CN113533716A - Preparation of double-index immunochromatography kit for detecting sST2/Gal3 - Google Patents
Preparation of double-index immunochromatography kit for detecting sST2/Gal3 Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention particularly relates to an immunochromatography kit for simultaneously detecting soluble ST2(sST2) and beta-Galectin-3 (Galectin3, Gal3), which mainly comprises an immunochromatography test strip, wherein the immunochromatography test strip is formed by sequentially adhering Fusion5, a nitrocellulose membrane and absorbent paper on a PVC (polyvinyl chloride) bottom plate. Wherein Fusion5 adsorbs sST2 antibody-microsphere and Gal3 antibody-microsphere, and cellulose nitrate membrane fixes sST2 antibody to form detection line 1(T1 line), fixes Gal3 antibody to form detection line 2(T2 line), and fixes anti-IgG antibody to form quality control line (C line). The invention also discloses a coupling microsphere process of the sST2 antibody and the Gal3 antibody. The invention establishes an sST2/Gal3 immunochromatographic kit, and simultaneously detects the concentrations of sST2 and Gal3 in blood or other body fluids, so that the kit is used for diagnosing myocarditis and monitoring the progress of myocarditis.
Description
Technical Field
The invention belongs to the field of immunology, and particularly relates to a process for preparing an sST2 antibody-fluorescent microsphere and a Gal3 antibody-fluorescent microsphere and establishing an sST2/Gal3 immunochromatography kit.
Background
Myocarditis (myocardis) is an inflammatory response that damages the heart muscle, affects myocardial contractility and electrical conduction systems of the heart, decreases the ability of the heart to pump blood or causes arrhythmias, causing heart failure, and is one of the leading causes of sudden and unexpected death in children or young adults. Viral infection is the leading cause of myocarditis, and secondly drugs induce myocardial inflammatory responses. Symptoms in patients with myocarditis range from flu-like conditions to dilated cardiomyopathy and heart failure.
At present, no specific biomarker for screening or diagnosing myocarditis exists, the golden standard for diagnosing myocarditis is to examine the existence of myocardial tissue injury and inflammatory reaction through cardiac biopsy, but different myocardial parts are injured and have different inflammation degrees, the injured or inflammatory parts can be missed in the biopsy, the cardiac biopsy method is not sensitive enough, the positive rate is only 30%, and a new method for diagnosing myocarditis needs to be found.
It has been found that the detection of soluble ST2(sST2) and β -Galectin-3 (Galectin3, Gal3) levels in blood can be used as biomarkers for the diagnosis of myocarditis.
ST2 has two main isoforms, namely transmembrane receptor type ST2L and soluble type sST2, ST2 is a receptor of interleukin-33 (IL-33), damaged myocardial cells secrete IL-33 into blood, and IL-33 can effectively block myocardial hypertrophy induced by angiotensin II or phenylephrine after IL-33 is combined with transmembrane receptor type ST2L, so that myocardial fibrosis and apoptosis can be reduced, and myocardial function can be improved. Under the condition of myocardial cell injury or inflammation, ST2 gene expression sST2 is strongly induced and secreted into blood circulation, sST2 can compete with ST2L and efficiently combine IL-33, so that IL-33/ST2L system signal conduction is blocked, IL-33 heart protection effect is eliminated, myocardial injury or inflammation progression is promoted, and heart failure can be developed. High levels of sST2 in the blood circulation represent myocardial cell injury or inflammatory response and continue to worsen.
Animal experiments show that mouse myocarditis is caused by virus infection, compared with uninfected healthy mouse hearts, the myocarditis mouse heart has higher sST2 expression level and higher sST2 blood level, and the detection of the sST2 blood level of the mouse can be used for judging the occurrence of mouse myocarditis.
In a clinical study, the level of sST2 in blood is detected, and compared with a healthy control group, the level of sST2 in the blood of a male myocarditis patient under the age of 50 years is remarkably increased, so that the level of sST2 in the blood is used as a new index for diagnosing myocarditis.
