while not increasing the number of false-positive results and, thus Thus, while maintaining a high level of specificity, an increase in the sensitivity of the determination of troponin I.

Table 3 Data diagnostic indicators of the inventive test system (second and third options) Time watch 1-3 3-6 6-12 12-24 24-48 Sensitivity SBSJK 88 93 99 98 Not determined Troponin I Not determined 59 94 97 98 Specificity SBSJK 94 97 one hundred 98 Not determined Troponin I Not determined 73 93 95 95

The simultaneous determination of the specific sBCLC and troponin I in the claimed test system allows for a reliable and quick diagnosis and an increase in the diagnostic time interval up to 48 hours. The combination of these markers avoids the disadvantages of tests containing only one of these markers. In the presence of renal failure in a patient, non-specific (false positive) reactions can be avoided. The use of the test will allow the doctor, in the absence of a clearly defined clinical picture of myocardial infarction in the patient and the absence of clear signs in the cardiogram, regardless of the time interval from the onset of pain, to decide on urgent hospitalization of the patient. For the same reasons, it is necessary to have combined tests for the diagnosis of myocardial infarction in feldsher-obstetric centers. A positive analysis will allow you to reasonably call an ambulance or emergency crew from the nearest location. The small size and weight of immunochromatographic tests allow their use by a local doctor or general practitioner when visiting a patient at home.

Claims (9)

1. Test system for immunochromatographic determination of cardiac protein that binds fatty acids and troponin I in whole blood for the rapid diagnosis of myocardial infarction, characterized in that it contains an immunological plate (1) with a window (2) for making a test blood sample and window (3) to view the results, inside which there is a substrate (4) for laminating porous materials, on which a pillow (5) with conjugates for applying a blood sample is placed sequentially along the length and lap a litration cushion (6) for separating the test blood sample, a porous membrane (7) for carrying out an immunochromatographic reaction, and an absorption cushion (8) for creating a fluid flow, and the first strip (9) of the particle conjugate is formed on the pillow (5) with conjugates and across the latter colloidal gold with the first monoclonal mouse antibodies specific for a cardiac fatty acid binding protein and a second strip (10) of a conjugate of colloidal gold particles with the first murine monoclonal antibodies specific for t oponin I, on the porous membrane (7) across the latter, a competing band (11) is formed containing a layer of adsorbed first or second monoclonal mouse antibodies specific for a heart protein that binds fatty acids, the first test strip (12) containing adsorbed second monoclonal murine antibodies specific for a cardiac fatty acid binding protein, a second test strip (13) containing adsorbed second monoclonal antibodies to troponin I, and a control strip (14) containing adsorbed s monospecific polyclonal rabbit antibodies against mouse immunoglobulin (RAM). 2. A test system for immunochromatographic determination of cardiac fatty acid binding protein and troponin I in whole blood for the rapid diagnosis of myocardial infarction, characterized in that it contains an immunological plate (1) with a window (2) for applying the test blood sample and window (3) to view the results, inside which there is a substrate (4) for laminating porous materials, on which a pillow (5) with conjugates for applying a blood sample is placed sequentially along the length and lap a litration cushion (6) for separating the test blood sample, a porous membrane (7) for carrying out an immunochromatographic reaction, and an absorption cushion (8) for creating a fluid flow, the first strip (9) of the conjugate of particles being formed on the pillow (5) with conjugates and across the latter colloidal gold with the first monoclonal mouse antibodies specific for a cardiac fatty acid binding protein and a second lane (10) of a conjugate of colloidal gold particles with the first murine monoclonal antibodies specific for tro onin I, on the filter pad (6) and across the latter, a strip (16) of the conjugate of the second monoclonal antibodies to troponin I associated with biotin is applied, a competing band (11) is formed across the latter on the porous membrane (7), containing a layer of adsorbed first or second monoclonal mouse antibodies specific for a heart protein fatty acid binding protein, a first test strip (12) containing adsorbed second monoclonal mouse antibodies specific for a heart protein fatty acid binding, second t a test strip (13) containing adsorbed avidin or streptavidin specific for biotin, and a control strip (14) containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM). 3. A test system for immunochromatographic determination of cardiac fatty acid binding protein and troponin I in whole blood for the rapid diagnosis of myocardial infarction, characterized in that it contains an immunological plate (1) with a window (2) for introducing the test blood sample and window (3) to view the results, inside which there is a substrate (4) for laminating porous materials, on which a pillow (5) with conjugates for applying a blood sample is placed sequentially along the length and lap a litration cushion (6) for separating the test blood sample, a porous membrane (7) for carrying out an immunochromatographic reaction, and an absorption cushion (8) for creating a fluid flow, and on the pillow (6) with conjugates and across the latter, the first strip (9) of the conjugate of particles is formed colloidal gold with the first monoclonal mouse antibodies specific for a cardiac fatty acid binding protein and a second strip (10) of a conjugate of colloidal gold particles with the first murine monoclonal antibodies specific for t oponin I, on the filter pad (6) and across the latter, a competing band (11) of the second monoclonal murine antibodies specific for the cardiac fatty acid binding protein and a band (16) of the conjugate of the second monoclonal antibodies to troponin I associated with biotin are applied to a first test strip (12) containing adsorbed second monoclonal murine antibodies specific for a cardiac protein binding fatty acids, a second test strip (13) containing adsorbents, was formed across the last porous membrane (7) biotin-specific avidin or streptavidin; and control band (14) containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM). 4. The test system according to claims 1 to 3, characterized in that the strips on the pillow (5) with conjugates, on the filter pad (6) and on the porous membrane (7) are formed in parallel and at a distance from each other by impregnation with appropriate reagents followed by moisture removal. 5. Test system according to claims 1 to 3, characterized in that the pillow (5) for applying the conjugate is made of cellulose or glass microfiber, or polyester microfiber 15 mm long with a pore size of 20-25 microns, a thickness of 350 microns and an absorption capacity not less than 55 mg / cm ² . 6. The test system according to claims 1 to 3, characterized in that the filter cushion (6) is made of a porous polysulfone material with a length of 20 mm, a thickness of 600-650 μm and a pore size of 55-57 μl / cm ² . 7. The test system according to claims 1 to 3, characterized in that the porous membrane (7) is made of nitrocellulose or polyethersulfone 20 mm long, 125-155 μm thick, absorption capacity at least 30 mg / cm ² , flow rate 90- 110 s / 40 mm. 8. The test system according to claims 1 to 3, characterized in that the absorption cushion (8) is made of porous material of cotton fluff or glass cellulose fiber with a length of 15 mm and a thickness of 400-450 microns. 9. The test system according to claims 1 to 3, characterized in that the lamination substrate (4) is made of polystyrene or acrylic adhesive material 60 mm long, 230-270 microns thick.

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