CN117420300A - Immunochromatography detection technology without mediated release through binding pad and application thereof - Google Patents

Immunochromatography detection technology without mediated release through binding pad and application thereof Download PDF

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CN117420300A
CN117420300A CN202210809240.0A CN202210809240A CN117420300A CN 117420300 A CN117420300 A CN 117420300A CN 202210809240 A CN202210809240 A CN 202210809240A CN 117420300 A CN117420300 A CN 117420300A
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detection
nitrocellulose membrane
sample
indicator
loading
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刘凤鸣
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Jiaxing Kangyuan Ketai Technology Development Co ltd
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Jiaxing Kangyuan Ketai Technology Development Co ltd
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract

The invention discloses an immunochromatography detection technology without mediated release through a bonding pad and application thereof. The technology is suitable for the rapid clinical detection of various immunochromatography such as an immune colloidal gold method, a fluorescent immunochromatography method, a latex particle immunochromatography method and the like, has various characteristics of high detection efficiency, convenience, accuracy, rapidness and the like, and has important clinical significance.

Description

Immunochromatography detection technology without mediated release through binding pad and application thereof
Technical Field
The invention relates to the technical field of medical instruments, in particular to an immunochromatography detection technology without mediated release through a bonding pad 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 design of the immunological principle, and is widely used for samples which are derived from human bodies and animal bodies and can be used for disease diagnosis and health detection and for environmental, pharmaceutical analysis, food and industrial analysis. The common methods include immunonephelometry, immunochromatography, enzyme-linked immunosorbent assay, time-resolved immunofluorescence, quantum dot immunofluorescence chromatography, latex-enhanced immunonephelometry, fluorescence immunochromatography, colloidal gold immunochromatography, fluorescence immunochromatography, latex immunochromatography, and the like. High sensitivity, rapidness, convenience, miniaturization, full quantification and automation are the development trend of the current clinical immunodetection technology.
The immunonephelometry technique, also called immunonephelometry, is a technique in which soluble antigens and antibodies are specifically combined in a liquid phase to generate a complex with a certain size, refraction or absorption of light is formed, transmitted light or scattered light after refraction or absorption is measured as a calculation unit, and the quantitative detection is performed, but the detection sensitivity is low and the method is not suitable for trace detection. The immunochromatography assay technology is based on solid-phase membranization of an antigen or an antibody and an indicator mark of the antigen or the antibody, color microspheres such as colloidal gold particles, latex microspheres and the like, fluorescent substances such as fluorescent microspheres, fluorescein and the like, the antigen or the antibody bound on the surface of a solid-phase carrier keeps the immunological activity, and an indicator mark conjugate of the antigen or the antibody keeps the immunological activity and also keeps the indicator characteristic. The immunochemistry detection technology is a high-sensitivity micro and trace analysis technology, has the remarkable advantages of convenient operation, high sensitivity, wide linear response range, easy automation realization and the like, is widely applied to environment, clinical, pharmaceutical analysis, food and industrial analysis, is also a solid-phase separation means based on antigen or antibody and a luminescent reagent labeling technology of antigen or antibody, but has long detection reaction time and high requirements on detection equipment and also affects the use of the detection technology. The ELISA technique, the time-resolved immunofluorescence technique and the latex-enhanced turbidimetric immunoassay are also commonly used detection techniques, but the prior art has the corresponding defects of long detection reaction time or sensitivity and accuracy deficiency, so that the detection technique with high sensitivity, rapidness, miniaturization, full quantification, automation, simple and convenient operation, rapid use and low cost is developed, the medical quality and efficiency are improved, and the detection technique has important clinical significance and application value.
Disclosure of Invention
The invention aims to provide a technology for rapid immunochromatography detection and application thereof, and compared with the prior art, the technology has the advantages of high detection sensitivity, rapidness, low cost and the like, and improves the detection quality.
