CN117420301A - Immunochromatography detection structure without mediated release through binding pad and application thereof - Google Patents

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

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Publication number
CN117420301A
CN117420301A CN202210809778.1A CN202210809778A CN117420301A CN 117420301 A CN117420301 A CN 117420301A CN 202210809778 A CN202210809778 A CN 202210809778A CN 117420301 A CN117420301 A CN 117420301A
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detection
nitrocellulose membrane
liquid
sample
membrane
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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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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

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Abstract

The invention discloses an immunochromatography detection structure which does not mediate release through a bonding pad and application thereof, which is a combination set of a chromatography detection structure, an indicator marker loading structure and a detection-aid loading structure, is characterized in that a nitrocellulose membrane is used as a carrier for dispersing and carrying an indicator marker and a detection structure for a carrier for coating a capturing agent, is suitable for 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 structure 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 structure 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 rapid immunochromatography detection structure and application thereof, which have the advantages of high detection sensitivity, rapidness, low cost and the like and improve the detection quality compared with the prior art.
In view of the above, the present invention provides an immunochromatography detection structure not mediated by a conjugate pad, characterized in that,
1) The immunochromatography detection structure is a combined set of a chromatography detection structure, an indicator marker loading structure and a detection-assisting liquid loading structure;
2) The chromatographic detection structure comprises a detection shell and a detection reagent strip positioned in the detection shell, wherein a conventionally used combination pad for mediating the dispersion and release of the indicator marker is not arranged on the detection reagent strip, and the chromatographic detection structure consists of a nitrocellulose membrane and a water absorption pad which are sequentially stuck on a support negative; the nitrocellulose membrane is characterized by being used as a detection structure of a carrier which is carried by dispersing and releasing the indicator marker and a carrier which is carried by coating the capturing agent, the indicator marker is a first immune conjugate which is marked by the indicator and is specific to a detected object, the capturing 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 at the proximal end of the nitrocellulose membrane, the capturing agent is coated at the middle section or the distal end of the nitrocellulose membrane, the immune conjugate comprises at least one of an antibody, an antigen, biotin, avidin and the like, the nitrocellulose membrane comprises a nitrocellulose membrane and a similar membrane structural material thereof, and the indicator is at least one of a color microsphere and a fluorescent microsphere;
3) The indicator marker loading structure comprises an indicator marker, and the preparation form of the indicator marker is preferably freeze-dried powder;
4) The auxiliary detection liquid loading structure contains auxiliary detection liquid, and the auxiliary detection liquid is water-soluble buffer salt solution.
The nitrocellulose membrane comprises one or more combinations of nitrocellulose membrane, polyvinylidene fluoride membrane, nylon membrane and DEAE cellulose membrane. 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 structure, the chromatography detection structure comprises an upper cover, an observation window, a sample adding hole, a base, a bracket for placing the detection reagent strip and a detection reagent strip, wherein the observation window and the sample adding hole are arranged on the upper cover, the bracket is positioned on the base, the detection reagent strip is placed on the bracket, 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.
In the immunochromatography detection structure, 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, wherein the sample adding hole of the immunodetection chromatography structure corresponds to the position of the blood cell filtering structure, and the observation window corresponds to the position of the nitrocellulose membrane.
In the immunochromatography detection structure, a liquid-phase dispersion film is stuck on a supporting bottom sheet of the detection reagent strip and is connected with the proximal end of the nitrocellulose membrane, and the liquid-phase dispersion film is at least one of a glass cellulose membrane and a polyester cellulose membrane, wherein the sample adding hole of the immunochromatography detection structure corresponds to the position of the liquid-phase dispersion film, and the observation window corresponds to the position of the nitrocellulose membrane.
In the immunochromatography detection structure, the nitrocellulose membrane is not coated with the indicator marker, the indicator marker is fully loaded in the indicator marker loading structure and is mixed with a liquid phase sample to be detected to form a mixed liquid to be detected, and the mixed liquid to be detected is directly loaded on the detection reagent strip, or the mixed liquid to be detected and the auxiliary detection liquid are further mixed to form a secondary mixed liquid to be detected, and then the secondary mixed liquid to be detected is loaded on the detection reagent strip.
In the immunochromatography detection structure, the auxiliary detection liquid is a water-soluble buffer salt solution containing a surfactant, and is directly loaded onto the detection reagent strip, or is mixed with the liquid to be detected to form a secondary auxiliary detection liquid, and then is loaded onto the detection reagent strip.
The detection reagent strip is a sample pad, a hemocyte filtration structure, a nitrocellulose membrane and a water absorption pad which are sequentially stuck on a PVC bottom sheet, but no marker binding pad is arranged in a detection shell, and the detection shell is of a rigid structure or a flexible structure.
