CN115524483A - Rapid detection kit for cardiac marker for combined detection and application thereof - Google Patents

Rapid detection kit for cardiac marker for combined detection and application thereof Download PDF

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Publication number
CN115524483A
CN115524483A CN202211196973.8A CN202211196973A CN115524483A CN 115524483 A CN115524483 A CN 115524483A CN 202211196973 A CN202211196973 A CN 202211196973A CN 115524483 A CN115524483 A CN 115524483A
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detection
marker
membrane
cardiac
diluent
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刘冰
李亚楠
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Changzhou Bowendi Pharmaceutical Co ltd
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Changzhou Bowendi Pharmaceutical Co ltd
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    • G01N33/54386—Analytical elements
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Abstract

The invention discloses a myocardial marker rapid detection kit for combined detection and application thereof, which is a combination of a detection buckle, a myocardial marker specific reagent strip, a myocardial marker specific immunity detection marker, an immunity detection tube, a diluent tube and a quantitative detector.

Description

Rapid detection kit for myocardial markers through combined detection and application of rapid detection kit
Technical Field
A rapid detection kit for a cardiac marker by combined detection and application thereof.
Background
The immunological detection technology is an experimental means for determining antigens, antibodies, immune cells, chemical components and the like by applying the immunological principle, and is widely applied to samples which are derived from human bodies and animal bodies and can be used for disease diagnosis and health detection and samples for environmental, pharmaceutical, food and industrial analysis. The conventional methods include immunochemiluminescence, enzyme linked immunosorbent assay, fluorescence immunochromatography, colloidal gold immunochromatography, latex immunochromatography, and the like. The detection of blood cardiac markers is a detection means commonly used in clinic, and commonly used cardiac markers comprise cardiac troponin I, cardiac troponin T, cardiac fatty acid binding protein, myoglobin, creatine kinase isozyme, myeloperoxidase, N-terminal brain natriuretic peptide precursor and the like. For clinical detection of cardiac markers, generally, the detection speed is required to be as fast as possible, and the time from sampling to result should be controlled within 10 minutes, but the existing detection cannot meet the requirement at present, and the clinical detection of cardiac markers mainly adopts immunological detection technologies, including colloidal gold immunochromatography, fluorescence immunochromatography, immunochemiluminescence detection and the like. The chemiluminescence detection time needs more than 3 hours before, and is shortened to more than 20 minutes through various technical improvements. Colloidal gold immunochromatography detection various products were developed in succession since the 1998 troponin T rapid detection kit, but the detection time was about 15-20 minutes, namely the time from sampling to result is more than 20 minutes. Therefore, the rapid detection technology product for the cardiac marker, which has the advantages of high sensitivity, rapidness, miniaturization, full quantification, automation, simple and convenient operation, rapid use and detection time within 10 minutes, is developed, is favorable for improving the medical quality and efficiency, and has important clinical significance and application value.
Disclosure of Invention
The invention aims to develop a myocardial marker rapid detection technology product with detection time controlled within 10 minutes, and compared with the prior art, the product has the advantages of high detection sensitivity, rapidness, low cost and the like so as to improve the clinical detection quality.
In order to achieve the above object, the present invention provides a kit for rapidly detecting a cardiac marker by combined detection, comprising: detect discount card (1), cardiac marker specificity reagent strip (2), cardiac marker specificity immunodetection marker (3), immunoassay tube (4), diluent (5), diluent pipe (6) and quantitative determination appearance (7), wherein:
the detection buckle (1), the immunity detection tube (4), the diluent tube (6) and the quantitative detector (7) are different combined structures which are independently arranged; the detection buckle card (1) comprises a detection shell (8) and the myocardial marker specific reagent strip (2) positioned in the detection shell (8); the myocardial marker specific reagent strip (2) consists of a supporting negative film (9), a nitrocellulose membrane (10) and absorbent paper (11), wherein the nitrocellulose membrane (10) and the absorbent paper (11) are sequentially stuck on the supporting negative film (9) and are coated with a myocardial marker specific antihuman myocardial marker secondary antibody; the myocardial marker specific immunodetection marker (3) is a first anti-human myocardial marker antibody marked by an indicator, and is loaded in the immunodetection tube (4), wherein the indicator is a colored microsphere and comprises at least one of a colloidal gold particle and a latex particle; the diluent (5) is a water-soluble buffer salt solution and is loaded in the diluent pipe (6); the quantitative detection instrument (7) is a quantitative detection unit of the rapid detection kit for the cardiac marker, and comprises at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer; the detection shell (8) comprises an upper layer structure (12) and a lower layer structure (13), wherein a sample adding hole (14) and an observation window (15) are formed in the upper layer structure (12), the lower layer structure (13) is provided with a bracket (16) for placing the myocardial marker specific reagent strip (2), the sample adding hole (14) corresponds to the near end of the nitrocellulose membrane (10), and the observation window (15) corresponds to the far end of the nitrocellulose membrane (10).
The nitrocellulose membrane comprises one or more of nitrocellulose membrane, polyvinylidene fluoride membrane, nylon membrane and DEAE cellulose membrane.
In the combined detection structure, a blood cell filtering structure (19) is adhered to the support bottom sheet (9) and is connected with the near end of the nitrocellulose membrane (10), and the blood cell filtering structure (19) comprises at least one of a blood cell filtering membrane or a membrane pad treated by erythrocyte antibodies.
In the combined detection structure, a liquid phase dispersion membrane (20) is pasted on a support bottom sheet (9) of the detection reagent strip and is connected with the near end of the nitrocellulose membrane, wherein the liquid phase dispersion membrane (20) is preferably a glass cellulose membrane or a polyester cellulose membrane, and the position of the detection reagent strip corresponding to the sample adding hole is the liquid phase dispersion membrane.
In the combined detection structure, the quantitative detection instrument (7) configured in the rapid detection kit for the cardiac marker comprises at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer.