In view of the above, the present invention provides an immunochromatography detection technique not mediated by a binding pad, comprising a chromatography detection structure, a detection reagent strip positioned in the detection structure, a nitrocellulose membrane positioned on the reagent strip, and an indicator marker, a loading structure, a detection assisting liquid and a loading structure which are combined, wherein the nitrocellulose membrane comprises a nitrocellulose membrane and a membrane structure material similar to the nitrocellulose membrane, and is characterized in that:
1) The detection reagent strip is not provided with a binding pad for mediating the dispersion and release of the indicator marker which is conventionally used, and consists of a nitrocellulose membrane and a water absorption pad which are sequentially stuck on a support bottom sheet;
2) The nitrocellulose membrane is characterized by being used as a detection structure of the carrier of the indicator marker dispersed release carrier and the carrier of the capture agent coating carrier, the indicator marker is a first immune conjugate of the indicator marker specificity of the to-be-detected object, the capture agent is a second immune conjugate which is not marked and has specific pairing binding characteristics with the first immune conjugate, the indicator marker is coated on the proximal end of the nitrocellulose membrane, and the capture agent is coated on the middle section or the distal end of the nitrocellulose membrane; the immunoconjugate comprises at least one of an antibody, an antigen, biotin, avidin, and analogs thereof;
3) The chromatographic detection structure comprises a detection shell and a detection reagent strip positioned in the detection shell, wherein a sample adding hole and an observation window are formed in the upper layer of the shell, the sample adding hole corresponds to the near-end part of the nitrocellulose membrane, and the observation window corresponds to the middle section and the far-end part of the nitrocellulose membrane;
4) When in use, firstly, the indicator marker is mixed with a liquid phase sample to be detected, loaded to the starting end of the nitrocellulose membrane, loaded once or repeatedly loaded more than once, and flows through the nitrocellulose membrane and the water absorption pad to start a first-stage detection reaction;
5) Then loading a detection aid which does not contain the indicator marker and the sample to be detected to the starting end of the nitrocellulose membrane, loading once or repeatedly loading more than once, and starting a second-stage detection reaction;
6) The detection process is completed by observing the amount of the indicator marker captured on the nitrocellulose membrane and reading the detection result.
A first-stage detection reaction, wherein a to-be-detected object is specifically captured by a capture agent coated and fixed on the nitrocellulose membrane; a second detection reaction for removing the indicator remaining on the nitrocellulose membrane and not specifically bound; the nitrocellulose membrane and the similar membrane structure thereof comprise one or more combinations of nitrocellulose membranes, polyvinylidene fluoride membranes, nylon membranes and DEAE cellulose membranes. The color microsphere comprises at least one of colloidal gold particles and latex particles. The chromatographic detection structure comprises at least one of an immune colloidal gold method, a fluorescent immunochromatography method and a latex particle immunochromatography method which are used in the current market.
In the immunochromatography detection technology, the indicator in the indicator marker is at least one of color microspheres and fluorescent microspheres, and the form of the indicator marker in the indicator marker loading structure is preferably freeze-dried powder.
In the immunochromatography detection technology, a blood cell filtering structure is stuck on a supporting bottom sheet of the detection reagent strip and is connected with the proximal end of the nitrocellulose membrane, the blood cell filtering structure comprises at least one of a blood cell filtering membrane and a membrane pad treated by erythrocyte antibodies, the blood cell filtering structure is positioned at a position corresponding to the sample adding hole, and a detection liquid phase is directly loaded on the blood cell filtering structure for detection reaction.
In the immunochromatography detection technique described above, the nitrocellulose membrane is not coated with the indicator label, and the indicator label is entirely loaded in the indicator label loading structure.
In the immunochromatography detection technology, a liquid-phase dispersion film is stuck on a supporting bottom sheet of the detection reagent strip, is connected with the proximal end of the nitrocellulose film and is positioned at a position corresponding to the sample adding hole of the chromatography detection structure, and the detection liquid phase is directly loaded onto the liquid-phase dispersion film for detection reaction. The liquid-phase dispersion film is preferably a glass cellulose film or a polyester cellulose film.
In the immunochromatography detection technology, the preparation of the detection reagent strip further comprises a biotin/avidin reaction system, the first antibody marked by the indicator and the second antibody marked by the biotin are arranged in the indicator marker loading structure, and the nitrocellulose membrane is coated with non-marked avidin and analogues thereof.
In the immunochromatography detection technology, the rapid immunochromatography detection technology is provided with a quantitative detection instrument, and the quantitative detection instrument comprises at least one of a chromaticity quantitative analyzer and a fluorescence quantitative analyzer.
