CN113607944B - Colloidal gold chromatography reagent strip, preparation method and neocorona antigen detection kit - Google Patents
Colloidal gold chromatography reagent strip, preparation method and neocorona antigen detection kit Download PDFInfo
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- CN113607944B CN113607944B CN202110988511.9A CN202110988511A CN113607944B CN 113607944 B CN113607944 B CN 113607944B CN 202110988511 A CN202110988511 A CN 202110988511A CN 113607944 B CN113607944 B CN 113607944B
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- 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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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a colloidal gold chromatography reagent strip for detecting a neocorona antigen, a preparation method and a kit. The colloidal gold chromatography reagent strip comprises a bottom plate, a nitrocellulose membrane, a coupling pad, a sample pad and water absorption paper, wherein the sample pad, the coupling pad, the nitrocellulose membrane and the water absorption paper are sequentially connected on the bottom plate along the horizontal direction, avidin biotin amplified colloidal gold nanoflowers are coupled on the coupling pad, the nitrocellulose membrane is modified by nanocellulose, a detection line coated with a neocorona antigen capture antibody and a quality control line coated with a quality control molecule capture antibody are arranged on the nitrocellulose membrane, and the detection line and the quality control line are sequentially distributed along the chromatography direction. The colloidal gold chromatography reagent strip can be used for conveniently, quickly and highly sensitively detecting the new crown antigen of the nasopharynx swab and the pharynx swab.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a colloidal gold chromatography reagent strip for rapidly detecting a neocorona antigen with high sensitivity, a preparation method and a neocorona antigen detection kit.
Background
In biological assays such as new coronavirus assays, the existing assay techniques include fluorescence immunochromatographic test strips, colloidal gold chromatography test strips, and the like. The fluorescence immunochromatographic test paper is characterized in that a complex formed by combining a fluorescent microsphere and an antigen or an antibody is placed in a conjugate pad, when a sample passes through the conjugate pad, the antibody or the antigen in the sample and the complex are subjected to immunoreaction to generate a fluorescent microsphere-antigen antibody complex, and the fluorescent microsphere-antigen antibody complex is captured by a nitrocellulose membrane, a quality control line coated on the membrane and a detection limit antibody, so that a detection strip is formed. However, the fluorescence chromatography test paper usually needs a matched instrument, the fluorescence intensity is read by a professional instrument and converted into a signal value, the result cannot be obtained directly by naked eyes, and the difference between different instruments exists.
The colloidal gold chromatography test paper is characterized in that a complex formed by combining colloidal gold particles and an antigen or an antibody is placed in a conjugate pad, when a sample passes through the conjugate pad, the antibody or the antigen in the sample and the complex are subjected to immunoreaction to generate a colloidal gold particle-antigen-antibody complex, and the colloidal gold particle-antigen-antibody complex is captured by a nitrocellulose membrane, a quality control line coated on the nitrocellulose membrane and a detection limit antibody, so that a detection strip is formed. Most of the existing colloidal gold chromatography test paper adopts the design of nano-gold particles, but the optical scattering property and the antibody binding capacity of the existing nano-colloidal gold particles are not outstanding, and the nitrocellulose membrane is not modified, so that the problem of virus antigen omission is caused.
According to the invention, colloidal gold nanoparticles are upgraded into colloidal gold nanoflowers with higher antibody affinity and better natural light scattering in the colloidal gold immunochromatographic test paper, more aggregated large-particle colloidal gold is obtained through an avidin biotin signal amplification system, more colloidal gold particles are combined to the surface of the membrane through surface modification of the nitrocellulose membrane, and triple signal amplification is performed, so that the sensitivity and color development of the colloidal gold immunochromatographic test paper are greatly improved, the sensitivity of the colloidal gold immunochromatographic test paper is enhanced, a user can conveniently recognize positive results, and the omission is reduced.
Disclosure of Invention
Therefore, it is necessary to provide a colloidal gold chromatographic reagent strip for detecting neocorona antigen, which aims at the problems that the traditional chromatographic test paper needs a matched professional instrument to read the fluorescence intensity and convert the fluorescence intensity into a signal value, and the antibody binding capacity does not highlight the omission of the pathogenic virus antigen.
The utility model provides a detect colloidal gold chromatography reagent strip of neo-corona antigen, includes bottom plate, nitrocellulose membrane, coupling pad, sample pad and absorbent paper, the sample pad the coupling pad the nitrocellulose membrane with absorbent paper is connected along horizontal direction order on the bottom plate, the coupling is gone up the coupling and is had the enlarged colloidal gold puffed rice of avidin biotin, the nitrocellulose membrane is decorated through the nanometer cellulose, be equipped with the detection line of peridium neo-corona antigen capture antibody and the quality control line of peridium quality control molecule capture antibody on the nitrocellulose membrane, the detection line with quality control line is along chromatography direction sequence distribution.
