CN115290884A - Neocorolla antigen detection kit and preparation method thereof - Google Patents
Neocorolla antigen detection kit and preparation method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to a neocorolla antigen detection kit and a preparation method thereof, and the method comprises the following steps: s1 preparation of an antibody-coated nitrocellulose membrane: respectively preparing the concentration of a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and a quality control antibody goat anti-chicken IgY by using membrane coating solutions, and respectively coating the membrane coating solutions on nitrocellulose membranes; s2, processing the sample pad; s3, marking and preparing a metal spraying bonding pad: respectively combining the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microspheres, respectively diluting into a gold spraying solution of the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and a gold spraying solution of the chicken IgY-bicolor microsphere conjugate, spraying the gold spraying solutions onto a conjugate pad, and drying; and S4, cutting and adhering the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection plate, and putting the adhered detection plate into a corresponding card shell for assembling to obtain the detection card.
Description
Technical Field
The invention relates to the technical field of fluorescence immunoassay, and relates to a neocorona antigen detection kit and a preparation method thereof.
Background
The novel coronavirus is a novel coronavirus which can cause pneumonia/pulmonary infection of human, belongs to a coronavirus family with common cold, severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), and is named as 2019 novel coronavirus, namely 2019-nCoV, by the World Health Organization (WHO). The novel coronavirus pneumonia is a serious II infectious disease, has strong infectivity and a death rate of 3-6 percent, and causes huge damage to the world economic development in 2020. There are currently three types of novel coronavirus assays, which are: nucleic acid detection, antibody detection and antigen detection. The window periods of the three detection methods are different, nucleic acid/antibody/antigen detection is respectively emphasized and cannot be replaced mutually, and the detection window periods can be effectively shortened, the positive detection rate is improved and double guarantees are provided for various possible risk groups by applying multiple methods for combined detection.
Chinese patent document (application number: 202120754366.3) discloses a novel coronavirus S antigen detection kit, which consists of a colloidal gold immunochromatography detection test strip and a detection clamp, wherein the detection clamp is detachably connected with a detection result observation assembly by a surface tension weakening device; the detection clamp is of a hollow structure, and the detection test strip is detachably placed in the detection clamp.
Disclosure of Invention
The prepared new corona antigen detection kit adopts a fluorescence immunoassay technology to provide a quick and accurate detection result, can be seen qualitatively by eyes and can also be used for detecting a fluorescence signal by an instrument by simultaneously dyeing polystyrene microspheres by fluorescent dye and color dye, and has the advantages of low cost, high sensitivity, more flexible detection and wider application range, thereby better coping with the spread of novel coronavirus under a new situation.
In order to solve the technical problems, the technical scheme adopted by the invention is that the preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a membrane coating solution for a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1, preparing the concentration of a membrane coating solution for a quality control antibody goat anti-chicken IgY, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
s2 treatment of the sample pad: treating the sample pad by using the treatment solution, and drying the treated sample pad in an oven for later use;
s3, marking and preparing a metal spraying bonding pad: respectively marking the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microspheres, respectively diluting the marked monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microsphere combinations into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere combinations gold spraying solution and a chicken IgY-bicolor microsphere combinations gold spraying solution by using microsphere preservation solution, spraying the gold spraying solutions onto a combination pad, placing the combination pad in a drying chamber, drying the combination pad in an oven, and drying the combination pad for later use;
s4, assembling the detection card: cutting and sticking the coated nitrocellulose membrane, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection plate, and putting the stuck detection plate into a corresponding card shell for detection card assembly to obtain the detection card.
As a preferred technical solution of the present invention, the method further comprises step S5 of preparing a sample extract: preparing and subpackaging a sample extracting solution; the extract is used for extracting a sample and is used as a component of a neocorona antigen detection kit for sample detection.
As a preferred technical solution of the present invention, the step S1 specifically comprises the steps of:
s11: preparing a detection line coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 into the detection line coating solution to ensure that the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 is 1.0-2.5 mg/mL;
s12: preparing a quality control line coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating the detection line coating solution added with the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control line coating solution added with the quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm;
s14: drying in a 45 ℃ oven for 12-20 h for later use.
The novel coronavirus (2019-nCoV) antigen detection kit adopts the principle of antigen-antibody reaction, and a reaction antibody is a specific antibody aiming at the novel coronavirus (2019-nCoV) N protein. Due to capillary action, the test sample will move forward and then the analyte of the sample will bind to the antibody attached to the red carboxyl microspheres. This labeled complex is attached to the detection zone where the antibody is immobilized, while the blue carboxyl microspheres are attached to the control zone. If the sample contains a novel coronavirus (2019-nCoV) antigen, a red strip appears at the position of a T line, goat anti-chicken IgY marked by blue carboxyl microspheres is combined with goat anti-chicken IgY antibodies on a C line, and a blue strip appears at the position of the C line and is used as a quality control line. The detection methods commonly used in clinic and laboratory include colloidal gold method, immunofluorescence method, etc.