In the immunochromatographic detection technology, the operation of the rapid immunochromatographic detection technology comprises the following steps:
1) Taking out the chromatographic detection structure, and horizontally placing the chromatographic detection structure on a plane;
2) Collecting a blood sample to be detected, adding the blood sample to be detected into a loading tube containing an indicator marker, adding a proper amount of auxiliary detection liquid, and mixing to prepare a sample mixed liquid to be detected;
3) Adding the sample mixed solution to be detected into a blood cell filtering structure through a sample adding hole, and allowing the sample mixed solution to flow through the blood cell filtering structure, a nitrocellulose membrane and a water absorption pad forwards, and repeating the sample for more than one time;
4) Adding the auxiliary detection liquid to the blood cell filtering structure through the sample adding hole, and allowing the auxiliary detection liquid to flow forward through the blood cell filtering structure, the nitrocellulose membrane and the water absorbing pad, and repeating the sample more than once;
5) And detecting the read result from the observation window to finish detection.
Use of the rapid immunochromatographic assay technique of claim 1 in the development of an immunoassay reagent product.
Due to the adoption of the technical scheme, the invention has the following advantages:
1. according to the invention, an immunochromatography detection technology without mediated release of a binding pad is designed, the immunochromatography detection technology is reduced from the existing detection structure requiring the loading and release of the marker by the marker binding pad to the detection reaction completed by using the nitrocellulose membrane and the absorbent paper pad, so that the detection cost is reduced, the detection time is shortened, the timeliness of clinical detection of an immunodetection technology product and the feasibility of popularization and popularization are effectively improved, and the clinical diagnosis and treatment effect is improved.
2. The invention is characterized in that a hemocyte filter membrane or a membrane pad structure treated by erythrocyte antibodies is attached to the front of the nitrocellulose membrane, and the membrane pad structure can be used for one-step detection of whole blood, thereby improving the convenience and practicality of detection and use.
3. According to the invention, the mixed solution of the sample to be detected containing the indicator marker is firstly loaded, and then the step-by-step structure using state of the auxiliary detection solution without the indicator 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 indicator marker on the nitrocellulose membrane is effectively reduced, and the detection efficiency is improved.
4. The invention provides the quantitative detector which simultaneously comprises at least one of a chromaticity quantitative analyzer and a fluorescence quantitative analyzer, can be used for immunity quantitative detection in various environments, and improves the clinical application value.
5. The invention has simple operation steps, convenient use, reduced cost of raw materials, and obviously improved working efficiency, and can be applied to various fields of medical treatment, environment, food and the like.
Drawings
FIG. 1 is a schematic flow chart of the detection technique of the present invention;
FIG. 2 is a schematic diagram of the basic structure of the indicator label of the present invention loaded onto a test reagent strip;
FIG. 3 is a schematic diagram of the basic structure of the test strip loaded with a liquid phase of the present invention;
FIG. 4 is a schematic diagram of a blood cell filtration structure of a test strip of the present invention;
FIG. 5 is a schematic diagram of the chromatographic detection structure of the present invention.
The figures are labeled as follows:
nitrocellulose membrane 1; an indicator marker coated region 2; detection line (capture agent) 3; a quality control line 4; a water absorbing pad 5; a support backsheet 6; freeze-dried indicator marker powder 7; an indicator marker loading structure 8; a liquid phase traffic mediating structure 9; a liquid phase for aiding examination 10; a test-aid liquid-phase loading structure 11; a hemocyte filtration structure 12; a chromatography detection structure upper cover 13; a chromatography detection structure base 14; a sample addition hole 15; a viewing window 16.
Detailed Description
In order to further illustrate the technical means and effects adopted by the present invention to achieve the preset purpose, the following embodiments are used for further illustrating the present invention with reference to the accompanying drawings, but the present invention is not limited to the following description.
As shown in fig. 1, the technical flow diagram of the invention is that a PVC support film is taken, a nitrocellulose film is stuck, a water absorbing paper pad is stuck, an indicator marker is coated on the nitrocellulose film, a capturing agent is coated on the nitrocellulose film, and a chromatography detection structure is assembled; preparing indicator marker freeze-dried powder, adding a liquid phase sample to be detected to prepare a mixed liquid to be detected, and adding a chromatographic detection structure to start a first-stage detection reaction; preparing a detection assisting liquid, adding the detection assisting liquid into the chromatographic detection structure, starting a second-stage detection reaction, reading a detection result, and finishing detection.