In one embodiment, the coupling pad and the absorbent paper are respectively positioned on two sides of the nitrocellulose membrane and are partially overlapped with the nitrocellulose membrane, and the sample pad is partially overlapped with the coupling pad.
In one embodiment, the detection device further comprises a housing, the housing is provided with a containing cavity, a detection window and a sample adding hole, the detection window and the sample adding hole are communicated with the containing cavity, the bottom plate, the nitrocellulose membrane, the coupling pad, the sample pad and the absorbent paper are all arranged in the containing cavity, the detection line and the quality control line are located at the detection window, and part of the sample pad is located at the sample adding hole.
In one embodiment, the outer surface of the shell is provided with a detection identification area and a quality control identification area which respectively correspond to the detection line and the quality control line.
In one embodiment, the sample application aperture tapers from an outwardly facing end to an inwardly facing end.
The invention also aims to provide a preparation method of the colloidal gold chromatography reagent strip for detecting the neocorona antigen.
The preparation method of the colloidal gold chromatography reagent strip for detecting the neocorona antigen comprises the following steps:
a) preparing a colloidal gold particle solution: HAuCl is added4Heating to 120 ℃ while stirring, adding 1/20-1/4 volumes of sodium citrate, continuing heating for 10-30 min until the color of the solution is stable and is ruby, and preserving at 2-8 ℃ after cooling;
b) preparing a colloidal gold nanoflower solution: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume of 60-100 times, heating to 60-80 ℃, and adding HAuCl4Stirring the solution, centrifuging the solution to remove supernatant, collecting the synthesized colloidal gold nanoflowers solution, and storing the colloidal gold nanoflowers solution at the temperature of between 2 and 8 ℃;
c) preparing colloidal gold-labeled streptavidin: taking the colloidal gold nanoflower solution prepared in the step b), adjusting the pH value to 9-10 and the concentration to 0.01-0.05 wt%, adding a streptavidin solution, carrying out oscillation reaction for 5-30 min, adding BSA until the final concentration of BSA is 1-2 wt%, oscillating for 5-30 min, centrifuging, removing a supernatant to obtain colloidal gold labeled streptavidin, and re-dissolving with a gold labeled diluent for later use;
d) preparing a biotinylation new coronavirus antigen labeled antibody and a biotinylation quality control molecule antibody: respectively diluting the labeled antibody and the quality control antibody to 0.5-2 mg/mL by adopting buffer solution, respectively dialyzing to obtain a new coronavirus antigen labeled antibody solution and a quality control molecule capture antibody solution, respectively adding biotin solution with the concentration of 0.5-2 mg/mL for reacting for 1-6 h at room temperature, and respectively adding biotin solution with the concentration of 0.5-2 mol/LNH4Stirring and reacting the Cl solution for 5-30 min at room temperature, and respectively dialyzing and purifying by a column to obtain a biotinylated new coronavirus antigen labeled antibody and a biotinylated quality control molecular antibody;
e) preparing a colloidal gold labeled streptomycin-biotinylation new coronavirus antigen labeled antibody and a colloidal gold labeled streptomycin-biotinylation quality control molecule antibody: mixing and oscillating the biotinylated new coronavirus antigen-labeled antibody and biotinylated quality control molecule antibody prepared in the step d) with the colloidal gold-labeled streptavidin prepared in the step c) for 10-30 min, centrifuging and removing supernate to respectively obtain a colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and a colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody, and re-dissolving the colloidal gold-labeled streptavidin-labeled antibody and the colloidal gold-labeled streptavidin-biotinylated quality control molecule antibody respectively for later use;
f) preparation of coupling pad: diluting the colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and the colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody prepared in the step e) with a conjugate diluent to prepare a conjugate treatment solution, wherein the volume ratio of the colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody is 18-20%, the volume ratio of the colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody is 20-25%, and the conjugate treatment solution is uniformly coated on a coupling pad;
g) the method comprises the following steps Preparing a nitrocellulose membrane: diluting the nano cellulose gel to the concentration of 0.1 wt% -0.5 wt%, scribing on a nitrocellulose membrane to form a detection line and a quality control line, and drying; diluting the new crown antigen capture antibody and the quality control molecule capture antibody to 1-3 mg/mL and 0.1-2 mg/mL respectively by using NC membrane coating solution, scribing on a detection line and a quality control line, and drying;
h) preparation of sample pad: uniformly coating the sample pad treatment solution on the sample pad;
i) assembling the reagent strip: the reagent strip of claim 1 or 2 assembled in the configuration.
In one embodiment, step b) comprises: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume of 60-100 times, heating to 60-80 ℃, and adding HAuCl with the volume of 0.5-3 times4And 1-7 times of sodium citrate by volume, stirring for 15-60 s, adding 10-40 times of hydroquinone solution by volume, stirring for 3-6 h, and centrifuging.