Gal3 consists of 201 amino acids, has a molecular weight of 30kD, and consists of an N-terminal proline, glycine and tyrosine rich structural region and a C-terminal sugar Recognition region (CRD). Gal3 is expressed in a variety of tissue organs and is produced and secreted primarily by activated macrophages. Gal3 is linked to different ligands through CRD, and plays a wide range of biological functions, and is involved in regulating cell proliferation and differentiation, inhibiting cell apoptosis, mediating cell adhesion, cell activation and chemotaxis. Gal3 can mediate macrophage and mast cell infiltration in cardiac muscle, promote cell release medium, enhance monocyte chemotaxis, stimulate monocyte macrophage and fibroblast proliferation and secrete procollagen I, form irreversible mutual binding collagen, thus leading to myocardial fibrosis formation, increase myocardial interstitial and perivascular fibrosis and cardiac collagen deposition, and cause myocardial hypertrophy and impaired myocardial compliance to cause heart failure. It can be seen that Gal3 plays an important role in the course of the myocarditis response.
There are studies on myocarditis, dilated cardiomyopathy and congestive heart failure in animals infected with meningitis virus. Histology detects and analyzes heart pathological condition and time process of Gal3 expression of each part of heart, after hours of virus inoculation, myocardium has inflammatory reaction, macrophage infiltration and heart tissue degeneration fibrosis pathological change are observed, along with the up-regulation of Gal3 expression level, the expression level of heart Gal3 is positively correlated with the heart fibrosis pathological change, and the Gal3 level in serum is increased within 96 hours and is consistent with the expression level of heart Gal 3. The results show that the expression of cardiac Gal3 is a biomarker for myocardial fibrosis caused by acute myocarditis, and the determination of the Gal3 level in the serum of animals can be used as a diagnostic indicator for the development of early acute myocarditis. The study is carried out to collect biopsy tissues and blood samples of patients with dilated cardiomyopathy and myocarditis, the expression of the Gal3 in the myocardial tissue is obviously and positively correlated with the number of inflammatory cells in the biopsy myocardial tissue in the myocarditis patient group, and the further detection shows that the Gal3 level in plasma is positively correlated with the Gal3 level in the myocardial tissue and the number of inflammatory cells in the biopsy myocardial tissue, so that the Gal3 in the blood can be used as a marker of myocarditis.
When myocarditis occurs, the levels of sST2 and Gal3 in myocardial tissues are increased, the levels of sST2 and Gal3 in blood circulation are also increased, and the dual indexes of sST2 and Gal3 in blood are detected at the same time, so that the positive rate and specificity of detecting myocarditis can be improved, and a kit for simply, quickly and quantitatively detecting the levels of sST2 and Gal3 in blood needs to be developed.
The invention establishes a double-index immunochromatographic kit for simultaneously detecting sST2 and Gal3, measures the levels of sST2 and Gal3 in blood, and is used for diagnosing myocarditis and monitoring the progress of myocarditis.
Disclosure of Invention
The invention discloses an sST2/Gal3 immunochromatography kit which contains an immunochromatography test strip, a chromatographic solution and a calibrator. The test strip is composed of a PVC bottom plate, wherein Fusion5, a nitrocellulose membrane and absorbent paper are sequentially connected and fixed on the PVC bottom plate, and a schematic diagram of the immunochromatographic test strip is shown in figure 1. Fusion5 adsorbed sST2 antibody-microspheres and Gal3 antibody-microspheres; the nitrocellulose membrane was provided with an immobilized sST2 antibody forming detection line 1(T1 line), an immobilized Gal3 antibody forming detection line 2(T2 line) and an immobilized anti-IgG antibody forming quality control line (C line).
The microsphere can be one of fluorescent microsphere, colorful microsphere, gold labeled microsphere, magnetic microsphere and up-conversion luminescent microsphere, and the Europium Chelate (Europium Chelate) fluorescent microsphere is selected, the excitation light wavelength is 360-420nm, and the emission light wavelength is 600-650 nm.