As shown in fig. 2 and 3, the basic structure of the detection reagent strip of the present invention is that a nitrocellulose membrane 1 and a water absorbing pad 5 are sequentially adhered to a PVC support backsheet 6 from the near to the far, the nitrocellulose membrane 1 is not provided with an indicator marker binding pad adopted in the prior art, and a detection line (capturing agent) 3 for a to-be-detected object, a quality control line 4 and an indicator marker coating area 2 are provided, wherein the indicator marker coating area 2 can be used as an area for coating and dispersing and bearing the indicator marker at the same time, or can only be used for dispersing and bearing the indicator marker without coating the indicator marker thereon. The indicator marker loading structure comprises an indicator marker freeze-dried powder 7, a sample to be tested, an indicator marker loading tube 8 and a liquid phase traffic mediating structure 9. The liquid-assisted detection loading structure comprises a liquid-assisted detection 10, a liquid-assisted detection loading pipe 11 and a liquid-phase traffic mediating structure 9. When the detection method is used, firstly, a liquid phase sample to be detected is added into an indicator marker loading tube 8 and is mixed with an indicator marker freeze-dried powder 7 to prepare a mixed liquid to be detected, the mixed liquid is added into a chromatographic detection structure through a liquid phase traffic mediating structure 9 to start a first-stage detection reaction, then, a detection aid is directly added into the chromatographic detection structure to start a second-stage detection reaction, and the detection result is read by observing the captured quantity of the indicator marker on the nitrocellulose membrane in a capture agent 3 coating area, so that the detection process is completed.
As shown in FIG. 4, the detection reagent strip of the present invention comprises a hemocyte filtration structure, a hemocyte filtration membrane 12, a nitrocellulose membrane 1 and a water absorption pad 5 are sequentially adhered on a PVC support bottom plate 16 from the near to the far, and no indicator marker binding pad adopted in the prior art is arranged, so that the chromatographic detection structure, the indicator marker loading structure and the auxiliary detection liquid phase loading structure are used in the flow, the mixed liquid of the sample to be detected is directly loaded on the hemocyte filtration membrane 12, and the detection process is started and completed.
As shown in fig. 5, the chromatography detection structure of the present invention comprises a chromatography detection structure upper cover 13, a chromatography detection structure base 14, a detection reagent strip 16, an observation window 16 and a sample application hole 15 which are positioned on the chromatography detection structure upper cover 13, a detection line (capturing agent) 3 and a quality control line 4 which are positioned on the detection reagent strip 16.
In the practical operation, when the immunochromatography detection technology is a colloidal gold immunoassay structure, a detection reagent strip is prepared by a colloidal gold method, a hemocyte filtration membrane 12, a nitrocellulose membrane 1 and a water absorption pad 5 are sequentially adhered on a PVC negative film 6, a colloidal gold-marked object-to-be-detected specific antibody or antigen is arranged in an indicator marker loading structure 7, a non-marked matched object-to-be-detected specific antibody or antigen is coated on the nitrocellulose membrane 1, and after the detection reaction is finished, a detection result is quantitatively analyzed by a colorimetry quantitative analyzer; when the immunochromatography detection technology is prepared by a latex microsphere immunization method, a detection reagent strip is prepared by the latex microsphere immunization method, a hemocyte filtration membrane 12, a nitrocellulose membrane 1 and a water absorption pad 5 are sequentially adhered on a PVC negative film 6, a latex microsphere marked object to be detected specific antibody or antigen is arranged in an indicator marker loading structure 7, a non-marked matched object to be detected specific antibody or antigen is coated on the nitrocellulose membrane 1, and after the detection reaction is finished, a colorimetric quantitative analyzer is used for quantitatively analyzing a detection result quantitatively; when the immunochromatography detection technology is prepared by a fluorescent microsphere immunoassay method, a detection reagent strip is prepared by the fluorescent microsphere immunoassay method, a hemocyte filtration membrane 12, a nitrocellulose membrane 1 and a water absorption pad 5 are sequentially adhered on a PVC negative film 6, a fluorescent microsphere-marked object-to-be-detected specific antibody or antigen is arranged in an indicator marker loading structure 7, a non-marked matched object-to-be-detected specific antibody or antigen is coated on the nitrocellulose membrane 1, and after the detection reaction is finished, a fluorescent quantitative analyzer is used for quantitatively analyzing the detection result quantitatively.
Experimental study of the invention: the following experiments illustrate the detection method and the effect of the present invention, but are not limiting of the present invention. The experimental methods used in the following experiments are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available.