In one embodiment, in step c), the final concentration of the streptavidin solution is 10-80. mu.g/mL.
In one embodiment, the NC film coating solution is prepared from 0.01-0.5 mol/L PBS with pH of 6.5-7.5 and 3-10 wt% trehalose solution.
In one embodiment, the absorption amount of the sample pad treatment solution on the sample pad is 25-50 +/-3 mL, and the sample pad is dried for 24 hours at 50-70 ℃ or naturally dried at room temperature.
In one embodiment, the assembled reagent strips are cut to 2-4 mm/strip and are respectively put into a shell.
The invention also aims to provide a new crown antigen detection kit.
A new corona antigen detection kit comprises virus lysate and the colloidal gold chromatography reagent strip or the colloidal gold chromatography reagent strip prepared by the preparation method.
The colloidal gold chromatography reagent strip for detecting the neocorona antigen can be used for conveniently, quickly and highly sensitively detecting the neocorona antigen of a nasopharynx swab and a pharynx swab. The colloidal gold chromatography reagent strip for detecting the neocorona antigen comprehensively uses triple amplification signals of colloidal gold nanoflowers, an avidin biotin amplification system and a nanocellulose modified nitrocellulose membrane, so that the detection sensitivity and stability are improved, and the missing detection phenomenon of a neocorona pathogen is reduced. Specifically, compared with the traditional colloidal gold nanoparticles, in the colloidal gold chromatographic reagent strip for detecting the neocorona antigen, the colloidal gold nanoflowers have larger space surface area and can be more fully adsorbed with the coupled antibody, so that the detection sensitivity and the result stability are improved, and the surface of the colloidal gold nanoflowers has a tip-graded flower-shaped structure to show strong light signal sensitivity, so that the color sense under natural light is more obvious and the naked eye identification is facilitated; the invention can obtain more antigen-antibody binding epitopes through an avidin biotin signal amplification system; according to the invention, the nano cellulose gel is pre-coated near the detection line, and the micropores on the nitrocellulose membrane are filled, so that more capture antibodies coated later are attached to the surface of the nitrocellulose membrane, and therefore, colloidal gold nanoparticles captured at a later stage are more remained on the surface of the nitrocellulose membrane, the color development effect is enhanced, and the visual effect of naked eyes is enhanced.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold chromatography reagent strip for detecting neocorona antigen according to an embodiment of the present invention;
fig. 2 is a schematic view of a colloidal gold chromatographic reagent strip for detecting neocorona antigen according to an embodiment of the present invention.
Description of the reference numerals
10. A colloidal gold chromatography reagent strip for detecting the neocorona antigen; 101. a base plate; 102. a nitrocellulose membrane; 1021. detecting lines; 1022. a quality control line; 103. a coupling pad; 104. a sample pad; 105. absorbent paper; 106. a housing; 1061. detecting a window; 1062. a sample application hole; 1063. detecting the identification area; 1064. and a quality control identification area.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and the like as used herein are for illustrative purposes only and do not denote a unique embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the application provides a colloidal gold chromatography reagent strip 10 for detecting a neocorona antigen, so as to solve the problems that the conventional traditional chromatography test paper needs a matched professional instrument to read fluorescence intensity and convert the fluorescence intensity into a signal value, and the antibody binding capacity does not highlight the omission of the pathogenic virus antigen. The following description will be made with reference to the accompanying drawings.
Fig. 1 shows an exemplary structure of a colloidal gold chromatography reagent strip 10 for detecting a corona neoantigen provided in an embodiment of the present application, and fig. 1 is a schematic structural diagram of the colloidal gold chromatography reagent strip 10 for detecting a corona neoantigen provided in an embodiment of the present application. The colloidal gold chromatography reagent strip 10 for detecting a new corona antigen can be used for detecting a new corona virus antigen.
In order to more clearly illustrate the structure of the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen, the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen will be described below with reference to the drawings.
Exemplarily, please refer to fig. 1, and fig. 1 is a schematic structural diagram of a colloidal gold chromatography reagent strip 10 for detecting neocorona antigen according to an embodiment of the present disclosure. A colloidal gold chromatography reagent strip 10 for detecting neocorona antigen comprises a bottom plate 101, a nitrocellulose membrane 102, a coupling pad 103, a sample pad 104 and absorbent paper 105, wherein the sample pad 104, the coupling pad 103, the nitrocellulose membrane 102 and the absorbent paper 105 are sequentially connected to the bottom plate 101 along the horizontal direction, avidin biotin amplified colloidal gold popcorn is coupled to the coupling pad 103, the nitrocellulose membrane 102 is modified by nanocellulose, a detection line 1021 coated with a neocorona antigen capture antibody and a quality control line 1022 coated with a quality control molecule capture antibody are arranged on the nitrocellulose membrane 102, and the detection line 1021 and the quality control line 1022 are sequentially distributed along the chromatography direction.