In the above-mentioned combination assay structure, the cardiac marker includes at least one of cardiac troponin I, cardiac troponin T, cardiac fatty acid binding protein, myoglobin, creatine kinase isoenzyme, myeloperoxidase, and N-terminal brain natriuretic peptide precursor.
In the above-mentioned combined detection structure, the operation of the rapid detection kit for cardiac marker uses a blood cell filtration membrane structure mode, and comprises the following steps:
1) Taking out the detection buckle card containing the hemocyte filtering membrane, and horizontally placing the detection buckle card on a table board;
2) Collecting a blood sample to be detected, adding the blood sample into an immunodetection tube (4) containing the antihuman myocardial marker first antibody immunodetection marker (3), adding a proper amount of diluent, and mixing to prepare a sample mixed solution to be detected;
3) Adding the sample mixed solution to be detected to a hemocyte filtering membrane through a sample adding hole, and enabling the mixed solution to flow through the hemocyte filtering membrane, a nitrocellulose membrane and a water absorption pad forwards;
4) Adding the diluent to the hemocyte filtering membrane through the sample adding hole, and enabling the diluent to flow through the hemocyte filtering membrane, the nitrocellulose membrane and the water absorption pad forwards;
5) And detecting the reading result from the observation window to finish the detection.
The use of the rapid detection kit for cardiac markers according to claim 1 in the development of human cardiac marker immunodetection reagent products.
Due to the adoption of the technical scheme, the invention has the following advantages:
1. the invention reduces the immunodetection buckle of the cardiac marker from the prior sample pad, the marker combination pad, the nitrocellulose membrane and the absorbent paper pad to the prior nitrocellulose membrane and the absorbent paper pad which are only used for completing detection reaction, thereby not only reducing the detection cost, but also shortening the detection time, realizing the purpose of controlling the detection time within 10 minutes, effectively improving the timeliness and popularization feasibility of clinical detection of products after the clinical detection of the cardiac marker is completed, and improving the clinical diagnosis and treatment effect.
2. The cardiac marker detection buckle is stuck in front of the nitrocellulose membrane and is adhered with a blood cell filtering membrane or a membrane pad structure treated by an erythrocyte antibody, so that the cardiac marker detection buckle can be simultaneously used for one-step detection of whole blood, the sample processing time is further shortened, and the detection speed, the detection convenience and the detection practicability are improved;
3. the myocardial marker detection buckle card adopts a step-by-step structural use state that the mixed solution of a sample to be detected containing the immunodetection marker is loaded firstly, and then the diluent not containing the immunodetection marker is loaded, so that the binding efficiency of the detected substance on the nitrocellulose membrane is improved, the cleaning effect is exerted, the non-specific binding of the immunodetection marker on the nitrocellulose membrane is effectively reduced, and the detection efficiency is improved.
4. The myocardial marker detection buckle card is provided with the quantitative detector simultaneously comprising at least one of the colorimetric quantitative analyzer and the fluorescent quantitative analyzer, can be used for immune quantitative detection in various environments, and improves the clinical application value.
Drawings
FIG. 1 is a schematic sectional view of the overall structure of the present invention;
FIG. 2 is a schematic view of the overall structure of the present invention;
FIG. 3 is a schematic view of the basic structure of the reagent strip of the present invention;
FIG. 4 is a schematic view of a reagent strip of the present invention including a hemocyte filtration structure;
FIG. 5 is a schematic view of a reagent strip of the present invention including a liquid phase dispersion film;
FIG. 6 is a schematic view of an immunoassay tube of the present invention;
FIG. 7 is a schematic view of a diluent tube of the present invention;
FIG. 8 shows a quantitative measuring apparatus according to the present invention.
The figures are labeled as follows:
detecting the buckling card 1; a myocardial marker-specific reagent strip 2; an immunodetection marker 3; an immunoassay tube 4; a diluent 5; a diluent tube 6; a quantitative detection instrument 7; a chromatographic detection housing 8; a support base sheet 9; a nitrocellulose membrane 10; a water absorbent pad 11; detecting the upper cover 12 of the buckle; a detection buckle base 13; a sample addition hole 14; an observation window 15; a reagent strip bracket 16; detection line (capture agent) 17; a quality control line 18; a blood cell filtering structure 19; a liquid-phase dispersion film 20; an immunodetection marker tube cap 21; an immunodetection label sample addition head 22; a diluent pipe cover 23; the diluent loads the sample addition head 24; a quantitative detection chamber 25.
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined purpose, the following embodiments are further described with reference to the accompanying drawings, but the present invention is not limited to the following description.
As shown in fig. 1, fig. 2 and fig. 3, the overall structure of the combined detection structure for cardiac markers of the present invention comprises a detection buckle 1, a cardiac marker specific reagent strip 2, an immunodetection marker 3, an immunodetection tube 4, a diluent 5, a diluent tube 6, a quantitative detection instrument 7, a chromatography detection shell 8, a support base plate 9, a nitrocellulose membrane 10, a water absorption pad 11, a detection buckle upper cover 12, a detection buckle base 13, a sample adding hole 14, an observation window 15, a reagent strip bracket 16, a detection line (capture agent) 17 and a quality control line 18, wherein the detection buckle 1 comprises the chromatography detection shell 8 and the cardiac marker specific reagent strip 2 located in the chromatography detection shell 8, and the cardiac marker specific reagent strip 2 comprises the support base plate 9 and the nitrocellulose membrane 10 and the water absorption pad 11 adhered to the support base plate 9; the chromatography detection shell 8 comprises a detection buckle upper cover 12, a detection buckle base 13, a sample adding hole 14 and an observation window 15 on the detection buckle upper cover 12, a reagent strip bracket 16 arranged on the detection buckle base 13 and a myocardial marker specific reagent strip 2 arranged on the reagent strip bracket 16; when the combined detection structure is used, the immunodetection marker 3 is mixed with a sample to be detected in the immunodetection tube 4, the mixture is loaded to the starting end of the nitrocellulose membrane 10 through the sample adding hole 14 and flows through the nitrocellulose membrane 10 and the water absorption pad 11, and the object to be detected is specifically captured and fixed by a capture agent (detection line, non-labeled anti-human cardiac marker second antibody) 17 coated and fixed on the nitrocellulose membrane 10; then loading a diluent 5 which does not contain the immunodetection marker 3 and the sample to be detected to the starting end of the nitrocellulose membrane 10, and loading for one or more times to remove the indicator which remains on the nitrocellulose membrane 10 and is not specifically bound; and detecting the amount of the immune detection marker 3 captured on the nitrocellulose membrane 10 by using a quantitative detection instrument 7, reading the detection result and finishing the detection process.