Experiment one: immune colloidal gold method blood insulin rapid detection comparison experiment:
1. preparing a detection reagent strip:
the detection reagent strip is prepared by adopting a double-antibody sandwich method by adopting a conventional immune colloidal gold detection technology, an insulin detection experiment is carried out by adopting the detection reagent kit prepared by adopting the immune chromatography detection technology and the detection reagent kit prepared by adopting the conventional detection method, wherein the colloidal gold mark of a detection line T of the detection reagent strip indicates that an antibody is an anti-insulin monoclonal antibody of 10ug/ml, colloidal gold particles with the particle size of 50nm are adopted, a complex solution of the colloidal gold mark antibody is 30mM Tris, 0.4% casein sodium, 0.1% Tween 20, 1% trehalose and 3% sucrose aqueous solution, and a coated non-marked antibody is diluted by adopting 50mM PBS (phosphate buffer saline) PH 7.4. The conventional method group experiment colloidal gold labeled antibody is coated on a glass cellulose membrane colloidal gold binding pad by 3 ul/detection reagent strip; the method comprises the steps of coating an experimental colloidal gold labeled antibody on a nitrocellulose membrane at the proximal end with 1 ul/detection reagent strip, adding 3 ul/tube into a freezing tube, and freeze-drying and preserving for later use; the capture antibody of the detection line T of the detection reagent strip is 1.0mg/ml of non-labeled anti-insulin monoclonal antibody with pairing binding property, and the non-labeled anti-insulin monoclonal antibody is coated on a nitrocellulose membrane pad; the capture antibody of the quality control line C of the detection reagent strip is a 1.0mg/ml unlabeled goat anti-mouse IgG polyclonal antibody, and the unlabeled goat anti-mouse IgG polyclonal antibody is coated on a nitrocellulose membrane pad and used for capturing a colloidal gold labeled anti-insulin monoclonal antibody which is not specifically captured. The conventional method comprises adhering a water absorption pad and a blood cell filter membrane pad at two ends of a nitrocellulose membrane pad, adhering a colloidal gold marker binding pad at one side of the blood cell filter membrane pad, and adhering a dispersion membrane pad with a glass cellulose membrane as matrix at one side of the colloidal gold marker binding pad. The method is characterized in that the two ends of the nitrocellulose membrane pad are respectively stuck with the water absorption pad and the blood cell filtering membrane pad, and the colloidal gold marker combination pad and the dispersion membrane pad are not stuck. Placing the stuck detection sheet on a slitter, and cutting into detection reagent strips with the thickness of 3.5 mm.
2. The immunochromatography detection technology of the invention is as follows:
a commercially available detection port card with an upper cover and a base is used as a chromatography detection structure, a 2ml preservation tube is used as an indicator marker loading structure and a detection assisting liquid phase loading structure for experiments, a cellulose nitrate membrane is adopted as a Sidoris CN140, a hemocyte filtration membrane is adopted as a FUSION 5 of Whatman, and a glass cellulose membrane is adopted as SB08 of Shanghai gold mark. The colorimetric quantitative analyzer adopts an European SkanFlexi multichannel immunochromatographic quantitative analyzer. The assay aid was an aqueous solution containing 30mM Tris pH8.5, 1% NP40, 1M sodium chloride, and 0.5% sodium caseinate.
3. Experimental method and results:
and in the experiment, the prepared detection reagent strip and immunochromatography detection technology are adopted, the assembled detection structure is placed into an aluminum foil sealing bag with a drying agent, and the aluminum foil sealing bag is sealed on a sealing machine and labeled. 10 uIU/ml of recombinant insulin glargine injection (100 IU/ml) was prepared with 10mM phosphate buffer salt solution. The experiment is divided into a conventional method detection and a method detection according to the invention. The detection is carried out by a conventional method, 50ul of the insulin difatty solution is directly dripped into a chromatographic detection structure sample adding window, and the sample is kept stand for 20 minutes, and during the detection, the colorimetric signal values are respectively measured once at 3, 5, 8, 10, 15 and 20 minutes by a colorimetric quantitative analyzer. The method detects, takes 50ul of insulin detention solution, adds the insulin detention solution into a colloidal gold marker tube, mixes the insulin detention solution evenly, takes 25ul of insulin detention solution, directly drops the insulin detention solution into a chromatographic detection structure sample adding window, starts timing, adds 25ul of insulin detention mixed solution at 1 minute, adds 25ul of auxiliary detection solution at 2 minutes, adds 25ul of auxiliary detection solution at 2.5 minutes, and the total standing time is 20 minutes, and the colorimetric signal values are measured once at 3, 5, 8, 10, 15 and 20 minutes respectively by using a colorimetric quantitative analyzer. As a result, the chroma signal value is basically stable after 15 minutes of detection by the conventional method, and the chroma signal value is basically stable after 4 minutes of detection by the method (Table 1), which shows that the detection time can be shortened and the detection sensitivity can be improved by the method.