As a preferred technical solution of the present invention, the step S3 specifically comprises the steps of:
s31: mixing the microsphere marking solution and the bicolor microsphere particles, respectively carrying out marking reaction on the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY, and respectively preparing a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and a chicken IgY-bicolor microsphere marker after centrifugation;
s32: and then diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and the chicken IgY-bicolor microsphere marker into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution respectively by using microsphere preservation solutions, spraying the gold spraying solutions onto the conjugate pad treated in the step S2 through a film-scribing gold spraying instrument, putting the conjugate pad into a 45-DEG C oven for drying, drying for 4 hours, and drying for later use. As a preferred technical scheme of the invention, the microsphere marking solution in the step S31 comprises 5mM MES or 5mM HEPES or 10mM PB, and the particle size of the bicolor microsphere particles is 100-300 nm; the mass ratio of the monoclonal antibody 2019-nCoV N protein antibody 2 labeled antibody to the bicolor microspherical particles is (10-20): 1.
In the step S32, the 2019-nCoV N protein antibody 2 labeled antibody-two-color microsphere conjugate is diluted into 1-2.5 mg/mL of antibody-microsphere conjugate gold spraying solution; the concentration of the chicken IgY-bicolor microsphere conjugate is 0.3-1 mg/mL; and the film-scribing metal spraying instrument sprays metal according to the spraying amount of 2.0 mu L/cm.
As a preferred technical solution of the present invention, the formulation of the extraction solution in the step S5 includes, by mass or volume percentage: PBS buffer solution 10nM, emulsifier 1-2% and Proclin300 0.01-0.03%, pH value is 7.4-7.8 + -1.
As a preferred technical solution of the present invention, in the step S31, the microsphere labeling solution is 10mM PB labeling buffer solution, the two-color microsphere particles are polystyrene microspheres in which a fluorescent dye and a chromatic dye coexist, the particle size is 300nm, and the usage amount by mass ratio of the 2019-nCoV N protein antibody 2 labeled antibody to the two-color microsphere particles is 15; in the step S32, the 2019-nCoV N protein antibody 2 labeled antibody-bicolor microsphere conjugate is diluted into a 1.5mg/mL monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere marker is 0.5mg/mL.
As a preferred technical solution of the present invention, the emulsifier in the extraction solution in the step S5 is Tween20 or/and Tween80; in the step S11, 2019-nCov (I09C 3M) antibody is added into the detection line coating solution, so that the final concentration of the antibody is 1.5mg/mL.
In a preferred embodiment of the present invention, the recipe for preparing 1000mL of the treatment solution in step S2 includes: 6-9 g of S9 surfactant, 9-11 g of BSA, 25-35 mL of LS009 intermediate solution, 0.5-0.6 mL of blocking agent, 0.15-0.25 mL of ProClin, and the balance of LS002 intermediate solution.
In a preferred embodiment of the present invention, the formula for preparing 100mL of the microsphere preservation solution in step S3 is: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water.
As a preferable technical scheme of the invention, the formula of the blocking agent is as follows: sodium azide at 0.02% mass to volume and 10mM PBS, pH 7.4; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL Tween20, 0.02mL ProClin300, and the balance purified water.
As a preferred technical scheme of the present invention, the two-color microsphere in step S31 is a polystyrene microsphere in which Eu (DBM) 3Phen fluorescent dye and oil-soluble yellow R color dye coexist, and the preparation method thereof includes the following steps:
s311: preparation of fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a ratio, reacting the DBM and ethanol mixed solution and the Phen and ethanol mixed solution with the rare earth ion and ethanol mixed solution, and filtering and drying to obtain fluorescent dye powder;
s312 preparation of polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid;
s313 preparation of polystyrene color microspheres: mixing the polystyrene microsphere solution obtained in the step S312 with a color dye, emulsifying, and centrifuging to remove supernatant to obtain polystyrene color microspheres;
s314 preparation of polystyrene PS bicolor microspheres: and (2) mixing the fluorescent dye powder prepared in the step (S1) with the polystyrene colorful microspheres prepared in the step (S313), emulsifying, and centrifuging to remove supernatant to obtain the polystyrene PS double-color microspheres.