As shown in FIG. 3, in the basic structure of the myocardial marker specific reagent strip of the present invention, a nitrocellulose membrane 10 and a water absorbent pad 11 are sequentially adhered to a PVC support plate 9 from the near side to the far side, and the nitrocellulose membrane 10 is coated with a detection line (capture agent, non-labeled anti-human myocardial marker secondary antibody) 17 and a quality control line 18 for capturing an object to be detected.
As shown in fig. 4, the reagent strip for specificity of cardiac marker of the present invention comprises a blood cell filtration structure, a blood cell filtration membrane 19, a nitrocellulose membrane 10 and an absorbent pad 11 are sequentially adhered to a PVC support base sheet 9 from near to far, so that when the detection buckle 1, the immunoassay tube 4 and the diluent tube 6 are in a combined use state, first, the first antibody marker-immunoassay marker 3 of anti-cardiac marker labeled by an indicator is mixed with a blood sample to be detected, and is loaded to the blood cell filtration membrane 19, blood cells are filtered, and a blood plasma part flows through the nitrocellulose membrane 10 and the absorbent pad 11, and the cardiac marker in the blood sample to be detected is specifically captured and fixed by the second antibody (capture agent) 11 of anti-cardiac marker coated and fixed on the nitrocellulose membrane 10; then loading the diluent without the immunodetection marker and the sample to be detected to the blood cell filtering membrane 19, and loading one or more times to remove the indicator remained on the nitrocellulose membrane 10 and not specifically bound; and detecting the amount of the captured immunodetection marker on the nitrocellulose membrane by using the quantitative detection structure 7, reading a detection result and finishing the detection process.
As shown in fig. 5, the myocardial marker specific reagent strip of the present invention comprises a liquid phase dispersion membrane structure, a liquid phase dispersion membrane 20, a nitrocellulose membrane 10 and a water absorption pad 11 are sequentially adhered to a PVC support base sheet 9 from near to far, so that when the detection buckle 1, the immunoassay tube 4 and the diluent tube 6 are in a combined use state, first, an indicator-labeled anti-human myocardial marker first antibody marker-immunoassay marker 3 is mixed with a sample to be detected, loaded onto the liquid phase dispersion membrane 20, and flows through the nitrocellulose membrane 10 and the water absorption pad 11, and the myocardial marker in the sample to be detected is specifically captured and fixed by an anti-human myocardial marker second antibody (capture agent) 11 coated and fixed on the nitrocellulose membrane 10; then, loading the diluent which does not contain the immunodetection marker and the sample to be detected onto the liquid phase dispersion membrane 20, and loading for one or more times to remove the indicator which is remained on the nitrocellulose membrane 10 and is not specifically combined; and detecting the amount of the captured immunodetection marker on the nitrocellulose membrane by using the quantitative detection structure 7, reading the detection result and finishing the detection process.
As shown in fig. 6, the immunoassay tube of the present invention comprises an indicator-labeled anti-human cardiac marker first antibody label-immunoassay marker 3, an immunoassay marker tube 4, an immunoassay marker tube cap 21, and an immunoassay marker sample application head 22, wherein the immunoassay marker 3 is preferably lyophilized powder, and a sample to be tested is added before detection, and mixed to obtain a sample mixture to be tested.
As shown in fig. 7, the diluent tube of the present invention includes a diluent 5, a diluent tube 6, a diluent tube cover 23, and a diluent loading head 24, and the diluent 5 is directly loaded on the reagent strip in use.
As shown in FIG. 8, the quantitative detection device of the present invention comprises a quantitative detection structure 7 and a quantitative detection chamber 25 for placing a detection buckle therein.
Thus, in practical operation, the detection buckle 1, the myocardial marker specific reagent strip 2, the immunodetection marker 3, the immunodetection tube 4, the diluent 5, the diluent tube 6 and the quantitative detection structure 7 of the combined detection structure are integrated into a combined use structure, when in use, the immunodetection marker 3 is mixed with a sample to be detected in the immunodetection tube 4, the mixture is loaded to the starting end of the nitrocellulose membrane 10 through the sample adding hole 14 and flows through the nitrocellulose membrane 10 and the water absorption pad 11, and the sample to be detected is specifically captured and fixed by a capture agent (detection line, a non-labeled anti-human myocardial marker second antibody) 17 coated and fixed on the nitrocellulose membrane 10; and then loading a diluent 5 which does not contain the immunodetection marker 3 and the sample to be detected to the starting end of the nitrocellulose membrane 10, loading for one time or more, removing the indicator which is remained on the nitrocellulose membrane 10 and is not specifically combined, putting the washed nitrocellulose membrane 10 into a quantitative detection chamber 24 of a quantitative detection device, starting detection, capturing the amount of the immunodetection marker 3 fixed on the nitrocellulose membrane 10, quantitatively detecting, reading a detection result, and finishing the detection process.
Experimental study of the invention: the following experiments illustrate the detection method of the present invention and its effects, but are not intended to limit the present invention. The experimental methods used in the following experiments are all conventional methods unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
Experiment one: the immune colloidal gold method detects the comparison experiment of human myoglobin of blood:
1. the invention discloses a material for a combined detection structure experiment:
the detection buckle card adopts a commercially available detection buckle card with an upper cover and a base, a 2ml storage tube is used as an immunity detection tube and a diluent tube for experiments, a nitrocellulose membrane adopts Sidoris CN140, a hemocyte filtration membrane adopts Whatman FUSION 5, a glass cellulose membrane adopts Shanghai gold-labeled SB08, a chromaticity quantitative analyzer adopts an Ouplan SkanFlexii multi-channel immunochromatography quantitative analyzer, a membrane printing instrument adopts an ISOF Flow membrane spraying and printing integrated machine of Imagen Technology company, USA, a freezing dryer adopts an LC-10N freezing dryer of Shanghai dynasty company, and a strip cutting machine adopts a Korean-sense Chinese CE strip cutting machine.