TABLE 1 comparison of fast detection experiments of blood insulin by immune colloidal gold method
Detection result (signal value) of structural type detection line
3 5 8 10 15 20*
21 23 42 65 81 85 of conventional construction
The technical structure 62 87 90 93 93 97
* The reaction time (minutes) was measured.
Experiment II: emulsion microsphere immunochromatography insulin rapid detection comparison experiment:
1. preparing a detection reagent strip:
the detection reagent strip is prepared by adopting a double-antibody sandwich method of a conventional latex microsphere immunochromatography detection technology, the detection reagent kit is prepared by adopting the immunochromatography detection technology of the invention and the detection reagent kit prepared by adopting the conventional detection method is used for insulin detection experiments, wherein the latex microsphere mark of a detection line T of the detection reagent strip indicates that an antibody is an anti-insulin monoclonal antibody of 50ug/ml, the red microsphere with the biological particle size of 300nm is adopted, the latex microsphere mark antibody complex solution is an aqueous solution of 10mM Tris PH8.5, 0.4% casein sodium, 0.02% Tween 20 and 10% trehalose, and the coated non-marked antibody is diluted by using 50mM PBS (phosphate buffer salt solution) PH 7.4. The latex microsphere labeled antibody is coated on a glass cellulose membrane colloidal gold binding pad by 3 ul/detection reagent strips in the conventional method group experiment; the method comprises the steps of coating an experimental latex microsphere marked antibody on the near end of a nitrocellulose membrane with 1 ul/detection reagent strip, adding 3 ul/tube into a freezing tube, and freeze-drying and preserving for later use; the capture antibody of the detection line T of the detection reagent strip is 1.0mg/ml of non-labeled anti-insulin monoclonal antibody with pairing binding property, and the non-labeled anti-insulin monoclonal antibody is coated on the middle section position of the nitrocellulose membrane pad; the capture antibody of the quality control line C of the detection reagent strip is a 1.0mg/ml unlabeled goat anti-mouse IgG polyclonal antibody, and the capture antibody is coated on the far end position of the nitrocellulose membrane pad and is used for capturing the colloidal gold labeled anti-insulin monoclonal antibody which is not specifically captured. The conventional method comprises adhering water absorption pad and blood cell filter membrane pad at two ends of nitrocellulose membrane pad, adhering latex microsphere marker binding pad at one side of blood cell filter membrane pad, and adhering dispersion membrane pad with glass cellulose membrane as matrix at one side of latex microsphere marker binding pad. The method is characterized in that the two ends of the nitrocellulose membrane pad are respectively stuck with the water absorption pad and the blood cell filtering membrane pad, and the latex microsphere marker combination pad and the dispersion membrane pad are not stuck. Placing the stuck detection sheet on a slitter, and cutting into detection reagent strips with the thickness of 3.5 mm.
2. The immunochromatography detection technology of the invention is as follows:
prepared as in experiment one.
3. Experimental method and results:
the experimental method is the same as "experiment one".
As a result of the experiment, the chroma signal value is basically stable after 15 minutes of detection by the conventional method, and the chroma signal value is basically stable after 4 minutes of detection by the method (Table 2), which shows that the detection time can be shortened and the detection sensitivity can be improved by the method.
TABLE 2 comparison of blood insulin Rapid detection experiments by latex microsphere immunochromatography
Detection result (signal value) of structural type detection line
3 5 8 10 15 20*
11 23 52 71 89 96 of conventional construction
The technical structure 62 91 97 93 95 99
* The reaction time (minutes) was measured.