As a preferred embodiment of the present invention, the step S311 specifically includes:
s3111: weighing raw materials including Eu 3+ Salt, DBM and Phen for standby;
s3112: respectively dissolving DBM and Phen in absolute ethyl alcohol to obtain a mixed solution of DBM and ethyl alcohol and a mixed solution of Phen and ethyl alcohol;eu is mixed 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s3113: adding a DBM and ethanol mixed solution into an anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, refluxing and stirring the mixture for reaction, dropwise adding a Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel for continuous reaction, adjusting the pH value of a reaction solution, and then continuously refluxing and stirring the mixture for reaction until the reaction is finished;
s3114: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder.
As a preferred technical solution of the present invention, the step S312 specifically includes:
s3121: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring;
s3122: when the temperature of the water bath is raised to 70-80 ℃, adding initiator ammonium persulfate, and continuously stirring for reaction to obtain uniform white emulsion, namely the polystyrene microsphere liquid.
As a preferred embodiment of the present invention, the step S313 specifically includes:
s3131: dispersing the polystyrene microsphere liquid obtained in the step S312 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
s3132: mixing the microsphere solution and the dye swelling medium mixed solution in the step S3131, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature;
s3133: and (3) removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, and finally dissolving the obtained precipitate in the pure water to obtain the PS colored microsphere liquid.
As a preferred embodiment of the present invention, the step S314 specifically includes:
s3141: dispersing the PS color microsphere solution obtained in the step S3133 in the swelling solutionIn a medium; eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium to obtain a fluorescent dye swelling medium mixed solution;
s3142: mixing the microsphere solution and the mixed solution of the fluorescent dye swelling medium in the step S3141, emulsifying, namely carrying out oscillation reaction at room temperature, adding methacrylic acid (MAA) after reacting for a period of time (the time needs to be considered according to the preparation amount of the microspheres, the particle size and other factors, generally 1-4 h), and continuing to react for 3-6h;
s3143: and (3) removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, and finally dissolving the obtained precipitate in the pure water to obtain the carboxyl PS double-color microsphere liquid. The microspheres are stored by using pure water as a storage solution and cannot be directly stored, so that the prepared carboxyl PS bicolor microspheres are dissolved in the pure water.
As the preferred technical scheme of the invention, the preparation method of the neocorona antigen detection kit further comprises the following steps of packaging in a detection card S6: putting the detection card and the drying agent into an aluminum foil bag for packaging, and making a mark; s7, detection card outer package: the detection card, the instruction book and the extraction tube are put into an external packing box, and the self-adhesive sticker of the packing box is pasted.
The invention also aims to solve the technical problem of providing a neocorona antigen detection kit.
In order to solve the technical problems, the detection card of the neocorona antigen detection kit comprises a PVC base plate, a nitrocellulose membrane, a combination pad, a water absorption pad and a sample pad from bottom to top in sequence, wherein the combination pad and the water absorption pad are respectively arranged at two ends of the nitrocellulose membrane and are positioned on the same horizontal plane, the sample pad is arranged on the combination pad, the nitrocellulose membrane is coated with a detection line and a quality control line through an antibody, and the widths of the nitrocellulose membrane, the sample pad, the water absorption paper and the PVC base plate are all 3mm.
Compared with the prior art, the invention has the beneficial effects that: the new corona antigen detection kit prepared by the preparation method of the new corona antigen detection kit can provide a quick and accurate detection result by adopting a fluorescence immunoassay technology, can be seen qualitatively by eyes and can detect a fluorescence signal by an instrument so as to perform quantitative calculation by simultaneously dyeing polystyrene microspheres by fluorescent dye and color dye, and has the advantages of low cost, high sensitivity, more flexible detection and wider application range, thereby better coping with the new coronavirus transmission under a new situation.
Drawings
FIG. 1 is a structural diagram of a neocorona antigen detection kit prepared by the method of the present invention.
Detailed Description
Example (b): as shown in figure 1, this neocoronal antigen detection kit detects card includes PVC bottom plate, nitrocellulose membrane, combination pad, water absorption pad and sample pad from supreme down in proper order, wherein the combination pad is established respectively with the water absorption pad the both ends of nitrocellulose membrane just are in on the same horizontal plane, the sample pad is established on the combination pad, nitrocellulose membrane has detection line and quality control line through the antibody coating, the width size of nitrocellulose membrane, sample pad, absorbent paper and PVC bottom plate is 3mm.
The preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 by using a film coating solution, preparing the concentration of a quality control antibody goat anti-chicken IgY by using a film coating solution, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
the step S1 comprises the following specific steps:
s11: preparing a detection line (T line) coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 into the detection line coating solution to ensure that the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 is 1.0-2.5 mg/mL; in the step S11, the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 added into the detection line coating solution is 1.5mg/mL;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating the detection line coating solution added with the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control line coating solution added with the quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 12-20 h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (prepared 1000 mL) of the treatment solution in step S2 includes: 6-9 g of S9 surfactant, 9-11 g of BSA, 25-35 mL of LS009 intermediate solution, 0.5-0.6 mL of blocking agent, 0.15-0.25 mL of ProClin, and the balance of LS002 intermediate solution; the formula of the blocking agent is as follows: 0.02% (mass/volume) of sodium azide and 10mM of PBS, and the pH thereof is 7.4; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300, and the balance of purified water.
S3, marking and preparing a metal spraying bonding pad: respectively marking the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microsphere particles, respectively diluting the marked monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microsphere conjugates into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solution, spraying the gold spraying solutions onto a conjugate pad, placing the conjugate pad in a drying room, drying the conjugate pad in an oven, and drying the conjugate pad for later use;
the specific steps of the step S3 are as follows:
s31: mixing the microsphere marking solution and the bicolor microsphere particles, respectively carrying out marking reaction on the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY, and respectively preparing a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and a chicken IgY-bicolor microsphere conjugate after centrifugation; the microsphere marking solution in the step S31 comprises 5mM MES or 5mM HEPES or 10mM PB, and the particle size of the bicolor microsphere particles is 100-300 nm; the mass ratio of the monoclonal antibody 2019-nCoV N protein antibody 2 labeled antibody to the bicolor microspherical particles is (10-20): 1; the bi-color microspherical particles are polystyrene microspheres with coexisting fluorescent dye and chromatic dye, the particle size is 300nm, and the dosage mass ratio of the 2019-nCoV N protein antibody 2 labeled antibody to the bi-color microspherical particles is 15; diluting the 2019-nCoV N protein antibody 2 labeled antibody-bicolor microsphere label into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution with the concentration of 1.5mg/mL in the step S32; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere marker is 0.5mg/mL;
s32: respectively diluting the monoclonal anti-2019-nCoV N protein antibody 2-bicolor microsphere conjugate and the chicken IgY-bicolor microsphere conjugate into a monoclonal anti-2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preserving fluid, spraying the gold spraying solution onto the conjugate pad treated in the step S2 through a film-scribing gold spraying instrument, putting the conjugate pad into a 45-DEG C oven for drying, drying for 4 hours, and drying for later use; in the step S32, the 2019-nCoV N protein antibody 2 labeled antibody-two-color microsphere conjugate is diluted into 1-2.5 mg/mL antibody-microsphere conjugate gold spraying solution; the concentration of the chicken IgY-bicolor microsphere conjugate is 0.3-1 mg/mL; the film-scribing metal spraying instrument sprays metal according to the spraying amount of 2.0 mu L/cm; diluting the antibody-microsphere marker into 1.5mg/mL antibody-microsphere conjugate gold spraying solution in the step S32; the concentration of the goat anti-chicken IgY-microsphere conjugate gold spraying solution is 0.5mg/mL; the formula for preparing 100mL of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s4, assembling a detection card: cutting and sticking the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection plate, and putting the stuck detection plate into a corresponding card shell for detecting card assembly to obtain a detection card;
s5, preparing a sample extract: preparing and subpackaging a sample extract; the formula of the extraction solution in the step S5 comprises the following components in percentage by mass or volume: PBS buffer solution 10mM, 1-2% emulsifier and 0.01-0.03% Proclin300, the pH value is 7.4-7.8 +/-1; the emulsifier in the extract in the step S5 is Tween20 or/and Tween80;
s6, packaging in the detection card: putting the detection card and the drying agent into an aluminum foil bag for packaging, and making a mark;
s7, detection card outer packaging: the detection card, the instruction book and the extraction tube are put into an external packing box, and the self-adhesive sticker of the packing box is pasted.