2. Preparing an immunodetection marker:
the method comprises the steps of adopting a conventional immune colloidal gold labeling and double-antibody sandwich method for detection, using a first monoclonal antibody of human myoglobin labeled by colloidal gold particles as an immune detection label, using a second monoclonal antibody of human myoglobin having specific immune binding property with the first monoclonal antibody of human myoglobin paired with the human myoglobin as a capture agent, coating the capture agent on a nitrocellulose membrane in a non-labeling state, binding the human myoglobin with the immune detection label through the immune detection label, binding the capture agent and capturing and fixing through the nitrocellulose membrane, and calculating the antigen concentration of a sample by measuring the capture amount of the colloidal gold label. The method comprises the following specific operations: taking 1.5ml centrifuge tube, adding 1ml colloidal gold solution with particle size of 60nm, adding 3.6ul 0.1M potassium carbonate, stirring uniformly, adding 10ug of first monoclonal antibody against human myoglobin, reacting for 10min, adding 20% BSA 1 ul/ml, reacting for 10min, centrifuging for 15min at 10000r/min, removing supernatant, suspending the precipitate with 1ml colloidal gold complex solution (30 mM Tris, 3% trehalose, 3% sucrose, 0.25% BSA), centrifuging for 15min at 10000r/min, removing supernatant, suspending the precipitate with colloidal gold complex solution again, and the final volume is 0.5ml.
3. Preparing a reagent strip:
conventional control group: and a colloidal gold combination pad, a blood cell filtering membrane, a nitrocellulose membrane and absorbent paper are sequentially adhered to the PVC support bottom sheet. Coating the colloidal gold marker on a colloidal gold bonding pad of a glass cellulose membrane, starting a membrane printing instrument, starting pressurized nitrogen, loading a colloidal gold marker solution, starting to spray membrane coating, and setting membrane printing conditions as follows: the moving speed of the spray pen is 30 mm/s, and the propelling speed of the liquid is 5.0 mu l/cm; and (3) putting the printed film into a drying oven, drying for 6 hours at 37 ℃, and then putting the film into a sealed bag containing a drying agent for storage. The capture antibody of the detection line T is a non-labeled paired anti-human myoglobin second monoclonal antibody of 1.0mg/ml, and is coated on the middle section of the nitrocellulose membrane; the goat anti-mouse IgG polyclonal antibody with the quality control line C of 1.0mg/ml is coated at the far end of the nitrocellulose membrane and is used for capturing the colloidal gold labeled anti-human myoglobin monoclonal antibody which is not captured by specificity.
Experimental group of the invention: and a hemocyte filtering membrane, a nitrocellulose membrane and absorbent paper are sequentially adhered to the PVC supporting bottom sheet. The experimental group of the invention subpackages 2.0 mul of colloidal gold marker into 2ml of preservation tube, freezes and dries, and the preservation is used at 4 ℃. The capture antibody of the detection line T is a non-labeled paired anti-human myoglobin second monoclonal antibody of 1.0mg/ml, and is coated on the middle section of the nitrocellulose membrane; the goat anti-mouse IgG polyclonal antibody with the quality control line C of 1.0mg/ml is coated at the far end of the nitrocellulose membrane and is used for capturing the colloidal gold labeled anti-human myoglobin monoclonal antibody which is not captured by specificity.
Preparing a reagent strip: starting a dehumidifier to reduce the humidity in the operating room to below 25%, placing the adhered detection sheet on a slitting machine, and cutting into reagent strips with the thickness of 4.0 mm. And (3) putting the reagent strip into an aluminum amber sealing bag with a drying agent, sealing the bag on a sealing machine, and adding a label for later use.
4. The experimental method comprises the following steps:
preparing a human myoglobin solution: a human myoglobin solution of known concentration was taken and diluted with a sample dilution buffer (1% BSA,100mM glycine, 50mM PBS, 150mM NaCl, pH 7.4) to prepare a human myoglobin solution of 100 ng/ml.
Experimental groups: the prepared human myoglobin solution is added into the prepared colloidal gold marker tube by 50 ul/tube to form a marker mixed solution. Taking the prepared reagent strip of the invention, dropwise adding 50ul of prepared marker mixed solution on a hemocyte filtration membrane, standing for 1 minute, dropwise adding 25ul of diluent on the hemocyte filtration membrane, standing for 30 seconds, dropwise adding 25ul of diluent, standing for 30 seconds, and placing on a colorimetric quantitative analyzer to read colorimetric values of 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
Control group: and taking the prepared conventional coated reagent strip, dropwise adding 50ul of the prepared human myoglobin marker mixed solution and 50ul of the prepared colloidal gold redissolution to a hemocyte filter membrane, standing, and reading the colorimetric values for 2, 3, 4, 5, 8, 10, 15 and 20 minutes on a colorimetric quantitative analyzer.
5. Results of the experiment
The colloidal gold marker and the reagent strip are in a separately established combined state, the colloidal gold marker and a sample to be detected are firstly mixed for a first-step reaction, a conventional colloidal gold combination pad is not arranged on the reagent strip, and a nitrocellulose membrane positioned on the reagent strip is used as a dispersion carrier for detection reaction. The experimental result is shown in table 1, the chroma signal value is basically stable after the conventional method detects for 15 minutes, and the chroma signal value is basically stable after the method detects for 4 minutes, which shows that the detection time can be shortened and the detection sensitivity can be improved by the method.