The surface modification functional group of the fluorescent microsphere is one of amino, carboxyl, hydroxyl or epoxy, and preferably the carboxyl modified fluorescent microsphere. The diameter range of the fluorescent microspheres is 100-300nm, and the fluorescent microspheres with the diameters of 100nm and 200nm are preferred.
The invention discloses a preparation process of an sST2 antibody and Gal3 antibody coupled fluorescent microsphere, which comprises the following steps:
(1)5-20mM carbodiimide (EDC) and 10-40mM N-hydroxyl thiosuccinimide (Sulfo-NHS) activated carboxyl modified fluorescent microspheres; (2) adding an sST2 antibody or a Gal3 antibody to perform coupling reaction; (3) adding 100-400mM Tris buffer solution to stop the coupling reaction; (4) after washing, sST2 antibody-fluorescent microspheres or Gal3 antibody-fluorescent microspheres were suspended in phosphate buffer and stored at 2-8 ℃ for further use.
The invention discloses a preparation process of Fusion5 adsorbing sST2 antibody-fluorescent microspheres and Gal3 antibody-fluorescent microspheres, which comprises the following steps:
diluting sST2 antibody-fluorescent microsphere to 0.02-0.25mg/ml with phosphate buffer solution, diluting Gal3 antibody-fluorescent microsphere to 0.02-0.25mg/ml, spraying the fluorescent microsphere onto Fusion5 at a speed of 4-10ul/cm, drying at 25-37 deg.C for 4-12 hr, drying, and sealing.
The invention discloses a preparation process of a T line and a C line on a nitrocellulose membrane, which comprises the following steps:
with NaHPO4Diluting sST2 antibody concentration to 0.5-2.5mg/ml with buffer solution, Gal3 antibody concentration to 0.5-2.5mg/ml and anti-IgG antibody concentration to 0.2-2.0mg/ml, spraying the antibody solution onto nitrocellulose membrane at 0.4-1.0ul/cm speed to form T1 line, T2 line and C line at 4-6mm interval, drying at 25-37 deg.C for 4-12 hr, drying and sealing.
The invention discloses an immunochromatographic test strip assembly process:
sticking a nitrocellulose membrane in the middle of the PVC bottom plate, sticking absorbent paper at one end, locating above the nitrocellulose membrane, and overlapping for 2-3 mm; and the other end is stuck with Fusion5 and is positioned above the nitrocellulose membrane and overlapped for 2-3mm to form a test paper board, and the test paper board is cut into test paper strips with the width of 3-5 mm.
The invention discloses a test paper card assembling process, which comprises the following steps:
the invention comprises a shell which is composed of a shell surface and a shell base. And (3) placing the test strip on a shell base, and closing the shell surface to form the test strip card. The shell surface is provided with a detection window corresponding to the position of the nitrocellulose membrane, and the shell surface is provided with a sample hole corresponding to the position of Fusion5, as shown in the front view of the immunochromatography test paper card in figure 2.
Chromatography liquid: 20-200mM Tris, pH7.0-8.0, 0.1-1.0% BSA, 50-500mM NaCl, 0.01-0.1% NaN30.2-2.0% Tween-20. Calibration products: 10-100mM NaHPO4，pH6.5-8.5，0.5-5.0mM EDTA，0.5-5.0mM DTT，20-200mM NaCl，0.2-2.0％BSA， 1.0-10％Sucrose，0.1-1.0％PVP，0.01-0.1％Tween-20，0.01-0.1％NaN30-100ng/ml ST2 protein and 0-200ng/ml Gal3 protein as sST2/Gal3 calibrator.