In some of these embodiments, the backplane 101 may be a PVC backplane.
In some embodiments, the coupling pad 103 and the absorbent paper 105 are respectively located on two sides of the nitrocellulose membrane 102 and both partially overlap the nitrocellulose membrane 102, and the sample pad 104 partially overlaps the coupling pad.
In some embodiments, referring to fig. 2, the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen further includes a casing 106. The housing 106 is provided with a containing cavity, a detection window 1061 and a sample adding hole 1062 which are communicated with the containing cavity, the bottom plate 101, the nitrocellulose membrane 102, the coupling pad 103, the sample pad 104 and the absorbent paper 105 are all arranged in the containing cavity, the detection line 1021 and the quality control line 1022 are located at the detection window 1061, and part of the sample pad 104 is located at the sample adding hole 1062.
In some embodiments, referring to fig. 2, a detection mark area 1063 and a quality control mark area 1064 are disposed on an outer surface of the housing 106 and respectively correspond to the detection line 1021 and the quality control line 1022.
In some embodiments, as shown in FIG. 2, the sample application hole 1062 narrows from the outward end to the inward end.
The colloidal gold chromatography reagent strip 10 for detecting neocorona antigen can be used for conveniently, quickly and highly sensitively detecting neocorona antigen of nasopharynx swab and pharynx swab. The colloidal gold chromatography reagent strip 10 for detecting the neocorona antigen comprehensively uses triple amplification signals of colloidal gold nanoflowers, an avidin biotin amplification system and the nanocellulose modified nitrocellulose membrane 102, so that the detection sensitivity and stability are improved, and the missing detection phenomenon of neocorona pathogens is reduced.
Another embodiment of the present invention provides a method for preparing a colloidal gold chromatography reagent strip 10 for detecting a neocorona antigen.
A preparation method of the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen comprises the following steps:
a) preparing a colloidal gold particle solution: adding HAuCl4Heating to 120 ℃ while stirring in an intelligent magnetic stirrer, adding 1/20-1/4 volume of sodium citrate (the adding amount of the sodium citrate is 1 wt%), continuing heating for 10-30 min until the color of the solution is stable and is ruby, considering that stable colloidal gold particles are generated, and cooling to room temperature and then storing at 2-8 ℃.
b) Preparing a colloidal gold nanoflower solution: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume of 60-100 times, heating to 60-80 ℃, and adding HAuCl4(make HAuCl4Adding 1 wt% of sodium citrate, adding 1 wt% of sodium citrate and hydroquinone solution, continuously stirring at room temperature, centrifuging at 9000-13000 rpm for 10-30 min, removing supernatant, collecting the synthesized colloidal gold nanoflowers solution, and storing at 2-8 ℃.
c) Preparing colloidal gold-labeled streptavidin: taking the colloidal gold nanoflowers prepared in the step b), adjusting the pH value to 9-10 and the concentration to 0.01-0.05 wt%, adding a streptavidin solution, wherein the concentration of the streptavidin solution is 10-15mg/mL, and the addition amount of the streptavidin solution and the colloidal gold solution meets the following requirements: and mixing the two solutions, wherein the final concentration of the streptavidin solution is 10-80 mug/mL, carrying out oscillation reaction for 5-30 min, adding BSA (bovine serum albumin) with the final concentration of 1-2 wt%, carrying out oscillation for 5-30 min, centrifuging, removing supernatant to obtain colloidal gold labeled streptavidin, and re-dissolving the colloidal gold labeled streptavidin by using a gold labeled diluent for later use.
d) Preparing a biotinylation new coronavirus antigen labeled antibody and a biotinylation quality control molecule antibody: respectively diluting the labeled antibody and the quality control antibody to 0.5-2 mg/mL by adopting buffer solution, respectively dialyzing to obtain a new coronavirus antigen labeled antibody solution and a quality control molecule capture antibody solution, respectively adding biotin solution with the concentration of 0.5-2 mg/mL, reacting for 1-6 h at room temperature, and respectively adding biotin solution with the concentration of 0.5-2 mol/LNH4And stirring and reacting the Cl solution at room temperature for 5-30 min, and respectively dialyzing and purifying by a column to obtain a biotinylated new coronavirus antigen labeled antibody and a biotinylated quality control molecule antibody.
e) Preparing a colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and a colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody: mixing and oscillating the biotinylated new coronavirus antigen-labeled antibody and biotinylated quality control molecule antibody prepared in the step d) with the colloidal gold-labeled streptavidin prepared in the step c) for 10-30 min, centrifuging and removing supernate to obtain a colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and a colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody respectively, and re-dissolving the antibodies with gold-labeled diluent for later use.