TABLE 1 comparison of fast blood myoglobin detection experiment by immunogold colloidal gold method
Group detection reaction time (min)/(color value)
2 3 4 5 8 10 15 20
Control group 06 12 15 23 58 91 89
Experimental group 0 38 91 89 93 97 。
Experiment two: quick detection and comparison experiment of troponin I by latex microsphere immunochromatography:
1. the invention discloses a material for a combined detection structure experiment:
the same as the experiment I.
2. Preparing an immunodetection marker:
the method comprises the steps of adopting a conventional immune latex microsphere labeling and double-antibody sandwich method for detection, using a latex microsphere labeled anti-human myoglobin first monoclonal antibody as an immune detection label, using an anti-human myoglobin second monoclonal antibody which has specific immune binding characteristics matched with the anti-human myoglobin first monoclonal antibody on human myoglobin as a capture agent, coating the anti-human myoglobin second monoclonal antibody on a nitrocellulose membrane in a non-labeled state, binding the human myoglobin with the immune detection label through the immune detection label, combining the nitrocellulose membrane with the capture agent, capturing and fixing the combination of the nitrocellulose membrane and the capture agent, and calculating the antigen concentration of a sample by measuring the capture amount of the latex microsphere label. Specifically, a 2.0ml tube is taken, 1ml of primary washing liquid (10 mM MES PH 5.5, T20.05%) is added, red latex microsphere stock solution with the particle size of 300nm of 12.5 microliter biological company is added, and the mixture is uniformly mixed and centrifuged at 10000rpm for 20min; preparing 50mg/ml EDC and NHS solution by using the primary washing solution, centrifuging, removing supernatant, adding 750 microliters of the primary washing solution, carrying out ultrasonic dissolution, adding 100 microliters of EDC solution and 150 microliters of NHS solution, mixing uniformly, activating at 37 ℃ for 15min, centrifuging at 10000rpm for 20min, centrifuging, removing supernatant, adding 1ml of coupling buffer solution (10 mM MES PH 5.0, PC300 0.04%) for ultrasonic mixing, centrifuging at 10000rpm for 20min, removing supernatant after centrifugation, adding 1ml of coupling solution for ultrasonic mixing, adding 50ug of first monoclonal antibody of human cardiac troponin I, coupling at 37 ℃ for 2h, carrying out ultrasonic mixing at 10000rpm for 20min, centrifuging, removing supernatant, adding 1ml of sealing solution for ultrasonic mixing, sealing at 37 ℃ for 30min, centrifuging at 10000rpm for 20min, removing supernatant after centrifugation, adding 1ml of final washing solution for ultrasonic mixing uniformly, centrifuging at 10000rpm for 20min, removing supernatant, adding 1ml of reconstituted solution (10 mM reconstituted solution for standby, PH8.5% Tween sodium, 0.4% for storage, 10% trehalose for ultrasonic mixing uniformly), and adding 10% aqueous solution of trehalose for ultrasonic mixing uniformly.
3. Preparing a reagent strip:
conventional control group: a latex microsphere combination pad, a hemocyte filtration membrane, a nitrocellulose membrane and absorbent paper are sequentially adhered to the PVC support bottom sheet. Coating the latex microsphere marker on a colloidal gold bonding pad of a glass cellulose membrane, starting a membrane printing instrument, starting pressurized nitrogen, loading a latex microsphere marker solution, starting to spray a membrane coating, and setting membrane printing conditions as follows: the moving speed of the spray pen is 30 mm/s, and the propelling speed of the liquid is 5.0 mu l/cm; and (3) putting the printed film into a drying box, drying for 6 hours at 37 ℃, and then putting the film into a sealed bag containing a drying agent for storage. The capture antibody of the detection line T is a non-labeled paired anti-human myoglobin second monoclonal antibody of 1.0mg/ml, and is coated on the middle section of the nitrocellulose membrane; the goat anti-mouse IgG polyclonal antibody with the quality control line C of 1.0mg/ml is coated at the far end of the nitrocellulose membrane and is used for capturing the latex microsphere labeled anti-human cardiac troponin I monoclonal antibody which is not captured by specificity.
Experimental group of the invention: and a hemocyte filtering membrane, a nitrocellulose membrane and absorbent paper are sequentially adhered to the PVC supporting bottom sheet. The experimental group of the invention subpackages 3.0 mul/tube of the latex microsphere marker into 2ml preservation tubes, freezes and dries, and the preservation is used at 4 ℃. The capture antibody of the detection line T is a non-labeled paired anti-human cardiac troponin I second monoclonal antibody of 1.0mg/ml, and is coated in the middle section of the nitrocellulose membrane; the goat anti-mouse IgG polyclonal antibody with the quality control line C of 1.0mg/ml is coated at the far end of the nitrocellulose membrane and is used for capturing the colloidal gold labeled anti-human cardiac troponin I monoclonal antibody which is not captured specifically.
Preparing a reagent strip: starting a dehumidifier to reduce the humidity in the operating room to below 25%, placing the adhered detection sheet on a slitting machine, and cutting into reagent strips with the thickness of 4.0 mm. And (3) putting the reagent strip into an aluminum amber sealing bag with a drying agent, sealing the bag on a sealing machine, and labeling for later use.
4. The experimental method comprises the following steps:
preparation of human cardiac troponin I solution: a human cardiac troponin I solution of known concentration was taken and diluted with a sample dilution buffer (1% BSA,100mM glycine, 50mM PBS, 150mM NaCl, pH 7.4) to prepare a human cardiac troponin I solution of 5.0 ng/ml.
Experimental groups: the prepared human cardiac troponin I solution is added into the prepared latex microsphere marker tube by 50 ul/tube to form a marker mixed solution. Taking the prepared reagent strip of the invention, dropwise adding 50ul of prepared marker mixed solution on a hemocyte filtration membrane, standing for 1 minute, dropwise adding 25ul of diluent on the hemocyte filtration membrane, standing for 30 seconds, dropwise adding 25ul of diluent, standing for 30 seconds, and placing on a colorimetric quantitative analyzer to read colorimetric values of 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
Control group: and (3) taking the prepared conventional coated reagent strip, dropwise adding a prepared detection mixed solution of 50ul of human cardiac troponin I solution and 50ul of latex microsphere complex solution to a blood cell filtering membrane, standing, and reading colorimetric values for 2, 3, 4, 5, 8, 10, 15 and 20 minutes on a colorimetric quantitative analyzer.