The invention adopts the principle of double-antibody sandwich immunochromatography: the sST2 and Gal3 in the sample hole of the sST2/Gal3 immunochromatography test paper card move forwards through chromatography, and are combined with the sST2 antibody-fluorescent microspheres or Gal3 antibody-fluorescent microspheres adsorbed by Fusion5 to form an sST2-sST2 antibody-fluorescent microsphere compound or Gal3-Gal3 antibody-fluorescent microsphere compound; under the action of capillary power, the diameter of a fluorescent microsphere of the Gal3-Gal3 antibody-fluorescent microsphere compound is 100nm, the fluorescent microsphere enters the nitrocellulose membrane firstly, and is combined with a fixed Gal3 antibody on a T2 line of the nitrocellulose membrane through a T1 line to form a fluorescent microsphere-Gal 3 antibody-Gal 3-Gal3 antibody compound which is gathered on the T2 line; the sST2-sST2 antibody-fluorescent microsphere complex has the diameter of 200nm, enters a nitrocellulose membrane, is combined with an sST2 antibody fixed by a nitrocellulose membrane T1 line to form a fluorescent microsphere-sST 2 antibody-sST 2-sST2 antibody complex, and is gathered on a T1 line; the free Gal3 antibody-fluorescent microspheres or sST2 antibody-fluorescent microspheres did not bind at the T1 line or T2 line, continued to move forward, and bound to the C-line immobilized anti-IgG antibody, and the unbound components continued to move to the absorbent paper position. The quantity of fluorescent microspheres captured by the T1 line is positively correlated with the concentration of sST2 in the sample, the quantity of fluorescent microspheres captured by the T2 line is positively correlated with the concentration of Gal3 in the sample, and the fluorescent microspheres captured by the C line indicates that the immunochromatographic process is completed. Scanning signals of a T1 line, a T2 line and a C line by an immunofluorescence analyzer, and obtaining the concentration of sST2 in the sample according to the positive correlation between the signal intensity of the T1 line and the concentration of sST2 in the sample; the concentration of Gal3 in the sample was obtained from the positive correlation between the intensity of the signal from the T2 line and the concentration of Gal3 in the sample.
Immunofluorescence analysis appearance, its characterized in that: the excitation wavelength is 350-430nm, and the receiving wavelength is 600-650 nm. The analyzer is used for interpreting the test paper card result, can realize automation, and provides a simple, quick and reliable detection result.
Adding the sST2/Gal3 calibrator into a sample well of an sST2/Gal3 test paper card, reacting for 5-20 minutes, measuring the fluorescence intensity of a T1 line, a T2 line and a C line on the test paper card by using an immunofluorescence analyzer, preparing an sST2 or Gal3 concentration standard curve, obtaining the relevant parameters of the concentration of the sST2 and Gal3 and a fluorescence intensity unit (RFU), setting up an immunofluorescence analyzer to automatically detect the concentration systems of the sST2 and Gal3, and further detecting the concentration of the sST2 and Gal3 in a sample.
The sST2/Gal3 immunochromatographic kit established by the invention is simple and rapid to operate, has high specificity, quantitatively determines the concentrations of sST2 and Gal3 in blood, and can meet the requirements of clinical examination.
Detailed Description
The principles and results of the present invention are further illustrated in conjunction with specific experiments, which set forth the multi-step experimental procedures of the present invention.
Preparation of sST2/Gal3 immunochromatography kit
The sST2/Gal3 immunochromatographic kit comprises an immunochromatographic test strip, a chromatographic solution and an sST2/Gal3 calibrator, and the preparation process is as follows.
(I) preparation of sST2 antibody-fluorescent microspheres
In the embodiment of the invention, Europium Chelate (Europium chemical) fluorescence-labeled carboxyl modified particles are selected, and are purchased from Bangs Laboratories Inc. microspheres with the diameter of 100nm-300 nm.
In the embodiment of the invention, an sST2 monoclonal antibody is purchased from Medix Biochemica and coupled to a europium chelate fluorescence-labeled carboxyl modified microsphere, the diameter of the microsphere is 200nm, and the method comprises the following steps:
1.10 mg/ml europium chelate fluorescence labeling carboxyl modified microsphere, 0.1ml is added into a 1.5ml centrifuge tube, centrifugation is carried out at 15000rpm for 15 minutes, and supernatant is removed.
