RU158294U1 - IMMUNOCHROMATOGRAPHIC TEST SYSTEM - Google Patents

Abstract

An immunochromatographic test system for determining an early marker of myocardial infarction - sBSLC and a late marker of myocardial infarction - troponin I, containing a cassette with windows for depositing a sample and observing the test result, inside which a test strip of polypropylene sheet is placed, on certain sections of which a filter is glued for the absorption and separation of the test sample of biomaterial, a membrane with deposited and dried conjugates of colloidal gold with monoclonal antibodies, a nitrocellulose membrane and ads absorbent paper for absorbing components of a sample of biomaterial, one control and two test lines are applied to the nitrocellulose membrane, characterized in that the control line is made from a solution of goat anti-mouse polyclonal antibodies, the first test line is made from a solution of the first monoclonal mouse antibodies specific for cardiac protein fatty acid binding clone F9, a second test line is drawn from a solution of the first monoclonal mouse antibodies specific for cardiac troponin I, cl it is IC19, a mixture of conjugates with colloidal gold of second monoclonal murine antibodies specific for cardiac fatty acid binding protein Clone F5 and second monoclonal murine antibodies specific for cardiac troponin I, clone IC4, is applied to the conjugate membrane.

Description

The utility model relates to diagnostic medicine and can be used to detect myocardial infarction in the early and late stages, both at home and by emergency doctors. The immunochromatographic test system is designed to equip inpatient medical facilities (emergency room, medical departments: resuscitation, department of cardiology, therapy, etc.), as well as to equip health workers at the prehospital stage (when examining a patient at home, at a clinic reception, in an ambulance, in a clinical diagnostic laboratory), in public transport (pre-trip examinations, first-aid kits on railway and motor vehicles), in personal vehicles (first-aid kit for motorists), in home conditions for self-diagnosis, when there is the greatest amount of difficulty in differential diagnosis of acute coronary syndrome.

Based on the practice established in modern diagnostic medicine, it is recommended to use two markers of myocardial necrosis - “early” and “late” at the same time for the diagnosis of acute myocardial infarction. As an early marker that meets modern requirements, use the cardiac form of a protein that binds fatty acids (sbcf). BSA is a low molecular weight (14-15 kDa) cytoplasmic protein that binds fatty acids, which is normally responsible for their transport into the cell. In acute myocardial infarction, when the death of cardiomyocytes occurs, sBSSF quickly enters the bloodstream, reaching diagnostically significant concentrations 1-2 hours after the onset of a pain attack. Its kinetics is similar to myoglobin, but BSA has more specificity for cardiac muscle. A significant increase in the concentration of this protein in the blood (~ 200-500 ng / ml) occurs 3 hours after acute myocardial infarction and returns to normal values (~ 5 ng / ml) after 12-24 hours. The indicated features of the sBCLC protein make it exclusively determined A convenient tool for the early diagnosis of myocardial infarction. A later marker is the marker troponin I. Cardiac troponin I in acute myocardial infarction usually reaches a diagnostically significant level in the blood of patients 6 hours after the onset of symptoms, its increased level persists for 7-14 days, which makes cardiac troponin I convenient for late diagnosis of myocardial infarction. Due to its high specificity and sensitivity, the definition of cardiac troponins has become the "gold standard" in the biochemical diagnosis of myocardial infarction. Identification of these two markers simultaneously on the same test will increase the period for diagnosing acute myocardial infarction from 1 to 72 hours from the onset of the symptoms of the disease, while maintaining high diagnostic value in medical practice for each individual marker.