f) Preparation of coupling pad: diluting the colloidal gold-labeled streptomycin-biotinylation new crown virus antigen-labeled antibody and the colloidal gold-labeled streptomycin-biotinylation quality control molecule antibody prepared in the step e) with a conjugate diluent to prepare a conjugate treatment solution, wherein the volume ratio of the colloidal gold-labeled streptomycin-biotinylation new crown virus antigen-labeled antibody is 18-20%, the volume ratio of the colloidal gold-labeled streptomycin-biotinylation quality control molecule antibody is 20-25%, uniformly coating the conjugate treatment solution on a coupling pad, and the coating mode usually adopts machine spraying, manual coating and the like.
g) The method comprises the following steps Preparation of the nitrocellulose membrane 102: diluting the nano cellulose gel (the addition amount of the nano cellulose gel is 0.92 wt%) to the concentration of 0.1-0.5 wt%, scribing on the nitrocellulose membrane 102 through a synovial membrane machine to form a detection line 1021 and a quality control line 1022, drying at 45 ℃ for 12 hours, diluting the neo-corona antigen capture antibody and the quality control molecule capture antibody to 1-3 mg/mL and 0.1-2 mg/mL respectively with an NC membrane coating solution, scribing on the detection line 1021 and the quality control line 1022 through the synovial membrane machine, and drying at 45 ℃ for 72 hours.
h) Preparation of sample pad 104: the sample pad 104 treatment solution is applied uniformly to the sample pad 104.
i) Assembling the reagent strip: and assembling according to the structure of the reagent strip, cutting the assembled reagent strip to 2-4 mm/strip, and respectively loading the reagent strip into the shell 106.
The invention further provides a new crown antigen detection kit.
A new crown antigen detection kit comprises virus lysate and a colloidal gold chromatography reagent strip or a colloidal gold chromatography reagent strip prepared by the preparation method.
Example 1
The embodiment provides a colloidal gold chromatography reagent strip 10 for detecting a neocorona antigen, and the preparation method comprises the following steps:
a) preparing a colloidal gold particle solution: adding HAuCl4Heating to 120 deg.C while stirring in intelligent magnetic stirrer, adding 1/10 volume of sodium citrate (1 wt%), heating for 20min until the solution color is stable and shows ruby color, and cooling to room temperature and storing at 4 deg.C.
b) Preparing a colloidal gold nanoflower solution: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume 80 times that of the solution, heating the solution to 60 ℃, and adding HAuCl4(1 wt%), sodium citrate (1 wt%) and hydroquinone solution, continuously stirring at room temperature, centrifuging at 10000rpm for 20min, removing supernatant, collecting the synthesized colloidal gold nanoflower solution, and storing at 4 ℃.
c) Preparing colloidal gold-labeled streptavidin: taking the colloidal gold nanoflowers prepared in the step b), adjusting the pH value to 9-10 and the concentration to 0.01 wt%, adding a streptavidin solution, and mixing the two solutions to obtain a streptavidin solution with the final concentration of 10-80 mu g/mL. Oscillating for 20min, adding BSA with the final concentration of 1 wt%, oscillating for 20min, centrifuging, removing supernatant to obtain colloidal gold-labeled streptavidin, and re-dissolving with gold-labeled diluent for later use.
d) Preparing a biotinylation new coronavirus antigen labeled antibody and a biotinylation quality control molecule antibody: respectively diluting the labeled antibody and the quality control antibody to 1mg/mL by adopting buffer solution, respectively dialyzing to obtain a new coronavirus antigen labeled antibody solution and a quality control molecule capture antibody solution, respectively adding biotin solution with the concentration of 1mg/mL, reacting for 4 hours at room temperature, and respectively adding NH with the concentration of 1mol/L4And stirring the Cl solution at room temperature for reaction for 20min, and respectively dialyzing and purifying by a column to obtain the biotinylated new coronavirus antigen labeled antibody and the biotinylated quality control molecule antibody.
e) Preparing a colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and a colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody: mixing and oscillating the biotinylated new coronavirus antigen-labeled antibody and biotinylated quality control molecule antibody prepared in the step d) with the colloidal gold-labeled streptavidin prepared in the step c) respectively for 20min, centrifuging and removing supernate to obtain colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody respectively, and re-dissolving the antibodies with gold-labeled diluent respectively for later use.
f) Preparation of coupling pad: diluting the colloidal gold-labeled streptomycin-biotinylation new crown virus antigen-labeled antibody and the colloidal gold-labeled streptomycin-biotinylation quality control molecule antibody prepared in the step e) with a conjugate diluent to prepare a conjugate treatment solution, wherein the volume ratio of the colloidal gold-labeled streptomycin-biotinylation new crown virus antigen-labeled antibody is 18%, the volume ratio of the colloidal gold-labeled streptomycin-biotinylation quality control molecule antibody is 20%, and the conjugate treatment solution is manually and uniformly coated on a coupling pad.