In the above immunochromatography detection structure, the use state structure in which the immunochromatography detection structure is provided with the blood cell filtration membrane includes:
1) The chromatographic detection structure comprises an upper cover provided with an observation window and a sample adding hole, a base provided with a bracket for placing the detection reagent strip, a blood cell filtering membrane, a nitrocellulose membrane and a water absorption pad are sequentially adhered on a support bottom plate of the detection reagent strip, an indicator marker is coated at the proximal end of the nitrocellulose membrane, a capturing agent is coated at the middle-far section of the nitrocellulose membrane, the blood cell filtering membrane is positioned at the corresponding position of the sample adding hole, and the nitrocellulose membrane is positioned at the corresponding position of the observation window;
2) The mixture containing the blood sample to be detected and the indicator marker forms a liquid phase traffic connection structure with a blood cell filtering membrane, a nitrocellulose membrane and a water absorption pad through a sample adding hole;
3) The auxiliary detection liquid forms a liquid phase traffic connection structure with the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad through the sample adding hole.
In the immunochromatography detection structure, the immunochromatography detection structure 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 above immunochromatography detection structure, the operation of the immunochromatography detection structure comprises the following steps:
1) Taking out the chromatographic detection structure, and horizontally placing the chromatographic detection structure on the table top;
2) Collecting a blood sample to be detected, adding the blood sample to be detected into a sample treatment 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 to a blood cell filtering membrane through a sample adding hole, and enabling the sample mixed solution to flow through the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad forwards;
4) Adding the auxiliary detection liquid to a blood cell filtering membrane through a sample adding hole, and enabling the auxiliary detection liquid to flow through the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad forwards;
5) And detecting the read result from the observation window to finish detection.
Use of the immunochromatographic assay structure 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 structure which is not released through the mediation of the binding pad is designed, the immunochromatography detection structure is reduced from the existing detection structure which needs the marker binding pad to load and release the indicator marker to the detection reaction which is 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 attaches the hemocyte filter membrane or the membrane pad structure treated by the erythrocyte antibody in front of the nitrocellulose membrane, can be simultaneously used for one-step detection of whole blood, and improves 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 diagram of the overall structure of the present invention;
FIG. 2 is a schematic diagram of the basic structure of the test strip of the present invention;
FIG. 3 is a schematic diagram of a blood cell filtration structure of a test strip of the present invention;
FIG. 4 is a schematic diagram of a chromatographic detection structure according to the present invention;
FIG. 5 is a schematic representation of the indicator marker loading structure of the present invention;
FIG. 6 is a schematic view of a loading structure of a liquid for assisting in detection according to the present invention;
FIG. 7 is a schematic diagram showing the structure of the indicator marker loading structure and the chromatography detection structure of the present invention in a detachable combined use state;
fig. 8 is a schematic structural diagram of a detachable combined use state of the liquid-assisted detection loading structure and the chromatographic detection structure of the present invention.
The figures are labeled as follows:
a chromatographic detection structure 1; an indicator marker loading structure 2; a test aid loading structure 3; a chromatography detection structure upper cover 4; a chromatography detection structure base 5; a detection reagent strip 6; a viewing window 7; a sample addition hole 8; a nitrocellulose membrane 9; a water absorbing pad 10; detection line (capture agent) 11; an indicator marker coated region 12; indicator marker lyophilized powder 13; a test aid 14; a liquid phase traffic mediating structure 15; a support backsheet 16; a quality control line 17; a hemocyte filtration structure 18; a sample 19 to be inspected; indicator marker-carrying container 20; indicator marker loading container lid 21; a test aid loading container 22; a test aid loading container cover 23; a structure 24 for indicating the liquid phase traffic usage status of the marker sample to be tested and the auxiliary test liquid; a mixed solution 25 of the indicator marker and the use state of the sample to be tested and the auxiliary detection solution; the structure 26 of the liquid phase traffic use state of the liquid phase of the auxiliary detection liquid.