Experiment III: immunofluorescence method insulin rapid detection comparison experiment:
1. preparing a detection reagent strip:
a double antibody sandwich method and a biotin/avidin reaction system are adopted to prepare a detection reagent strip, the immunochromatography detection technology is adopted to prepare a detection reagent kit and the detection reagent kit prepared by the conventional detection method are adopted to carry out insulin detection experiments, wherein a fluorescent microsphere mark of the detection reagent strip indicates that an antibody adopts a 20ug/ml first anti-insulin monoclonal antibody, a biotinylation mark antibody adopts a 20ug/ml second anti-insulin monoclonal antibody, the fluorescent microsphere adopts a biological particle size of 300nm, a latex microsphere mark antibody complex solution is an aqueous solution of 10mM Tris PH8.5, 0.2% BSA, 0.4% casein sodium, 0.02% Tween 20 and 10% trehalose, and a coated non-marked antibody is diluted by using 50mM PBS (phosphate buffer saline) PH 7.4. The latex microsphere labeled antibody is coated on a glass cellulose membrane colloidal gold binding pad by 3 ul/detection reagent strips in the conventional method group experiment; the method comprises the steps of coating an experimental latex microsphere marked antibody on the near end of a nitrocellulose membrane with 1 ul/detection reagent strip, adding 3 ul/tube into a freezing tube, and freeze-drying and preserving for later use; the capture antibody of the detection line T of the detection reagent strip is 1.0mg/ml of non-labeled anti-insulin monoclonal antibody with pairing binding property, and the non-labeled anti-insulin monoclonal antibody is coated on the middle section position of the nitrocellulose membrane pad; the capture antibody of the quality control line C of the detection reagent strip is a 1.0mg/ml unlabeled goat anti-mouse IgG polyclonal antibody, and the capture antibody is coated on the far end position of the nitrocellulose membrane pad and is used for capturing the colloidal gold labeled anti-insulin monoclonal antibody which is not specifically captured.
The fluorescent microsphere marking antibody and the biotinylated marking antibody are coated on a glass cellulose film marker binding pad, and the liquid of the fluorescent microsphere marking antibody and the biotinylated marking antibody is loaded into a freezing tube and is frozen and stored for standby. The capture antibody of the detection reagent strip is 1.0mg/ml recombinant streptavidin, and the recombinant streptavidin is coated on a nitrocellulose membrane; the capture antibody of the quality control line C of the detection reagent strip is a goat anti-mouse IgG polyclonal antibody of 1.0mg/ml, and the goat anti-mouse IgG polyclonal antibody is coated on a nitrocellulose membrane and used for capturing fluorescent microsphere marked anti-insulin monoclonal antibodies which are not specifically captured. The two ends of the nitrocellulose membrane are respectively stuck with a water absorption paper membrane pad and a fluorescent microsphere marking combination membrane pad, and one side of the combination membrane pad is stuck with a sample pad. Placing the stuck detection sheet on a strip cutting machine, and cutting into 3.5mm test strips.
2. The immunochromatography detection technology of the invention is as follows:
prepared as in experiment one.
3. Fluorescence detector: a commercially available Bos AFS-100 dry-type fluorescence immunoassay was used.
4. Experimental method and results:
the experimental method is the same as "experiment one".
As a result of the experiment, the chroma signal value is basically stable after 15 minutes of detection by the conventional method, and the chroma signal value is basically stable after 4 minutes of detection by the method (Table 3), which shows that the detection time can be shortened and the detection sensitivity can be improved by the method.
TABLE 3 comparison of fast detection experiments of blood insulin by immunofluorescence
Detection result of structure type detection line (fluorescence signal value)
3 5 8 10 15 20*
623 758 10250 56330 85840 81507 of conventional construction
The technical structure 59411 82673 88755 87456 91024 90475
* The reaction time (minutes) was measured.