2. Then, 1.2ml of 20mM MES buffer, pH6.0 was added thereto, and the microspheres were suspended in a centrifuge tube, centrifuged at 15000rpm for 15 minutes, and the supernatant was removed.
3. 0.2ml of the MES buffer containing 5mM EDC and 10mM Sulfo-NHS was added to the centrifuge tube, mixed well, and reacted at room temperature for 30 minutes.
4. 1.2ml of MES buffer was added to the centrifuge tube, and the mixture was centrifuged at 15000rpm for 15 minutes to remove the supernatant.
5. 0.2ml of PBS containing 35ug of the monoclonal antibody of ST2, pH7.0PBS was added to the centrifuge tube, and the reaction was carried out at room temperature for 2 hours.
6. 40ul of 100mM Tris, pH7.4 was added to the centrifuge tube and reacted at room temperature for 30 minutes.
7. 1.2ml of NaHPO was added4Buffer, 20mM NaHPO4pH7.0, 0.2% BSA, 0.01% Tween-20 into the centrifuge tube.
8. Centrifugation at 15000rpm for 15 minutes removed the supernatant.
9. With the above NaHPO4The sST2 antibody-fluorescent microspheres were suspended in buffer and stored at 4 ℃ until use.
(II) preparation of Gal3 antibody-fluorescent microspheres
In the embodiment of the invention, a Gal3 monoclonal antibody is purchased from Medix Biochemica and coupled to a europium chelate fluorescence-labeled carboxyl modified microsphere, the diameter of the microsphere is 100nm, and the method comprises the following steps:
1.10 mg/ml europium chelate fluorescence labeling carboxyl modified microsphere, 0.1ml is added into a 1.5ml centrifuge tube, centrifugation is carried out at 20000rpm for 20 minutes, and supernatant is removed.
2. Then, 1.2ml of 20mM MES buffer, pH6.0 was added thereto, and the microspheres were suspended in a centrifuge tube, centrifuged at 20000rpm for 20 minutes, and the supernatant was removed.
3. 0.2ml of the MES buffer containing 5mM EDC and 10mM Sulfo-NHS was added to the centrifuge tube, mixed well, and reacted at room temperature for 30 minutes.
4. 1.2ml of MES buffer was added to the centrifuge tube, and the mixture was centrifuged at 20000rpm for 20 minutes to remove the supernatant.
5. 0.2ml of PBS (pH7.0) containing 40ug of Gal3 monoclonal antibody was added to the above centrifuge tube, and the reaction was carried out at room temperature for 2 hours.
6. 40ul of 100mM Tris, pH7.4 was added to the centrifuge tube and reacted at room temperature for 30 minutes.
7. 1.2ml of NaHPO was added4Buffer, 20mM NaHPO4pH7.0, 0.2% BSA, 0.01% Tween-20 into the centrifuge tube.
8. Centrifugation at 20000rpm for 20 minutes removed the supernatant.
9. With the above NaHPO4Gal3 antibody-fluorescent microspheres were suspended in buffer and stored at 4 ℃ until use.
(III) spraying Fusion5 and nitrocellulose membrane
1. With NaHPO4Buffer, 20mM NaHPO4pH7.0, 10mM NaCl, 1.0% BSA, 10% Sucrose, 0.2% PVP, 0.5% Tween20, dilution of sST2 antibody-europium chelate fluorescent microspheres to 0.075mg/ml, dilution of Gal3 antibody-europium chelate fluorescent microspheres to 0.1mg/ml, using XYZ three-dimensional slide-coating gold spraying apparatus (HM3035), purchased fromThe method comprises the steps of spraying sST2 antibody-fluorescent microspheres and Gal3 antibody-fluorescent microspheres onto Fusion5 at a micro-quantitative spraying mode of 5ul/cm speed by Shanghai gold-labeled science and technology biological Limited, drying for 6 hours at 30 ℃, drying and sealing for later use.