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 whole structure of the invention comprises a chromatography detection structure 1, an indicator marker loading structure 2 and a detection aid loading structure 3, wherein the chromatography detection structure 1 comprises a chromatography detection structure upper cover 4, a chromatography detection structure base 5 and a detection reagent strip 6, and a viewing window 7 and a sample loading hole 8 which are positioned on the chromatography detection structure upper cover 4. The detection reagent strip 6 comprises a nitrocellulose membrane 9 and a water absorbing pad 10 which are sequentially stuck on a support bottom sheet 16, and an indicator marker binding pad adopted in the prior art is not arranged, wherein a detection line (capturing agent) 11 for a to-be-detected object and an indicator marker coating area 12 are arranged on the nitrocellulose membrane 9, wherein the indicator marker coating area 12 can be used as an area for coating and dispersing and bearing the indicator marker at the same time, or only the dispersing and bearing of the indicator marker can be realized, and the indicator marker is not coated on the indicator marker coating area. The chromatographic detection structure 1, the indicator marker loading structure 2 and the auxiliary detection liquid loading structure 3 are combined when in use, firstly, the indicator marker 13 and a detected sample are mixed and loaded to the initial end of the nitrocellulose membrane 9, and are loaded once or more times and flow through the nitrocellulose membrane 9 and the water absorption pad 10, and the detected object is specifically captured and fixed by the capture agent 11 coated and fixed on the nitrocellulose membrane 9; 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 one or more times, and removing the indicator which is remained on the nitrocellulose membrane and is not specifically combined; the detection process is completed by observing the amount of the indicator marker captured on the nitrocellulose membrane and reading the detection result.
As shown in fig. 2, the basic structure of the detection reagent strip of the present invention is that a nitrocellulose membrane 9 and a water absorbing pad 10 are sequentially adhered to a PVC support backsheet 16 from the near to the far, the indicator marker binding pad adopted in the prior art is not provided, a detection line (capturing agent) 11 for the object to be detected, a quality control line 17 and an indicator marker coating area 12 are provided on the nitrocellulose membrane 9, wherein the indicator marker coating area 12 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.
As shown in fig. 1 and 3, the detection reagent strip of the present invention comprises a hemocyte filtration structure, a hemocyte filtration membrane 18, a nitrocellulose membrane 9 and a water absorption pad 10 are sequentially stuck on a PVC support bottom sheet 16 from the near to the far, and no indicator marker binding pad adopted in the prior art is provided, so that when the chromatography detection structure 1, the indicator marker loading structure 2 and the auxiliary detection liquid loading structure 3 are in a combined use state, firstly, an indicator marker 13 and a detected sample are mixed, loaded on the hemocyte filtration membrane 18, loaded one or more times, and flow through the nitrocellulose membrane 9 and the water absorption pad 10, and the detected material is specifically captured and fixed by a capturing agent 11 coated and fixed on the nitrocellulose membrane 9; then loading a detection aid solution which does not contain the indicator marker and the sample to be detected to a hemocyte filter membrane 18, and loading the detection aid solution one or more times to remove the indicator which is not specifically combined and remains on the nitrocellulose membrane 9; the detection process is completed by observing the amount of the indicator marker captured on the nitrocellulose membrane and reading the detection result.
As shown in FIG. 4, the chromatography detection structure of the invention comprises a chromatography detection structure upper cover 4, a chromatography detection structure base 5, a detection reagent strip 6, an observation window 7 and a sample adding hole 8 which are positioned on the chromatography detection structure upper cover 4, a detection line (capture agent) 11 and a quality control line 17 which are positioned on the detection reagent strip 6.
As shown in fig. 5, the indicator marker loading structure of the present invention includes an indicator marker 13, a sample 19 to be tested, an indicator marker loading container 20, and an indicator marker loading container cover 21, wherein the indicator marker 13 is a lyophilized powder, and the sample 19 to be tested is added before the detection and mixed into a sample mixture to be tested.
As shown in fig. 6, the liquid-assisted detection loading structure of the present invention includes a liquid-assisted detection 14, a liquid-assisted detection loading container 22, and a liquid-assisted detection loading container cover 23, and in use, the liquid-assisted detection 14 is directly loaded onto the detection reagent strip.
As shown in fig. 7, the structure of the detachable combined use state of the indicator marker loading structure and the chromatographic detection structure of the present invention, wherein the indicator marker 13 is mixed with the sample 19 to be detected, directly loaded to the initial end of the nitrocellulose membrane 9 through the liquid phase traffic mediating structure 15, loaded once or more times, and flows through the nitrocellulose membrane 9 and the water absorbing pad 10, and the sample to be detected is specifically captured and fixed by the capturing agent 11 coated and fixed on the nitrocellulose membrane 9; if the detection reagent strip 6 is provided with a hemocyte filtration structure or a liquid-phase dispersion film, the indicator marker 13 is mixed with the detected sample 19, then is loaded to the hemocyte filtration structure or the liquid-phase dispersion film through the liquid-phase traffic mediation structure 15, and then flows through the nitrocellulose membrane 9 to be loaded one or more times; the method comprises the steps of further selecting mixed liquid 25 comprising the indicator marker 13 and the detected sample 19, then adding the auxiliary detection liquid for dilution to prepare mixed liquid to be detected, loading the mixed liquid 25 comprising the indicator marker, the detected sample and the auxiliary detection liquid, or diluting the indicator marker 13 by adding the auxiliary detection liquid, then adding the detected sample 19 for mixing to prepare mixed liquid to be detected, mixing the indicator marker, the detected sample and the auxiliary detection liquid in the mixed liquid 25, and then carrying out sample loading detection. The structure 24 of the indicator marker sample to be detected and the liquid phase traffic use state of the auxiliary detection liquid is the structure of the use state of the sample loading in the process. The liquid phase traffic mediating structure 15 selects various forms such as a catheter, a sample-adding gun head, a syringe, etc.
