RU87262U1 - TEST SYSTEM "CARDIOBSJK" FOR IMMUNOCHROMATOGRAPHIC DETERMINATION OF THE HEART PROTEIN BINDING FATTY ACIDS IN THE WHOLE BLOOD SAMPLE FOR EXPRESS DIAGNOSTICS OF MYOCARDIAL INFARCTION - Google Patents

Abstract

1. The test system for immunochromatographic determination of cardiac protein that binds fatty acids in whole blood for the rapid diagnosis of myocardial infarction contains an immunological plate with a window for introducing a sample and a window for viewing the results, inside which there is a substrate for laminating porous materials, on which length and overlap are a filtration pad for applying a test blood sample, a conjugate pad, a porous membrane and an absorption pad moreover, the pillow for applying the conjugate is impregnated with a conjugate of colloidal gold particles with the first monoclonal mouse antibodies specific for the cardiac fatty acid binding protein on the porous membrane, transverse to the latter and parallel to each other, a test strip containing adsorbed second monoclonal mouse antibodies and a control strip is formed containing adsorbed rabbit antibodies against mouse immunoglobulins, characterized in that on the porous membrane, a conc riruyuschaya strip located along the edge of the cushion and for applying comprising the conjugate adsorbed first layer of mouse monoclonal antibodies specific to cardiac proteins which bind fatty acids. ! 2. The test system according to claim 1, characterized in that the pillow for applying the conjugate is made of cellulose, or glass microfiber, or polyester microfiber with a pore size of 20-25 μm and an absorption capacity of at least 55 mg / cm2. ! 3. The test system according to claim 1, characterized in that the filter cushion is made of porous material

Description

The utility model relates to devices for immunochromatographic determination of a cardiac protein that binds fatty acids for rapid diagnosis of myocardial infarction and can be used in biotechnology, immunology and healthcare.

The inventive test system is designed for one-step detection in vitro of a cardiac protein that binds fatty acids (sBLC) in whole venous blood. It serves as an aid in the diagnosis of acute coronary syndrome (ACS) and is intended for use by healthcare professionals.

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.

For the first time, the proposal to use sBSLC as a marker for ACS was proposed by the German scientist J. Glatz et al. about 10 years ago. The advantage of sBSSF is its high cardiospecificity due to the maximum concentration of sBFSFA in myocardial tissue. The only muscle in which there is a relatively large amount of SBJFA is the diaphragm (approximately 25% of the content in the myocardial tissue). In skeletal musculature, sBLC is present in small quantities.

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 6 hours after myocardial damage and returns to its normal value after 12-16 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.

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 on its surface a chromatographic medium; and (2) means for mounting opposing blocks against each other and applying sufficient 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 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, localized on one of the opposing blocks and located so that when the opposing block with the applicator and the absorber is mounted on it opposite the opposing block containing the chromatographic medium, a second 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 reverses, in order to detect and / or determine the analyte, carried out after reverse flow through chromatographic medium.

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

The closest analogue (prototype) is a test system for immunochromatographic determination of cardiac protein that binds fatty acids for rapid diagnosis of myocardial infarction (Chinese patent No. 1704757, IPC G01N 33/558, publ. 07.12.2005), containing an immunological tablet with a window for introducing a sample and a window for viewing the results, inside of which there is a substrate for laminating porous materials, on which a filter pad is arranged sequentially along the length and lap for applying the test sample ovi, a conjugate application pad, a porous membrane and an absorption pad, wherein the conjugate application pad 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, transverse to the latter and parallel to each other, formed 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 rate of protein content varies from 5 to 15 ng / ml. Diagnostically, the value (level of pathological values) of SBJC varies from 10 to 20 ng / ml. As a result of these protein features, non-specific marker detection occurs.

In this regard, the immunochromatographic test proposed in the prototype allows the detection 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.

The technical result of the proposed utility model is to reduce the number of false positive results by increasing the specificity and accuracy of the test.

The specified technical result is achieved by the fact that in the test system for immunochromatographic determination of cardiac protein that binds fatty acids for rapid diagnosis of myocardial infarction, containing an immunological tablet with a window for introducing 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, a pillow for applying a conjugate, are located sequentially along the length and lap a porous membrane and an absorption pad, wherein the pad 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, a test strip containing adsorbed second monoclonal is formed across the porous membrane parallel to the latter mouse antibodies and a control strip containing adsorbed rabbit antibodies against mouse immunoglobulins, according to a utility model, in a porous mbrane transversely formed last contention strip located along the edge of the cushion and for applying comprising the conjugate adsorbed first layer of mouse monoclonal antibodies specific to cardiac proteins which bind fatty acids.