2. With NaHPO4Buffer, 10mM NaHPO4pH7.0, 1.5% Sucrose, 0.1% PVP, sST2 antibody from Medix Biochemica diluted to 1.2mg/ml, Gal3 antibody from Medix Biochemica diluted to 1.5mg/ml, anti-mouse IgG antibody diluted to 1.0mg/ml, nitrocellulose membrane on XYZ three-dimensional scribing gold spraying instrument (HM3035), using micro-quantitative scribing method, 0.8ul/cm speed, spraying the above 3 antibodies onto nitrocellulose membrane to form T1 line, T2 line and C line, two-line spacing 5mm, 30 ℃, drying for 8 hours, drying and sealing for standby.
(IV) assembling immunochromatographic test paper card
The test strip is assembled in a room with the humidity of less than 30 percent and the temperature of 25-37 ℃. An immunochromatographic test strip is shown in figure 1, and the immunochromatographic test strip comprises a PVC bottom plate, and 25mm Fusion5, 25mm nitrocellulose membrane and 32mm absorbent paper which are sequentially adhered to the bottom plate along the length direction of the bottom plate; wherein, the nitrocellulose membrane is adhered to the middle part of the bottom plate, and is provided with an sST2 antibody coating layer at intervals to form a T1 line, a Gal3 antibody coating layer to form a T2 line and an anti-mouse IgG antibody coating layer to form a C line; fusion5 was spray coated with sST2 antibody-europium chelate fluorescent microspheres and Gal3 antibody-europium chelate fluorescent microspheres.
In the embodiment of the invention, the T1 line and the T2 line are arranged in parallel, the distance between the T1 line and the T2 line is 5mm, the T2 line and the C line are arranged in parallel, and the distance between the T2 line and the C line is 5 mm.
The absorbent paper is positioned at one end of the nitrocellulose membrane close to the line C, is partially overlapped with the nitrocellulose membrane, and has the overlapping length of 2 mm; the absorbent paper was overlaid on top of the nitrocellulose membrane.
Fusion5 was located on the nitrocellulose membrane near the end of the T1 line; partially overlapping the nitrocellulose membrane, wherein the overlapping length is 2 mm; fusion5 was overlaid on top of the nitrocellulose membrane. And (3) assembling each group of parts into a test paper board, and cutting the test paper board into test paper strips with the width of 4 mm.
Putting the test strip into a shell base, and closing the shell surface to form a test strip card, wherein the shell surface is provided with a sample adding hole (8) and a detection window (9) to expose a local area of the test strip as a front view of the immunochromatography test strip card shown in figure 2; the sample hole was opened above Fusion5(2), exposing a part of Fusion 5; the detection window was placed on top of the nitrocellulose membrane (3) to expose all of the T1 line (4), T2 line (5) and C line (6).
(V) preparing chromatographic solution and sST2/Gal3 calibrator
1. Preparation of chromatography liquid
25mM Tris，pH7.4，120mM NaCl，0.5％BSA，1.0％Tween-20，0.02％NaN3As a chromatography liquid.
2. Preparation of a sST2/Gal3 calibrator
NaHPO4Buffer, 20mM NaHPO4，pH7.0，1.0mM EDTA，2.0mM DTT，100mM NaCl，1.0％BSA，2.0％Sucrose， 0.2％PVP，0.01％Tween-20，0.02％NaN3ST2 to 0, 5, 10, 20, 40, 80ng/ml and Gal3 to 0, 10, 20, 40, 80, 160ng/ml were diluted as sST2/Gal3 calibrators.
Second, the process of detecting the concentration of sST2 and Gal3 by an immunofluorescence analyzer
(I) sST2/Gal3 immunochromatography kit operation process
When the sST2 and Gal3 in a sample are quantitatively detected by using an sST2/Gal3 immunochromatography test paper card, 50ul of sST2/Gal3 calibrator solution or 50ul of serum/slurry is added to a sample hole, 50ul of chromatography solution is added to the sample hole, the reaction is carried out for 5-20 minutes, and corresponding fluorescence signals are generated by a T1 line, a T2 line and a C line and are detected by using an immunofluorescence analyzer.