g) The method comprises the following steps Preparation of the nitrocellulose membrane 102: diluting the nano cellulose gel (0.92 wt%) to a concentration of 0.5 wt%, scribing on the nitrocellulose membrane 102 by a synovial membrane machine to form a detection line 1021 and a quality control line 1022, drying at 45 ℃ for 12 hours, diluting the neo-corona antigen capture antibody and the quality control molecule capture antibody to 2mg/mL and 1mg/mL respectively by an NC membrane coating solution, scribing on the detection line 1021 and the quality control line 1022 by the synovial membrane machine, and drying at 45 ℃ for 72 hours.
h) Preparation of sample pad 104: the sample pad 104 treatment solution is applied uniformly to the sample pad 104.
i) Assembling the reagent strip: and assembling according to the structure of the reagent strip, cutting the assembled reagent strip to 4mm per strip, and respectively loading the cut reagent strip into the shell 106 to obtain the colloidal gold chromatography reagent strip 10 for detecting the neocorona antigen.
Example 2
In this embodiment, the colloidal gold chromatography reagent strip 10 for detecting a new corona antigen prepared in embodiment 1 is used to rapidly detect a new corona virus antigen in a fresh sample and match the new corona virus antigen with a PCR result, so as to evaluate the sensitivity and specificity of the colloidal gold chromatography reagent strip 10 for detecting a new corona antigen.
The results were compared with the PCR results by testing 514 fresh nasopharyngeal swabs at 3 different sites. Adding a nasopharyngeal swab into 450 mu L of virus lysate, performing up-and-down rotary extrusion for 3-10 times, taking 80 mu L of the lysate after full lysis, adding the resulting product into a sample adding hole 1062 of a colloidal gold chromatography reagent strip 10 for detecting new corona antigen for about 3 drops, and observing a test result after waiting for 15min, wherein the test result is shown in table 1, and the matching result is that the sensitivity is 96.49% as can be seen from table 1; the specificity was 99.25% and the accuracy 98.6%.
TABLE 1
Example 3
In this embodiment, the colloidal gold chromatography reagent strip 10 for detecting a neocorona antigen prepared in example 1 is used to rapidly detect a culture of a neocorona virus, so as to evaluate the minimum detection limit of the colloidal gold chromatography reagent strip 10 for detecting a neocorona antigen.
Sample preparation: inactivated Virus culture (ATCC) at a concentration of 1.6X 105TCID50and/mL, carrying out gradient dilution by using nasopharyngeal swab eluent of normal healthy people, firstly diluting by 10 times, and then diluting by 2 times from the previous concentration after testing a negative result. The concentration level of not less than 19 positive results out of 20 test results was selected as the lowest detection limit of the test.
As a result: performing heat inactivated virus culture gradient dilution test at a concentration of 2.0 × 102TCID50mL, the detection results were all positive, so the lowest detection limit of the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen prepared in example 1 was 2.0X 102TCID50and/mL. The color development of the cultures at different concentrations is shown in FIG. 2, with concentrations of 1.6X 103TCID50/mL、8.0×102TCID50/mL、4.0×102TCID50/mL、2.0×102TCID50/mL、1.0×102TCID50/mL。
Example 4
In this embodiment, the colloidal gold chromatography reagent strip 10 for detecting a new corona antigen prepared in embodiment 1 is used to rapidly detect a sample configured by a new corona virus culture, so as to evaluate the anti-interference capability of the colloidal gold chromatography reagent strip 10 for detecting a new corona antigen.
The method comprises the following steps: the negative sample is used to dilute the cell culture new type coronary virus liquid to 2 XLoD concentration level as a basic sample. Diluting the collected strains and virus culture stock solution by using negative samples to prepare cross samples with corresponding concentrations. Diluting the collected strains and virus culture stock solution by using a basic sample to prepare an interference sample with corresponding concentration. And diluting the negative sample by using the basic sample to prepare a control sample with a corresponding concentration. The detection is carried out by cross samples, interference samples and control samples.
The results show that: when the sample contains human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, adenovirus and parainfluenzaVirus, influenza A virus H1N1, influenza A virus H3N2, influenza B virus, enterovirus, respiratory syncytial virus less than or equal to 105TCID50mL, concentration of rhinovirus is less than or equal to 105PFU/mL, concentration of Mycoplasma pneumoniae is less than or equal to 106And in CFU/mL, the detection results of the cross samples meet the detection standard, and no cross reaction occurs. The detection result of the interference sample meets the detection standard, and when SARS-CoV-2 and other microorganisms (including various microorganisms, viruses and negative matrixes) exist in the sample, false negative does not occur. The specific results of the interference samples and the corresponding interference concentrations are shown in table 2 below.