An immunochromatographic diagnostic device is known (RF patent for utility model RU 107364, G01N33 / 53, 11/30/2010) for determining a protein that binds fatty acids (BSLC), for express diagnosis of myocardial infarction, containing a test strip consisting of a test strip consisting of combinations of porous materials and biological components, such as a conjugate pad containing antibodies conjugated to colloidal gold, a nitrocellulose membrane with test and control lines with immobilized antibodies, under shka for introducing the sample and the absorbent cushion, wherein a releasable protective housing provided with two openings, one of which is located over the test and control lines, and the other - over the cushion for introducing the sample. The specified diagnostic device is characterized in that the detachable protective case is made with guide elements designed for spatial fixation of the test strip, and is also equipped with a cover placed above the hole located above the pillow for making a sample, and this cover is made with a piston entering the hole, the end face of the piston is made beveled so that its maximum height faces the nearest edge of the test strip, and the minimum height faces the test and control lines. Thanks to this system design, testing time is reduced, the likelihood of operator contact with the patient’s blood decreases, and the amount of blood taken from the patient decreases.

This test system is a mono test system, which does not allow to detect the late marker troponin I, the level of this cardiomarker rises in the blood 6-8 hours after the onset of the disease.

The invention is known (according to the international application WO2003044531, G01N 33/543, 11/20/2002), which relates to cartridges and is used for quick diagnosis of heart attack based on the immunoassay for detecting the heart form of a fatty acid binding protein (sFLC) for personal use, storage and analysis of biological fluids, in particular blood samples. However, the disadvantage of this test system is that it is presented in the form of a tablet, which contains two separate test strips for determining sBSLC for some time. The sBSLC marker closes the time range up to 12 hours from the onset of a heart attack. The determination of the later stages of a heart attack using this system is not possible.

Thus, rapid immunodiagnostic systems are of practical interest, which, along with the determination of an early marker of AMI - BSAK - make it possible to control markers that are present in the blood for a long time after AMI.

The closest analogue is a test system for immunochromatographic determination of cardiac protein binding fatty acids and troponin I in whole blood for the rapid diagnosis of myocardial infarction (RU patent 2464572, G01N33 / 558, 12/20/2010). The test system contains an immunological tablet with a window for introducing the test blood sample and a window for viewing the results, inside which is placed a substrate for laminating porous materials. On this substrate, sequentially along the length and overlap are located:

- a pillow with conjugates for applying the test blood sample,

- filtration pad for the separation of the test blood sample,

- a porous membrane for carrying out an immunochromatographic reaction,

- an absorption pad to create fluid flow.

On the pillow with conjugates and across the latter, the first band of the conjugate of colloidal gold particles with the first monoclonal mouse antibodies specific for the cardiac fatty acid binding protein and the second band of the conjugate of colloidal gold particles with the first mouse monoclonal antibodies specific for troponin I were formed. On the porous membrane transverse to the last formed:

- a competing band containing a layer of adsorbed first or second monoclonal mouse antibodies specific for a heart protein that binds fatty acids,

- the first test strip containing adsorbed second monoclonal murine - the second test strip containing adsorbed second monoclonal antibodies to troponin I,

- a control strip containing adsorbed monospecific polyclonal rabbit antibodies against mouse immunoglobulins (RAM).

However, this test system determines the presence of cardiomarkers only in whole venous blood. The collection of whole blood from a vein should always be carried out by qualified personnel trained in the procedure for collecting venous blood in specialized conditions (clinical diagnostic laboratories, medical institutions, etc.). It should be noted that due to the use of venous blood as biological material, the time for preparation for analysis increases.

The technical result of the proposed utility model is to simplify the analysis procedure, reduce the preparation time for the analysis and expand the options for the tested biomaterial.

The indicated technical result is achieved by the fact that an immunochromatographic test system is proposed for determining an early marker of myocardial infarction - sBSLC and a late marker of myocardial infarction - troponin I. The test system contains a cassette with windows for depositing a sample and observing the test result. Inside the test system, a test strip of polypropylene sheet is placed, on certain areas of which a filter is glued to absorb and separate the test sample of biomaterial, a membrane with deposited and dried colloidal gold conjugates with monoclonal antibodies, a nitrocellulose membrane and adsorption paper to absorb components of the sample of biomaterial. One control and two test lines are applied to the nitrocellulose membrane. A distinctive feature of the claimed device is that the control line is drawn from polyclonal goat antibodies against mouse immunoglobulins. The first test line was plotted from a solution of the first monoclonal mouse antibodies specific for the cardiac fatty acid binding protein, clone F9. The second test line is drawn from the first monoclonal mouse antibodies specific for cardiac troponin I, clone IC19. A mixture of conjugates with colloidal gold from second monoclonal murine antibodies specific for cardiac fatty acid binding protein, clone F5, and second monoclonal murine antibodies specific for cardiac troponin I, clone IC4, is applied to the conjugate coating membrane.