(II) establishing an immunofluorescence analyzer system for detecting concentrations of sST2 and Gal3
1. Establishing standard curves of sST2 and Gal3 concentration
Adding 50ul of sST2/Gal3 calibrator into a sample well of an sST2/Gal3 immunochromatography test paper card, adding 50ul of chromatography liquid into the sample well, performing a membrane chromatography reaction, after 5-20 minutes, selecting an excitation wavelength of 365nm and a detection wavelength of 610nm by using an immunofluorescence analyzer, and measuring Relative fluorescence intensity units (RFU) of a T1 line, a T2 line and a C line of the test paper strip card. Calibration with sST2The product concentration is ordinate, RFU is abscissa on T1 line, and sST2 calibrator concentration standard curve is prepared to obtain equation, y is 0.002x-2.1426, R2Referring to fig. 3, the sST2 concentration and RFU correlation curve is shown in 0.9966, and the sST2 concentration standard card is obtained from the standard curve as a basis for quantitative analysis of the concentration of sST2 contained in the sample.
Preparing a Gal3 calibrator concentration standard curve by taking the concentration of the Gal3 calibrator as an ordinate and the RFU (transverse coordinate) of a T2 line as an abscissa to obtain an equation, wherein y is 0.0029 x-3.5524, and R is2Referring to fig. 4, a curve relating concentration of Gal3 to RFU, 0.998, a standard card of concentration of Gal3 was obtained from the standard curve, and this standard curve was used as a basis for quantitative analysis of concentration of Gal3 contained in the sample.
And inputting the standard card into an immunofluorescence analyzer, and establishing a system for automatically displaying the concentrations of sST2 and Gal3 in the detected sample by the immunofluorescence analyzer.
2. Determination of reproducibility and accuracy of sST2/Gal3 immunochromatography kit
The sST2 and Gal3 calibrators at 20ng/ml and 40ng/ml were prepared as samples, and the sST2 and Gal3 samples were assayed in duplicate 15 times using the immunochromatographic kit described above, and the mean (M), Standard Deviation (SD), SD/Mx 100% ═ Coefficient of Variation (CV), and (1-M/sample concentration) x 100% ═ relative deviation (Bias) were calculated, respectively, and the results are shown in the table below.
| Sample | sST2 | Gal3 | |
| 19.17 | 38.19 | ||
| 18.44 | 39.47 | ||
| 19.56 | 38.48 | ||
| 19.48 | 40.32 | ||
| 18.78 | 38.85 | ||
| 19.57 | 39.57 | ||
| 20.11 | 38.78 | ||
| 18.65 | 37.65 | ||
| 18.44 | 39.64 | ||
| 19.28 | 38.29 | ||
| 17.86 | 39.38 | ||
| 19.68 | 39.56 | ||
| 18.96 | 38.38 | ||
| 18.67 | 40.46 | ||
| 19.83 | 37.58 | ||
| 19.16 | 38.45 | ||
| 19.48 | 38.66 | ||
| 18.87 | 39.65 | ||
| 19.92 | 37.58 | ||
| 18.69 | 38.74 | ||
| M | 19.13 | 38.884 | |
| SD | 0.582 | 0.848 | |
| CV | 3.03％ | 2.18％ | |
| Bias | 3.80％ | 2.80％ |
The CV of sST2 was 3.03%, the Bias was 3.8%, the CV of Gal3 was 2.18%, and the CV of Bias was 2.8%, indicating that the sST2/Gal3 immunochromatographic kit had good reproducibility and accuracy.