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

The filter cushion is made of a porous polysulfone material with a layer thickness of 600-650 μm and a pore volume of 55-57 μl / cm ² .

The porous membrane is made of nitrocellulose, or polyethersulfone, or nylon with a layer thickness of 125-155 μm and

The absorption cushion is made of a porous material of cotton fluff, or glass cellulose fiber with a layer thickness of 400-450 microns.

The substrate for lamination is made of polystyrene or acrylic adhesive material with a layer thickness of 230-270 microns.

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.

Figure 1 shows a diagram of the appearance of the test system, made in the form of an immunological tablet, and figure 2 is a layout of the internal elements of the test system in the immunological tablet.

The CardioBSLC test system for immunochromatographic analysis contains an immunological plate 1 with an oval window 2 for depositing a sample and a rectangular window 3 for viewing the results. Inside the immunological plate 1, a substrate 4 for laminating porous materials is placed, on which a filter pad 5 for applying the whole venous blood sample, a pillow 6 for applying the conjugate, a porous membrane 7 and an absorption pad 8. are sequentially laced and overlapping 8. The pillow 6 is impregnated with a particle conjugate colloidal gold with F9 monoclonal mouse antibodies specific for BSA. A competing band 9, located along the edge of the pillow 6 and containing adsorbed monoclonal mouse antibodies F9 specific for sBSLC, test strip 10 containing adsorbed monoclonal mouse antibodies F10 and a control strip 11 containing adsorbed rabbit antibodies against mouse immunoglobulins.

The design of the immunological tablet 1 provides a contact between 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 through all porous materials due to the action of capillary forces.

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 composition of materials material characteristics Conjugate Pillow 6 cellulose or glass microfiber; or polyester microfiber; - pore size 22 microns; - absorption capacity of 55 mg / cm ² ; - release of the conjugate (after 90 seconds) 89%; Filter cushion 5 porous polysulfone material - layer thickness of 628 microns; - pore volume 56.8 μl / cm ² ; Porous Membrane 7 nitrocellulose or polyethersulfon, or nylon, or polyvinylidene chloride on a polystyrene substrate - the thickness of the membrane layer 125-155 microns; - the thickness of the substrate is 100 μm; - sample flow rate 110-165 sec / 40 mm; Absorption pad 8 porous material from cotton fluff, or fiberglass - layer 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%;

The inventive test system "CardioBSJK" works as follows.

The introduced whole blood sample through the oval window 2 of the tablet 1 is absorbed by the filter pad 5 (figure 1). Then, when passing through a porous pillow 6, under the action of capillary forces, the SBSC of the blood sample interacts with a conjugate of colloidal gold particles and the first F9 monoclonal antibodies deposited on pillow 6 (FIG. 2). As a result, a colored antigen-antibody complex is formed. The resulting colored immune complex moves under the action of capillary forces along the nitrocellulose membrane 7, crosses the competing band 9 and interacts with the adsorbed F9 monoclonal murine antibodies specific for sBSLC on it. The function of this band 9 is to reduce the analytical sensitivity of the test to 15 ng / ml due to the competitive binding of sBSLC with F9 monoclonal antibodies immobilized on this line. 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. Further, the immune complex moves under the action of capillary forces along the nitrocellulose membrane 7, crosses the test strip 10 and interacts with the second monoclonal mouse antibodies F10 immobilized on the indicated test strip 10. As a result, in the presence of sBSLC in the blood sample above the threshold value, test strip 10 is colored purple. This can be seen through the rectangular window 3 of tablet 1. If the blood sample does not contain sBSLC in a concentration above the threshold, test strip 10 remains unstained. Further, the conjugate not bound in the test strip 10 moves further along the nitrocellulose membrane 7 and inevitably interacts with rabbit antibodies to mouse immunoglobulins immobilized in the control strip 11. As a result, the control strip 11 is also colored purple (second colored strip). Control lane 11 is the internal control of the test and, if the analysis is carried out correctly, should always occur, regardless of the presence of sBSLC in the blood sample.