As shown in fig. 8, the structure of the detachable combination use state of the detection aid loading structure and the chromatography detection structure according to the invention, wherein the detection aid 14 is directly loaded to the initial end of the nitrocellulose membrane 9 through the liquid phase traffic mediating structure 15, is loaded one or more times, flows through the nitrocellulose membrane 9 and the water absorbing pad 10, eliminates the indicator which remains on the nitrocellulose membrane and is not specifically combined, and completes the detection process by observing the captured amount of the indicator marker on the nitrocellulose membrane 9 and reading the detection result. The liquid phase traffic usage pattern 26 of the liquid of the test aid fluid is the usage pattern of the process sample loading. The liquid phase traffic mediating structure 15 selects various forms such as a catheter, a sample-adding gun head, a syringe, etc.
In the practical operation, when the immunochromatography detection structure is a colloidal gold immunoassay structure, the detection reagent strip 6 is prepared by a colloidal gold method, a hemocyte filtration membrane 18, a nitrocellulose membrane 9 and a water absorption pad 10 are sequentially stuck on a PVC negative film, a colloidal gold-marked object-to-be-detected specific antibody or antigen is filled in the indicator marker loading structure 2, a non-marked matched object-to-be-detected specific antibody or antigen is coated on the nitrocellulose membrane 9, and after the detection reaction is finished, the detection result is quantitatively analyzed by a colorimetry quantitative analyzer; when the immunochromatography detection structure is prepared by a latex microsphere immunization method, a detection reagent strip 6 is prepared by the latex microsphere immunization method, a hemocyte filtration membrane 18, a nitrocellulose membrane 9 and a water absorption pad 10 are sequentially adhered on a PVC negative film, a latex microsphere marked object to be detected specific antibody or antigen is arranged in an indicator marker loading structure 2, a non-marked matched object to be detected specific antibody or antigen is coated on the nitrocellulose membrane 9, and after the detection reaction is finished, a colorimetric quantitative analyzer is used for quantitatively analyzing the detection result quantitatively; when the immunochromatography detection structure is prepared by a fluorescent microsphere immunization method, the detection reagent strip 6 is prepared by the fluorescent microsphere immunization method, a hemocyte filtration membrane 18, a nitrocellulose membrane 9 and a water absorption pad 10 are sequentially adhered on a PVC negative film, a fluorescent microsphere marked object specific antibody or antigen to be detected is arranged in the indicator marker loading structure 2, a non-marked matched object specific antibody or antigen to be detected is coated on the nitrocellulose membrane 9, 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 structure 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% T20, 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 structure of the invention is prepared by the following steps:
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 aid 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 structure are taken, 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, which shows that the detection time can be shortened and the detection sensitivity can be improved.
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*
15 18 38 66 79 82 of conventional construction
The technical structure 59 82 88 91 92 96
* The reaction time (minutes) was measured.

Claims (10)

1. An immunochromatography detection structure which does not mediate release through a bonding pad is characterized in that,
1) The immunochromatography detection structure is a combined set of a chromatography detection structure, an indicator marker loading structure and a detection-assisting liquid loading structure;
2) The chromatographic detection structure comprises a detection shell and a detection reagent strip positioned in the detection shell, wherein a conventionally used combination pad for mediating the dispersion and release of the indicator marker is not arranged on the detection reagent strip, and the chromatographic detection structure consists of a nitrocellulose membrane and a water absorption pad which are sequentially stuck on a support negative; the nitrocellulose membrane is characterized by being used as a detection structure of a carrier which is carried by dispersing and releasing the indicator marker and a carrier which is carried by coating the capturing agent, the indicator marker is a first immune conjugate which is marked by the indicator and is specific to a detected object, the capturing 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 at the proximal end of the nitrocellulose membrane, the capturing agent is coated at the middle section or the distal end of the nitrocellulose membrane, the immune conjugate comprises at least one of an antibody, an antigen, biotin, avidin and the like, the nitrocellulose membrane comprises a nitrocellulose membrane and a similar membrane structural material thereof, and the indicator is at least one of a color microsphere and a fluorescent microsphere;
3) The indicator marker loading structure comprises an indicator marker, and the preparation form of the indicator marker is preferably freeze-dried powder;
4) The auxiliary detection liquid loading structure contains auxiliary detection liquid, and the auxiliary detection liquid is water-soluble buffer salt solution.