TABLE 2
| Interfering substances | Concentration of |
| Mucins | 5mg/mL |
| Human red blood cell | 5％v/v |
| Zanamivir | 0.1mg/mL |
| Oseltamivir | 2.5μg/mL |
| Tobramycin | 1.0mg/mL |
| Interferon alpha | 3×105IU/mL |
| Ribavirin | 0.4mg/mL |
| Peramivir | 0.15mg/mL |
| Lopinavir | 0.2mg/mL |
| Levofloxacin | 0.2mg/mL |
| Azithromycin | 1μg/mL |
| Ritonavir | 0.3mg/mL |
| Histamine hydrochloride | 0.1mg/mL |
| Sodium chloride | 5％v/v |
| Oxymetazoline | 15％v/v |
| Dexamethasone | 1.2mg/dL |
| Fluticasone propionate | 5％v/v |
| Triamcinolone acetonide | 5％v/v |
In summary, in the colloidal gold chromatography reagent strip 10 for detecting neocorona antigen, the colloidal gold nanoflowers have larger space surface area, and can be more fully adsorbed with the coupled antibody, so that the detection sensitivity and the result stability are improved, and the surface of the colloidal gold nanoflowers have the pointed graded flower-shaped structures to show strong light signal sensitivity, so that the color sense under natural light is more obvious and the naked eye identification is facilitated; the invention can obtain more antigen-antibody binding epitopes through an avidin biotin signal amplification system; according to the invention, the nano-cellulose gel is pre-coated near the detection line 1021 to fill the micropores on the nitrocellulose membrane 102, so that more capture antibodies coated later are attached to the surface of the nitrocellulose membrane 102, and therefore, colloidal gold nanoparticles captured at a later stage are more remained on the surface of the nitrocellulose membrane 102, the color development effect is enhanced, and the visual effect of naked eyes is enhanced.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A preparation method of a colloidal gold chromatography reagent strip for detecting a neocorona antigen is characterized by comprising the following steps:
a) preparing a colloidal gold particle solution: adding HAuCl4Heating to 120 ℃ while stirring, adding 1/20-1/4 volumes of sodium citrate, and continuously heating for 10-30min, until the color of the solution is stable and is ruby, cooling and storing at 2-8 ℃;
b) preparing a colloidal gold nanoflower solution: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume of 60-100 times, heating to 60-80 ℃, and adding HAuCl4Stirring the solution, centrifuging the solution to remove supernatant, collecting the synthesized colloidal gold nanoflowers solution, and storing the colloidal gold nanoflowers solution at the temperature of between 2 and 8 ℃;
c) preparing colloidal gold-labeled streptavidin: taking the colloidal gold nanoflowers prepared in the step b), adjusting the pH value to 9-10 and the concentration to 0.01-0.05 wt%, adding a streptavidin solution, carrying out oscillation reaction for 5-30 min, adding BSA until the final concentration of BSA is 1-2 wt%, oscillating for 5-30 min, centrifuging, removing the supernatant to obtain colloidal gold labeled streptavidin, and re-dissolving the colloidal gold labeled streptavidin with a gold labeled diluent for later use;
d) preparing a biotinylation new coronavirus antigen labeled antibody and a biotinylation quality control molecule antibody: respectively diluting the labeled antibody and the quality control antibody to 0.5-2 mg/mL by adopting buffer solution, respectively dialyzing to obtain a new coronavirus antigen labeled antibody solution and a quality control molecule capture antibody solution, respectively adding biotin solution with the concentration of 0.5-2 mg/mL for reacting for 1-6 h at room temperature, and respectively adding biotin solution with the concentration of 0.5-2 mol/LNH4Stirring and reacting the Cl solution for 5-30 min at room temperature, and respectively dialyzing and purifying by a column to obtain a biotinylated new coronavirus antigen labeled antibody and a biotinylated quality control molecular antibody;
e) preparing a colloidal gold labeled streptomycin-biotinylation new coronavirus antigen labeled antibody and a colloidal gold labeled streptomycin-biotinylation quality control molecule antibody: mixing the biotinylated new crown virus antigen labeled antibody and biotinylated quality control molecule antibody prepared in the step d) with the colloidal gold labeled streptavidin prepared in the step c) respectively, performing oscillation reaction for 10-30 min, centrifuging, removing supernate, respectively obtaining colloidal gold labeled streptomycin-biotinylated new crown virus antigen labeled antibody and colloidal gold labeled streptomycin-biotinylated quality control molecule antibody, and re-dissolving with a gold labeled diluent respectively for later use;
f) preparation of coupling pad: diluting the colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody and the colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody prepared in the step e) with a conjugate diluent to prepare a conjugate treatment solution, wherein the volume ratio of the colloidal gold-labeled streptomycin-biotinylated new coronavirus antigen-labeled antibody is 18-20%, the volume ratio of the colloidal gold-labeled streptomycin-biotinylated quality control molecule antibody is 20-25%, and the conjugate treatment solution is uniformly coated on a coupling pad;
g) preparing a nitrocellulose membrane: diluting the nano cellulose gel to the concentration of 0.1 wt% -0.5 wt%, scribing on a nitrocellulose membrane to form a detection line and a quality control line, and drying; diluting the new crown antigen capture antibody and the quality control molecule capture antibody to 1-3 mg/mL and 0.1-2 mg/mL respectively by using NC membrane coating solution, scribing on a detection line and a quality control line, and drying;
h) preparation of sample pad: uniformly coating the sample pad treatment solution on the sample pad;
i) assembling the reagent strip: and assembling according to the structure of the reagent strip.