5. Results of the experiment
The latex microsphere marker and the reagent strip are in a combined state of being separately set, the latex microsphere marker and a sample to be detected are firstly mixed for a first-step reaction, the reagent strip is not provided with a conventional latex microsphere combination pad, and a nitrocellulose membrane positioned on the reagent strip is used as a dispersion carrier for detection reaction. The experimental results are shown in Table 2, the chroma signal value is basically stable after the conventional method detects for 15 minutes, and the chroma signal value is stable after the method detects for 4 minutes, which shows that the detection time of the latex microsphere marker can be shortened and the detection sensitivity can be improved.
TABLE 2 comparison of the present invention with conventional latex microsphere labeling experiments
Group detection reaction time (min)/(color value)
2 3 4 5 8 10 15 20
Experimental group 1 15 95 118 117 119 120
Control group 12 36 68 109 112。
Experiment three: fluorescence immunochromatography heart-type fatty acid binding protein rapid detection comparison experiment:
1. the invention discloses a material for a combined detection structure experiment:
the experimental fluorescence detector adopts a blue Bo AFS-100 dry type fluorescence immunoassay analyzer, and the rest is the same as 'experiment one'.
2. Preparing an immunodetection marker:
the method comprises the steps of adopting a preparation method of an experiment II immunodetection marker, using fluorescent microspheres as an indicator to mark a first monoclonal antibody of anti-human heart fatty acid binding protein, using a second monoclonal antibody of the anti-human heart fatty acid binding protein, which has specific immunological binding characteristics matched with the first monoclonal antibody, of the anti-human heart fatty acid binding protein as a capture agent, coating the capture agent on a nitrocellulose membrane in a non-labeled state, binding the human heart fatty acid binding protein with the immunodetection marker, binding the capture agent with the nitrocellulose membrane through the nitrocellulose membrane, capturing and fixing, and measuring the capture amount of the fluorescent microsphere marker to calculate the antigen concentration of a sample. Specifically, fluorescent microspheres with the particle size of 300nm of Toyobo company are adopted, the adding amount of the first monoclonal antibody of the anti-human heart fatty acid binding protein is 30ug/ml, and the compound solution is 10mM Tris PH8.5, 0.5% sodium caseinate and 10% trehalose aqueous solution.
3. Preparing a reagent strip:
conventional control group: the preparation method is characterized in that a conventional control group reagent strip of experiment two is prepared by adopting a fluorescent microsphere binding pad, a fluorescent microsphere marker and a second monoclonal antibody of the anti-human heart fatty acid binding protein. The nitrocellulose membrane coated antibody is a second monoclonal antibody of non-labeled paired anti-human heart fatty acid binding protein with 1.2 mg/ml.
Experimental group of the invention: the preparation method of the reagent strip of the conventional control group of experiment two is used, the fluorescent microsphere marker and the second monoclonal antibody of the anti-human heart fatty acid binding protein are used for preparation, a fluorescent microsphere binding pad is not used, the fluorescent microsphere marker is subpackaged into 2ml of storage tubes by 3.0 mu l/tube, and the reagent strip is freeze-dried and stored for use at 4 ℃. The nitrocellulose membrane coated antibody is a second monoclonal antibody of a non-labeled paired anti-human heart fatty acid binding protein with 1.2 mg/ml.
Preparing a reagent strip: the same as experiment two.
4. The experimental method comprises the following steps:
preparing a human heart-type fatty acid binding protein solution: a human heart-type fatty acid-binding protein solution of known concentration was diluted with a sample dilution buffer (1% BSA,100mM glycine, 50mM PBS, 150mM NaCl, pH 7.4) to prepare a 10.0ng/ml human heart-type fatty acid-binding protein solution.
Experimental groups: the prepared human heart-shaped fatty acid binding protein solution is added into the prepared fluorescent microsphere marker tube by 50ul per tube to form marker mixed solution. Taking the prepared reagent strip, dropwise adding the prepared marker mixed solution to a blood cell filtering membrane for 50ul, standing for 1 minute, dropwise adding a diluent for 25ul to the blood cell filtering membrane, standing for 30 seconds, dropwise adding the diluent for 25ul, standing for 30 seconds, and placing on a fluorescence detector to read colorimetric values of 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
Control group: taking 50ul of prepared human heart-shaped fatty acid binding protein liquid from the prepared conventional coated reagent strip, mixing the prepared human heart-shaped fatty acid binding protein liquid with 50ul of fluorescent microsphere redissolution to prepare mixed liquid, then dropwise adding 100ul of the mixed liquid onto a hemocyte filtering membrane, standing, and placing on a fluorescence detector to read colorimetric values of 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
5. Results of the experiment
The fluorescent microsphere marker and the reagent strip are in a combined state of being separately set, the fluorescent microsphere marker and a sample to be detected are firstly mixed for a first-step reaction, the reagent strip is not provided with a conventional fluorescent microsphere binding pad, and a nitrocellulose membrane positioned on the reagent strip is used as a dispersion carrier for detection reaction. The experimental result is shown in table 3, the fluorescence signal value is basically stable after the conventional method detects for 15 minutes, and the fluorescence signal value is basically stable after the method detects for 4 minutes, which shows that the detection time can be shortened and the detection sensitivity can be improved by the fluorescent microsphere marker method.
TABLE 3 comparison of the present invention with conventional fluorescent microsphere labeling experiments
Group detection reaction time (minute)/(luminescence value)
2 3 4 5 8 10 15 20
Experimental group 189 2972 3862 4022 4103 4121 4098 4172
Control group 102 157 275 1693 3506 4027 4321 4433 。
Experiment four: the invention relates to a comparison experiment for fast detecting creatine kinase isoenzyme (CKMB) of a biotin/avidin system, which comprises the following steps:
1. the invention discloses a material for a combined detection structure experiment:
the same as the experiment I.