2. The immunochromatographic detection structure according to claim 1, which comprises an upper cover, a viewing window and a sample addition hole which are arranged on the upper cover, a base, a bracket which is arranged on the base and is used for placing the detection reagent strip, and the detection reagent strip which is arranged on the bracket, wherein the sample addition hole corresponds to a proximal end part of the nitrocellulose membrane, and the viewing window corresponds to a middle section and a distal end part of the nitrocellulose membrane.
3. The immunochromatographic detection structure according to claim 1 or 2, wherein a blood cell filtration structure is attached to a support base sheet of the detection reagent strip and connected to a proximal end of a nitrocellulose membrane, the blood cell filtration structure comprising at least one of a blood cell filtration membrane and a membrane pad treated with an erythrocyte antibody, wherein the sample-applying hole of the immunodetection chromatographic structure corresponds to a portion of the blood cell filtration structure, and the observation window corresponds to a portion of the nitrocellulose membrane.
4. The immunochromatographic detection structure according to claim 1 or 2, wherein a liquid-phase dispersion film is attached to a support backsheet of the detection reagent strip and connected to a proximal end of a nitrocellulose film, and the liquid-phase dispersion film is at least one of a glass cellulose film and a polyester cellulose film, wherein the sample-loading hole of the immunochromatographic detection structure corresponds to a portion of the liquid-phase dispersion film, and the observation window corresponds to a portion of the nitrocellulose film.
5. The immunochromatographic assay structure of claim 1, wherein the nitrocellulose membrane is not coated with the indicator marker, the indicator marker is fully loaded in the indicator marker loading structure and is mixed with a liquid sample to be tested to form a mixed solution to be tested, and the mixed solution to be tested is directly loaded on the detection reagent strip, or the mixed solution to be tested and the auxiliary detection solution are further mixed to form a secondary mixed solution to be tested and then loaded on the detection reagent strip.
6. The immunochromatographic assay structure of claim 1, wherein the test-aid liquid is a water-soluble buffer salt solution containing a surfactant, and is directly loaded onto the test reagent strip, or is mixed with the test-aid liquid to form a secondary test-aid liquid, and is further loaded onto the test reagent strip.
7. The immunochromatographic detection structure according to claim 1, wherein the structure of the immunochromatographic detection structure provided with the blood cell filtration membrane in-use state comprises:
1) The chromatographic detection structure comprises an upper cover provided with an observation window and a sample adding hole, a base provided with a bracket for placing the detection reagent strip, a blood cell filtering membrane, a nitrocellulose membrane and a water absorption pad are sequentially adhered on a support bottom plate of the detection reagent strip, an indicator marker is coated at the proximal end of the nitrocellulose membrane, a capturing agent is coated at the middle-far section of the nitrocellulose membrane, the blood cell filtering membrane is positioned at the corresponding position of the sample adding hole, and the nitrocellulose membrane is positioned at the corresponding position of the observation window;
2) The mixture containing the blood sample to be detected and the indicator marker forms a liquid phase traffic connection structure with a blood cell filtering membrane, a nitrocellulose membrane and a water absorption pad through a sample adding hole;
3) The auxiliary detection liquid forms a liquid phase traffic connection structure with the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad through the sample adding hole.
8. The immunochromatographic detection structure 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.
9. The immunochromatographic detection structure according to 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 the table top;
2) Collecting a blood sample to be detected, adding the blood sample to be detected into a sample treatment 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 to a blood cell filtering membrane through a sample adding hole, and enabling the sample mixed solution to flow through the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad forwards;
4) Adding the auxiliary detection liquid to a blood cell filtering membrane through a sample adding hole, and enabling the auxiliary detection liquid to flow through the blood cell filtering membrane, the nitrocellulose membrane and the water absorption pad forwards;
5) And detecting the read result from the observation window to finish detection.
10. Use of the immunochromatographic assay structure of claim 1 in the development of an immunoassay reagent product.
CN202210809778.1A 2022-07-11 2022-07-11 Immunochromatography detection structure without mediated release through binding pad and application thereof Pending CN117420301A (en)

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