2. The method for preparing a colloidal gold chromatography reagent strip for detecting a neocorona antigen as claimed in claim 1, wherein the step b) comprises: adding the colloidal gold particle solution prepared in the step a) into purified water with the volume of 60-100 times, heating to 60-80 ℃, and adding HAuCl with the volume of 0.5-3 times4And 1-7 times of sodium citrate by volume, stirring for 15-60 s, adding 10-40 times of hydroquinone solution by volume, stirring for 3-6 h, and centrifuging.
3. The method for preparing the colloidal gold chromatography reagent strip for detecting neocorona antigen as claimed in claim 1, wherein in the step c), the final concentration of the streptavidin solution is 10-80 μ g/mL;
and/or the NC film coating solution is prepared from 0.01-0.5 mol/LpH 6.5.5-7.5 of PBS and 3-10 wt% of trehalose solution.
4. The method for preparing the colloidal gold chromatography reagent strip for detecting a neocorona antigen as claimed in any one of claims 1 to 3, wherein the absorption amount of the sample pad treatment solution on the sample pad is 25 to 50 ± 3mL, and the sample pad is dried at 50 to 70 ℃ for 24 hours or naturally dried at room temperature;
and/or cutting the assembled reagent strips to 2-4 mm/strip, and respectively filling the reagent strips into the shell.
5. The method for preparing the colloidal gold chromatography reagent strip for detecting neocorona antigen according to any one of claims 1 to 3, wherein in step i), when the reagent strip is assembled according to the structure of the reagent strip, the structure of the reagent strip comprises a bottom plate, a nitrocellulose membrane, a coupling pad, a sample pad and absorbent paper, the sample pad, the coupling pad, the nitrocellulose membrane and the absorbent paper are sequentially connected to the bottom plate along a horizontal direction, avidin-biotin-amplified colloidal gold popcorn is coupled to the coupling pad, the nitrocellulose membrane is modified by nanocellulose, a detection line coated with a neocorona antigen capture antibody and a quality control line coated with a quality control molecule capture antibody are arranged on the nitrocellulose membrane, and the detection line and the quality control line are sequentially distributed along a chromatography direction.
6. The method for preparing the colloidal gold chromatography reagent strip for detecting neocoronary antigen according to claim 5, wherein the reagent strip has a structure in which the coupling pad and the absorbent paper are respectively positioned on two sides of the nitrocellulose membrane and are partially overlapped with the nitrocellulose membrane, and the sample pad is partially overlapped with the coupling pad.
7. The method according to claim 4, wherein the housing has a receiving chamber, a detection window and a sample application hole, the detection window and the sample application hole are communicated with the receiving chamber, the bottom plate, the nitrocellulose membrane, the coupling pad, the sample pad and the absorbent paper are all disposed in the receiving chamber, the detection line and the quality control line are located at the detection window, and a portion of the sample pad is located at the sample application hole.
8. The method for preparing the colloidal gold chromatography reagent strip for detecting neocorona antigen as claimed in claim 7, wherein a detection identification region and a quality control identification region corresponding to the detection line and the quality control line respectively are provided on an outer surface of the housing.
9. The method for preparing a colloidal gold chromatography reagent strip for detecting a neocorona antigen as claimed in claim 7, wherein the well is tapered from an outward end to an inward end.
10. A new crown antigen detection kit, comprising virus lysate and the colloidal gold chromatography reagent strip prepared by the preparation method of any one of claims 1 to 9.
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| CN114414799A (en) * | 2021-12-27 | 2022-04-29 | 深圳德夏科技发展有限公司 | New coronavirus detection chromatography reagent strip, kit and preparation method thereof |
| CN115166237A (en) * | 2022-07-29 | 2022-10-11 | 复旦大学 | Colloidal gold chromatography reagent strip capable of controlling self-detection sampling procedure of neocorona antigen and preparation method and application thereof |
| CN116338179A (en) * | 2023-01-13 | 2023-06-27 | 安徽国泰国瑞医疗科技有限公司 | Acute severe respiratory syndrome coronavirus antigen detection kit and application thereof |
| CN116400070B (en) * | 2023-06-07 | 2023-08-18 | 济南德亨医学科技有限公司 | Preparation method of reagent for quantitatively detecting allergen-specific IgE antibody with high flux |
| CN117368464B (en) * | 2023-10-12 | 2024-09-17 | 科尼格沃斯(无锡)医学科技有限公司 | Nano gold microsphere compound and preparation method and application thereof |
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