Results are determined visually within 5-30 minutes. If the test result is positive, two colored stripes appear - test and control. The color intensity of the control and test stripes may be different.

With a negative result, only one band 11 appears - the control. If control band 11 does not appear, the test results are considered invalid.

Research data confirming the achievement of a technical result. Being an effective diagnostic tool, rapid tests allow you to visually identify and evaluate the content of antigens within a few minutes.

The quality of the test and, accordingly, the indicators of sensitivity and specificity, depend on the quality of the monoclonal antibodies used in the test and the individual characteristics of the marker under study. As noted above, the sBSLC cardiomarker is present in the blood of healthy people. The individual rate of protein content varies from 5 to 15 ng / ml. Diagnostically, the value (level of pathological values) of sBSLC and varies from 10 to 20 ng / ml. As a result of these protein features, non-specific marker detection occurs.

In the closest analogue (prototype), an immunochromatographic test involves applying 2 bands to the membrane - test and control and it allows you to detect 5 ng / ml. This test model was highly sensitive, 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.

However, when using the test for express diagnostics, high sensitivity is not always necessary, which allows them to be used only as an indicative test.

So according to the results of a study on 150 patients with suspected myocardial necrosis using this test design (prototype of the claimed object), there are the following diagnostic indicators, presented in table 2.

Table 2.
Data diagnostic indicators test system prototype Time watch 1-3 3-6 6-12 More than 12 Test prototype coated with two bands of antibodies Sensitivity 92 95 one hundred 87 Specificity 64 72 72 69 Accuracy 74 78 85 80

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 false negative

N number of all patients

To eliminate the shortcomings of the prototype test system, an additional (competing) lane 9 (Fig. 2), located in front of the test lane 10, was used. The function of this lane 9 is to reduce the analytical sensitivity of the test to 15 ng / ml due to the competitive binding of BSLC with F9 monoclonal antibodies immobilized in this lane 9. Since an increase in blood BSA levels 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 required level of diagnostic sensitivity. Indeed, the use of an additional competing band 9 made it possible to significantly reduce the number of false positive results, while not increasing the number of false negative and, thus, while maintaining the previous level of diagnostic sensitivity, the specificity and, as a result, the accuracy of the test were increased. Table 3 shows the data of diagnostic indicators of the claimed test system, which confirm the claimed technical result.

Table 3.
Data diagnostic indicators of the inventive test system Time watch 1-3 3-6 6-12 More than 12 The inventive test system Sensitivity 88 93 one hundred 90 Specificity 90 92 95 91 Accuracy 82 87 97 90

Claims (6)

1. The test system for immunochromatographic determination of cardiac protein that binds fatty acids in whole blood for the rapid diagnosis of myocardial infarction contains an immunological plate with a window for introducing a sample and a window for viewing the results, inside which there is a substrate for laminating porous materials, on which length and overlap are a filtration pad for applying a test blood sample, a conjugate pad, a porous membrane and an absorption pad moreover, the pillow for applying the conjugate is impregnated with a conjugate of colloidal gold particles with the first monoclonal mouse antibodies specific for the cardiac fatty acid binding protein on the porous membrane, transverse to the latter and parallel to each other, a test strip containing adsorbed second monoclonal mouse antibodies and a control strip is formed containing adsorbed rabbit antibodies against mouse immunoglobulins, characterized in that on the porous membrane, a conc riruyuschaya strip located along the edge of the cushion and for applying comprising the conjugate adsorbed first layer of mouse monoclonal antibodies specific to cardiac proteins which bind fatty acids. 2. The test system according to claim 1, characterized in that the pillow for applying the conjugate is made of cellulose, or glass microfiber, or polyester microfiber with a pore size of 20-25 μm and an absorption capacity of at least 55 mg / cm ² . 3. The test system according to claim 1, characterized in that the filter pad is made of a porous polysulfone material with a layer thickness of 600-650 μm and a pore volume of 55-57 μl / cm ² . 4. The test system according to claim 1, characterized in that the porous membrane is made of nitrocellulose, or polyethersulfone, or nylon with a layer thickness of 125-155 microns. 5. The test system according to claim 1, characterized in that the absorption cushion is made of a porous material of cotton fluff, or glass cellulose fiber with a layer thickness of 400-450 microns. 6. The test system according to claim 1, characterized in that the lamination substrate is made of polystyrene or acrylic adhesive material with a layer thickness of 230-270 microns.