3. Detection of concentrations of sST2 and Gal3 in serum by using sST2/Gal3 immunochromatographic kit
50ul of serum is added into a sample adding hole of an sST2/Gal3 immunochromatography test paper card, 50ul of chromatographic solution is added into the sample adding hole for membrane chromatography reaction, and after 5-20 minutes, the concentrations of sST2 and Gal3 are automatically detected by an immunofluorescence analyzer, and the results are as follows:
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| RFU | 2864 | 8793 | 11254 | 24356 | 4758 | 14625 | 3756 | 1827 | 7462 | 14738 |
| sST2 | 3.6ng/ml | 15.4ng/ ml | 20.1ng/ ml | 46.6ng/ ml | 7.4ng/ml | 27.1ng/ ml | 5.4ng/ml | 1.6ng/ml | 12.9ng/ ml | 27.3ng/ ml |
| RFU | 3758 | 17686 | 20247 | 31568 | 5168 | 17287 | 4857 | 2874 | 9265 | 11365 |
| Gal3 | 7.4ng/ml | 48ng/ml | 55.4ng/ ml | 88ng/ml | 11.5ng/ ml | 46.6ng/ ml | 10.5ng/ ml | 4.8ng/ml | 23.3ng/ ml | 29.4ng/ ml |
Claims (6)
1. A rapid quantitative detection sST2/Gal3 immunochromatography kit mainly comprises an immunochromatography test strip which is formed by sequentially connecting and fixing Fusion5, a nitrocellulose membrane and absorbent paper on a PVC base plate; the Fusion5 adsorbs sST2 antibody-microspheres and Gal3 antibody-microspheres; the cellulose nitrate membrane is fixed with sST2 antibody to form a detection line 1(T1 line), fixed with Gal3 antibody to form a detection line 2(T2 line), and fixed with anti-IgG antibody to form a quality control line (C line).
2. The microsphere of claim 1, which is a fluorescent microsphere, a colored microsphere, a gold-labeled microsphere, a magnetic microsphere, or an upconversion luminescent microsphere, wherein the fluorescent microsphere is Europium Chelate (Europium Chelate).
3. The microsphere of claim 1, wherein the surface modification functional group is one of amino, carboxyl, amino, hydroxyl or epoxy, the diameter of the microsphere is 100-300nm, and the fluorescent microsphere with 100nm and 200nm diameters and modified by carboxyl is selected.
4. The process for the preparation of sST2 antibody-microspheres and Gal3 antibody-microspheres according to claim 1, comprising the steps of:
(1) activating carboxyl groups to modify the fluorescent microspheres by using carbodiimide (EDC) and N-hydroxyl thiosuccinimide (Sulfo-NHS); (2) adding an sST2 antibody or a Gal3 antibody to perform coupling reaction; (3) adding Tris solution to terminate the coupling reaction; (4) after washing, sST2 antibody-fluorescent microspheres and Gal3 antibody-fluorescent microspheres were suspended in phosphate buffer and stored at 2-8 ℃ for further use.
The preparation process of the sST2/Gal3 fluorescent immunochromatography reagent card comprises the following steps:
(1) preparing sST2 antibody-fluorescent microsphere and Gal3 antibody-fluorescent microsphere liquid, spraying the sST2 antibody-fluorescent microsphere liquid on Fusion5, and drying the sST2 antibody-fluorescent microsphere liquid for 4 to 12 hours at the temperature of between 25 and 37 ℃; (2) preparing a detection line and a quality control line: spraying sST2 antibody liquid, Gal3 antibody liquid and anti-IgG antibody liquid onto nitrocellulose membrane to form a T1 line, a T2 line and a C line, and drying at 25-37 ℃ for 4-12 hours; (3) assembling an immunochromatography test strip: sequentially adhering Fusion5, a nitrocellulose membrane and absorbent paper on a PVC (polyvinyl chloride) bottom plate to form a test paper plate, and cutting the test paper plate into test paper strips with the thickness of 3-5 mm; (4) and putting the test strip into a shell to form the immunochromatographic test strip card.
6. The sST2/Gal3 immunochromatographic kit of claim 1, which simultaneously detects sST2 and Gal3 concentrations in blood or other body fluids for diagnosing myocarditis and monitoring myocarditis progression.
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