2. Preparing an immunodetection marker:
the detection method adopts a conventional immune colloidal gold labeling and double-antibody sandwich method, uses a colloidal gold particle labeled anti-human creatine kinase isoenzyme B monoclonal antibody (Au-CKBmAb) as an immune detection label, uses a biotin labeled anti-human creatine kinase isoenzyme M monoclonal antibody (Bio-CKMmAb) as a bridge connection label, uses non-labeled streptavidin coated on a nitrocellulose membrane as a capture agent, combines a B subtype in the CK-MB to be detected with the immune detection label Au-CKBmAb to form Au-CKBmAb-CKMB, then combines with the biotin labeled Bio-CKMmAb to form Au-CKBmAb-CKMmAb-Bio, and combines with Streptavidin (SV) through the nitrocellulose membrane to form Au-CKBmAb-CKMmAb-Bio-SV which is captured and fixed, and calculates the concentration of the sample CKMB by measuring the capture amount of the colloidal gold label. The method comprises the following specific operations:
preparing a colloidal gold marker: adding 1ml of colloidal gold solution with the particle size of 50nm into a 1.5ml centrifuge tube, adding 3.0ul 0.1M potassium carbonate, uniformly stirring, adding 10ug of anti-human creatine kinase isoenzyme B monoclonal antibody, reacting for 10min, adding 20-20% BSA 1 ul/ml, reacting for 10min, centrifuging for 15min at 10000r/min, removing supernatant, suspending the precipitate with 1ml of colloidal gold complex solution (30 mM Tris, 3% trehalose, 3% sucrose and 0.25% BSA), centrifuging for 15min at 10000r/min, removing supernatant, suspending the precipitate with the colloidal gold complex solution again, wherein the final volume is 0.5ml, subpackaging into 2ml storage tubes by 2.0 mul/tube, freeze-drying, and storing at 4 ℃ for use. Preparing a colloidal gold bonding pad from another part of the glass cellulose membrane, coating a colloidal gold marker on the colloidal gold bonding pad, starting a membrane printing instrument, starting pressurized nitrogen, loading a colloidal gold marker solution, starting membrane spraying and coating, and setting membrane printing conditions as follows: the moving speed of the spray pen is 30 mm/s, and the propelling speed of the liquid is 5.0 mu l/cm; and (3) putting the printed film into a drying box, drying for 6 hours at 37 ℃, and then putting the film into a sealed bag containing a drying agent for storage.
Preparation of biotin marker: taking an anti-human creatine kinase isoenzyme M monoclonal antibody, and diluting the monoclonal antibody to 1.0mg/ml by using 10mM PBS PH7.4; activated biotin (Sigma) was taken, weighed in an EP tube, dissolved in 10mM PBS PH7.4 to a final concentration of 20mM; adding 13.3ul of 20mM biotin into each 2mg of the monoclonal antibody of the anti-human creatine kinase isoenzyme M, uniformly mixing, and reacting at room temperature for 60min; after the reaction is finished, dialyzing with 10mM PBS PH7.4, wherein the volume of the dialyzate is 200 times that of the monoclonal antibody, changing the dialyzate for 3 times, subpackaging with 2.0 mu l/tube into 2ml preservation tubes, freeze-drying, and preserving at 4 ℃ for use. Preparing a biotin combined pad from another part, coating the biotin marker on the glass cellulose membrane biotin combined pad by adopting the method, and storing in a sealed bag for use.
3. Preparation of a coated solid-phase membrane:
streptavidin (Sigma) was taken and diluted with 50mM phosphate buffer pH7.4 to prepare a streptavidin-coated solution at a concentration of 100 ug/ml. Starting a film printing instrument, loading 100ug/ml of coating liquid, taking a PVC sheet pasted with a nitrocellulose membrane with the width of 20mm, starting scribing and printing the film, and setting the film printing conditions as follows: the moving speed of the spray pen is 10 mm/s, and the propelling speed of the liquid is 1.5 mul/cm. And (3) putting the sprayed film into a drying oven at 37 ℃, drying for 6 hours, and then putting the film into a drying container containing a drying agent for storage and use.
4. Assembling a semi-finished product:
the same as the experiment I. Conventional control group: a hemocyte filtering membrane, a colloidal gold combination pad and a biotin combination pad are sequentially adhered to the near end of the nitrocellulose membrane coated with the solid-phase membrane, and a water-absorbing paper pad is adhered to the far end. Experimental group of the invention: the near end of the nitrocellulose membrane coated with the solid phase membrane is adhered with a blood cell filtering membrane, and the far end is adhered with a water absorption paper pad. Starting a dehumidifier to reduce the humidity in the operating room to below 25%, placing the adhered detection sheet on a slitting machine, and cutting into reagent strips with the thickness of 4.0 mm. And (3) putting the reagent strips into an aluminum foil sealing bag with a drying agent, sealing the bag on a sealing machine, and labeling for later use.
6. The experimental method comprises the following steps:
preparation of human CKMB solution: human CKMB solution of known concentration was diluted with sample dilution buffer (1% BSA,100mM glycine, 50mM PBS, 150mM NaCl, pH 7.4) to prepare 10.0ng/ml human CKMB solution. .
Experimental groups: a colloidal gold marker and a biotin marker were mixed (the amount of the labeled antibody was mixed at a ratio of about 7 in terms of the yield of the marker). The prepared human CKMB solution is added into the prepared marker mixed solution pipe by 50 ul/pipe to form CKMB marker mixed solution.