The attached drawing (figure 1) shows the inventive device, where 1 is a strip of sheet propylene, 2 is a membrane of cellulose nitrate, 3 is adsorption paper, 4 is a membrane with deposited and dried colloidal gold conjugates with monoclonal antibodies against sBSLC and cTnI, 5 - filter for absorption and separation of the test sample of biomaterial, 6 - plastic cassette, 7 - window for entering the sample, 8 - window for viewing the result, A - control line of polyclonal goat antibodies against mouse immunoglobulins, B and C - first and second I test line of monoclonal antibodies specific to sBSZhK and cTnI.

The device operates as follows. A sample of biomaterial is introduced into the window for introducing a sample (7) of a plastic cartridge (6). A myocardial infarction marker (sBSLC or / and cTnI) present in the biosample (venous or capillary blood, blood plasma, blood serum) reacts with a colloidal gold conjugate with antibodies specific for sBCLC or / and cTnI deposited on the membrane (4). The result is a colored antigen-antibody complex. The resulting complex advances under the action of capillary forces along the membrane of cellulose nitrate (2) and interacts with specific monoclonal antibodies immobilized on cellulose nitrate (B, C). As a result, if SBBS is present in the biosample, then as a result of the immune response, test line 1 is colored pink, if cTnI is present in the biological sample, then 2 test lines are stained. Staining only the first test (B) line informs about the identification of the “early” marker of myocardial infarction sBCLC, and, accordingly, the early onset of the disease. Staining only the second test (C) line informs about the identification of a “late” marker of myocardial infarction cTnI, and, accordingly, about the long time that has passed since the onset of the disease. It is also possible that both cardiomarkers are present in the biosample, in which case both test lines (B and C) are colored pink. Colloidal gold conjugates not bound in the first and second test lines migrate further along the cellulose nitrate membrane (2) and interact with goat anti-mouse polyclonal antibodies immobilized on control line (A). As a result of the reaction, a colored control strip appears. The control band should always occur, regardless of the presence of cardiomarkers in the sample. The control strip is an internal control of the performance of the test. The absence of a control strip indicates that the test system is inoperative. Evaluation of the test result is carried out visually, through the result window in a plastic cassette (8) with a test strip (1).

Claims (1)

An immunochromatographic test system for determining an early marker of myocardial infarction - sBSLC and a late marker of myocardial infarction - troponin I, containing a cassette with windows for depositing a sample and observing the test result, inside which a test strip of polypropylene sheet is placed, on certain sections of which a filter is glued for absorption and separation of the test sample of biomaterial, a membrane with deposited and dried conjugates of colloidal gold with monoclonal antibodies, a nitrocellulose membrane and ads absorbent paper for absorbing components of a sample of biomaterial, one control and two test lines are applied to the nitrocellulose membrane, characterized in that the control line is made from a solution of goat anti-mouse polyclonal antibodies, the first test line is made from a solution of the first monoclonal mouse antibodies specific for cardiac protein fatty acid binding clone F9, a second test line is drawn from a solution of the first monoclonal mouse antibodies specific for cardiac troponin I, cl it is IC19, a mixture of conjugates with colloidal gold of second monoclonal murine antibodies specific for cardiac fatty acid binding protein Clone F5 and second monoclonal murine antibodies specific for cardiac troponin I, clone IC4, is applied to the conjugate membrane.

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