Claims (9)

1. The immunochromatography detection technology without the mediated release of the binding pad comprises a chromatography detection structure, a detection reagent strip positioned in the detection structure, a nitrocellulose membrane positioned on the reagent strip, and an indicator marker, a loading structure, a detection assisting liquid and a loading structure which are arranged in a combined way, wherein the nitrocellulose membrane comprises a nitrocellulose membrane and similar membrane structural materials, and is characterized in that:
1) The detection reagent strip is not provided with a binding pad for mediating the dispersion and release of the indicator marker which is conventionally used, and consists of a nitrocellulose membrane and a water absorption pad which are sequentially stuck on a support bottom sheet;
2) The nitrocellulose membrane is characterized by being used as a detection structure of the carrier of the indicator marker dispersed release carrier and the carrier of the capture agent coating carrier, the indicator marker is a first immune conjugate of the indicator marker specificity of the to-be-detected object, the capture agent is a second immune conjugate which is not marked and has specific pairing binding characteristics with the first immune conjugate, the indicator marker is coated on the proximal end of the nitrocellulose membrane, and the capture agent is coated on the middle section or the distal end of the nitrocellulose membrane; the immunoconjugate comprises at least one of an antibody, an antigen, biotin, avidin, and analogs thereof;
3) The chromatographic detection structure comprises a detection shell and a detection reagent strip positioned in the detection shell, wherein a sample adding hole and an observation window are formed in the upper layer of the shell, the sample adding hole corresponds to the near-end part of the nitrocellulose membrane, and the observation window corresponds to the middle section and the far-end part of the nitrocellulose membrane;
4) When in use, firstly, the indicator marker is mixed with a liquid phase sample to be detected, loaded to the starting end of the nitrocellulose membrane, loaded once or repeatedly loaded more than once, and flows through the nitrocellulose membrane and the water absorption pad to start a first-stage detection reaction;
5) Then loading a detection aid which does not contain the indicator marker and the sample to be detected to the starting end of the nitrocellulose membrane, loading once or repeatedly loading more than once, and starting a second-stage detection reaction;
6) The detection process is completed by observing the amount of the indicator marker captured on the nitrocellulose membrane and reading the detection result.
2. The immunochromatographic detection technique according to claim 1, wherein the indicator in the indicator label is at least one of a color microsphere and a fluorescent microsphere, and the morphology of the indicator label in the indicator label loading structure is preferably lyophilized powder.
3. The immunochromatographic assay technology according to claim 1, wherein a blood cell filtration structure is attached to a support substrate of the assay reagent strip and connected to a proximal end of a nitrocellulose membrane, the blood cell filtration structure comprises at least one of a blood cell filtration membrane and a membrane pad treated with an erythrocyte antibody, and a detection liquid phase is directly loaded onto the blood cell filtration structure at a position corresponding to the sample-loading hole of the chromatographic assay structure for detection reaction.
4. The immunochromatographic detection technique as defined in claim 1, wherein the nitrocellulose membrane is not coated with the indicator label, and the indicator label is entirely loaded in the indicator label loading structure.
5. The immunochromatographic detection technique as defined in claim 1, wherein a liquid-phase dispersion film is attached to the support base sheet of the detection reagent strip, and is connected to the proximal end of the nitrocellulose film, and is positioned at a position corresponding to the sample-loading hole of the chromatographic detection structure, and the detection liquid phase is directly loaded onto the liquid-phase dispersion film for detection reaction.
6. The immunochromatographic assay technique of claim 1, wherein the preparation of the assay reagent strip further comprises a biotin/avidin reaction system, the indicator-tag loading structure is filled with the first antibody and the second antibody, and the nitrocellulose membrane is coated with non-labeled avidin and the like.
7. The immunochromatographic detection technique according to claim 1, which is provided with a quantitative detection instrument including at least one of a colorimetric quantitative analyzer and a fluorescent quantitative analyzer.
8. The immunochromatographic detection technique as defined in claim 1, which is operatively used, comprising the steps of:
1) Taking out the chromatographic detection structure, and horizontally placing the chromatographic detection structure on a plane;
2) Collecting a blood sample to be detected, adding the blood sample to be detected into a loading tube containing an indicator marker, adding a proper amount of auxiliary detection liquid, and mixing to prepare a sample mixed liquid to be detected;
3) Adding the sample mixed solution to be detected into a blood cell filtering structure through a sample adding hole, and allowing the sample mixed solution to flow through the blood cell filtering structure, a nitrocellulose membrane and a water absorption pad forwards, and repeating the sample for more than one time;
4) Adding the auxiliary detection liquid to the blood cell filtering structure through the sample adding hole, and allowing the auxiliary detection liquid to flow forward through the blood cell filtering structure, the nitrocellulose membrane and the water absorbing pad, and repeating the sample more than once;
5) And detecting the read result from the observation window to finish detection.
9. Use of the immunochromatographic assay technique of claim 1 in the development of an immunoassay reagent product.
CN202210809240.0A 2022-07-11 2022-07-11 Immunochromatography detection technology without mediated release through binding pad and application thereof Pending CN117420300A (en)

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