Taking the prepared experimental reagent strip, dropwise adding 50ul of the prepared CKMB marker mixed solution on the hemocyte filtration membrane, standing for 1 minute, dropwise adding 25ul of diluent on the hemocyte filtration membrane, standing for 30 seconds, dropwise adding 25ul of diluent, standing for 30 seconds, and placing on a colorimetric quantitative analyzer to read colorimetric values of 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
Control group: taking the prepared conventional coated reagent strip, taking 50ul of the prepared human CKMB solution, mixing the prepared human CKMB solution with 50ul of the colloidal gold redissolution to prepare a mixed solution, dropwise adding 100ul of the mixed solution onto a hemocyte filtration membrane, standing, and placing on a chromaticity detector to read chromaticity values for 2, 3, 4, 5, 8, 10, 15 and 20 minutes.
6. Results of the experiment
The colloidal gold marker and the reagent strip are in a combined state of being separately set, a biotin/avidin detection system is adopted, streptavidin is used as a capture agent to detect the human CKMB level, the result is shown in Table 4, the fluorescence signal value is basically stable after the conventional method detects for 15 minutes, and the fluorescence signal value is basically stable after the method detects for 4 minutes, which shows that the detection time can be shortened, the detection sensitivity can be improved, and the method is simultaneously suitable for the biotin/avidin detection system.
TABLE 4 comparison of fast CKMB detection experiment by immunocolloidal gold method
Structure type detection reaction time (minute)/(colorimetric value)
2 3 4 5 8 10 15 20
Conventional structure 25 11 53 79 91 102 107
Structure 2 48 97 102 112 108 110 of the present technology
。

Claims (7)

1. A myocardial marker rapid detection kit for combined detection is characterized by comprising: detect discount card (1), cardiac marker specificity reagent strip (2), cardiac marker specificity immunodetection marker (3), immunoassay tube (4), diluent (5), diluent pipe (6) and quantitative determination appearance (7), wherein:
the detection buckle (1), the immunity detection tube (4), the diluent tube (6) and the quantitative detector (7) are different combined structures which are independently arranged; the detection buckle card (1) comprises a detection shell (8) and the myocardial marker specific reagent strip (2) positioned in the detection shell (8); the myocardial marker specific reagent strip (2) consists of a supporting negative plate (9), a nitrocellulose membrane (10) and absorbent paper (11), wherein the nitrocellulose membrane (10) is sequentially adhered to the supporting negative plate (9) and is coated with a myocardial marker specific antihuman myocardial marker secondary antibody; the myocardial marker specific immunodetection marker (3) is a first anti-human myocardial marker antibody marked by an indicator, and is loaded in the immunodetection tube (4), wherein the indicator is a colored microsphere and comprises at least one of a colloidal gold particle and a latex particle; the diluent (5) is a water-soluble buffer salt solution and is loaded in the diluent pipe (6); the quantitative detection instrument (7) is a quantitative detection unit of the rapid detection kit for the cardiac marker, and comprises at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer; the detection shell (8) comprises an upper layer structure (12) and a lower layer structure (13), wherein a sample adding hole (14) and an observation window (15) are formed in the upper layer structure (12), the lower layer structure (13) is provided with a bracket (16) for placing the myocardial marker specific reagent strip (2), the sample adding hole (14) corresponds to the near end of the nitrocellulose membrane (10), and the observation window (15) corresponds to the far end of the nitrocellulose membrane (10).
2. The rapid cardiac marker detection kit according to claim 1, wherein a blood cell filtering structure (19) is adhered to the support base sheet (9) and connected to the proximal end of the nitrocellulose membrane (11), and the blood cell filtering structure (19) comprises at least one of a blood cell filtering membrane or a membrane pad treated with erythrocyte antibodies.
3. The combined detecting structure of claim 1, wherein the supporting bottom sheet (9) of the detecting reagent strip is adhered with a liquid phase dispersion film (20) connected with the proximal end of the nitrocellulose membrane, wherein the liquid phase dispersion film (20) is preferably a glass cellulose membrane or a poly-ester cellulose membrane, and the position of the detecting reagent strip corresponding to the sample adding hole is the liquid phase dispersion film.
4. The rapid myocardial marker detection kit according to claim 1, wherein the rapid myocardial marker detection kit is provided with a quantitative detection instrument (7) comprising at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer.
5. The rapid cardiac marker assay kit of claim 1, wherein the cardiac marker comprises at least one of cardiac troponin I, cardiac troponin T, cardiac fatty acid binding protein, myoglobin, creatine kinase isozyme, myeloperoxidase, N-terminal pro-brain natriuretic peptide.
6. The rapid detection kit for cardiac markers according to claim 1, which is operated in a blood cell filtration membrane structure mode, and comprises the following steps:
1) Taking out the detection buckle card containing the blood cell filtering membrane, and horizontally placing the detection buckle card on a table top;
2) Collecting a blood sample to be detected, adding the blood sample into an immune detection tube (4) containing the anti-human myocardial marker first antibody immune detection marker (3), adding a proper amount of diluent, and mixing to prepare a sample mixed solution to be detected;
3) Adding the sample mixed solution to be detected to a hemocyte filtering membrane through a sample adding hole, and enabling the mixed solution to flow through the hemocyte filtering membrane, a nitrocellulose membrane and a water absorption pad forwards;
4) Adding the diluent to a hemocyte filtering membrane through a sample adding hole, and enabling the diluent to flow through the hemocyte filtering membrane, a nitrocellulose membrane and a water absorption pad forwards;
5) And detecting the reading result from the observation window to finish the detection.
7. The use of the rapid detection kit for cardiac markers according to claim 1 in the development of human cardiac marker immunodetection reagent products.
CN202211196973.8A 2022-09-29 2022-09-29 Rapid detection kit for cardiac marker for combined detection and application thereof Pending CN115524483A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117165416A (en) * 2023-11-03 2023-12-05 苏州雅睿生物技术股份有限公司 Dilution tube, nucleic acid detection device and detection method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117165416A (en) * 2023-11-03 2023-12-05 苏州雅睿生物技术股份有限公司 Dilution tube, nucleic acid detection device and detection method thereof
CN117165416B (en) * 2023-11-03 2024-01-26 苏州雅睿生物技术股份有限公司 Dilution tube, nucleic acid detection device and detection method thereof

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