The two-color microsphere in the step S31 is a polystyrene microsphere with Eu (DBM) 3Phen fluorescent dye and oil-soluble yellow R color dye coexisting, and the preparation method comprises the following steps:
preparation of S311 fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a ratio, reacting the DBM and ethanol mixed solution, the Phen and ethanol mixed solution and the rare earth ion and ethanol mixed solution, and filtering and drying to obtain fluorescent dye powder;
the specific steps of step S311 include:
s3111: weighing raw materials including Eu 3+ Salt, DBM, phen and absolute ethyl alcohol for later use; the ratio of the amounts of the substances in the step S11 is as follows: eu (Eu) 3+ Weighing raw materials according to the weight ratio of DBM to Phen = 1;
s3112: respectively dissolving DBM and Phen in absolute ethyl alcohol to obtain a mixed solution of DBM and ethyl alcohol and a mixed solution of Phen and ethyl alcohol, and adding Eu 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s3113: adding DBM and ethanol mixed solution into anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, carrying out reflux stirring reaction, dropwise adding the Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel, continuously reacting, adjusting the pH value of the reaction solution, and then continuously carrying out reflux stirring reaction until the reaction is finished; in the step S3113, heating at 60-70 ℃, carrying out reflux stirring reaction for 0.5-1.5 h, dropwise adding a mixed solution of Phen and ethanol into a three-neck flask by using a constant-pressure dropping funnel, continuously reacting for 20-40 min, adjusting the pH value of the reaction solution to 6-7, and continuously heating, carrying out reflux stirring reaction for 3-5 h until the reaction is finished;
s3114: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder; in the step S3114, the reaction solution is transferred into a clean open container, is kept stand at room temperature for 24 hours, is cooled for 20-40 min by using an ice water bath, is subjected to suction filtration, and is dried at the temperature of 60-70 ℃ to obtain Eu (DBM) 3 Phen fluorescent dye powder (namely fluorescent dye complex Eu (DBM) 3 The Phen-fluorescence intensity is high, and the preparation cost is low);
s312 preparation of polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid (PSt);
the specific step of step S312 includes:
s3121: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring; the capacity of the three-mouth bottle in the step S3121 is 500mL, and the three-mouth bottle is provided with a condensation pipe and a mechanical stirrer;
s3122: heating in a water bath to 70-80 ℃, adding an initiator Ammonium Persulfate (APS) (dissolving 100mg of APS in 5mL of pure water), and continuously stirring for reaction for 20 hours to obtain a uniform white emulsion, namely polystyrene microsphere liquid (PSt);
s313 preparation of polystyrene color microspheres: mixing and emulsifying the polystyrene microsphere liquid obtained in the step S312 by using a color dye, and centrifuging to remove supernatant liquid to obtain polystyrene color microspheres;
the step S313 specifically includes:
s3131: dispersing the polystyrene microsphere solution obtained in the step S312 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
in the step S3131, 50 to 100 μ L of polystyrene microsphere solution with a mass fraction of 10% is weighed and dispersed in a swelling medium (50% acetone aqueous solution), and 5.2mg of oil-soluble yellow R dye is dissolved in the swelling medium (50% acetone aqueous solution);
s3132: mixing the microsphere solution and the dye swelling medium mixed solution in the step S3131, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature; mixing the microsphere solution and the dye swelling medium mixed solution in the step S3131 in the step S3132, and then performing ultrasonic treatment for 0-15 min, preferably for 5min; ultrasonic treatment is carried out for 0-10 min every 1-2 h in the emulsification reaction; preferably, the ultrasonic treatment is carried out for 3min every 1 h;
s3133: removing swelling medium (50% acetone aqueous solution) by rotary evaporation at 40 deg.C, performing ultrasonic treatment, diluting the rest liquid gradually, centrifuging (rotation speed of 16500rpm, centrifuging for 15 min), and observing whether the centrifuged precipitate is complete and the color of supernatant remains; discarding the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, removing the swelling medium solution, washing for multiple times by using the pure water until no color residue exists in the supernatant after centrifugation, removing the oil-soluble yellow R dye which does not enter the interior of the microsphere, and finally dissolving the obtained precipitate in 500 mu L of pure water to obtain PS colored microsphere solution; the ultrasonic treatment time in the step S3133 is 1-15 min, preferably 5min;
s314 preparation of polystyrene PS bicolor microspheres: mixing the fluorescent dye powder prepared in the step S311 with the polystyrene colorful microspheres prepared in the step S313, emulsifying, and centrifuging to remove supernatant to obtain polystyrene PS double-color microspheres;
the step S314 specifically includes:
s3141: dispersing the PS colored microsphere liquid (namely 5 mg-10 mg) obtained in the step S313 into 500 mu L of swelling medium (50% acetone aqueous solution); eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium (50% acetone aqueous solution) to obtain a fluorescent dye swelling medium mixed solution; said step (c) isS3141, 50 or 100 mu L of polystyrene colorful microsphere liquid with the mass fraction of 10 percent is weighed and dispersed in a swelling medium;
s3142: mixing the microsphere solution obtained in the step S3141 with the mixed solution of the fluorescent dye swelling medium, emulsifying, namely carrying out oscillation reaction at room temperature, adding 100 mu L of methacrylic acid (MAA) after reacting for 1h, and continuing to react for 4h; the step S3142 of mixing the microsphere solution and the dye swelling medium mixed solution in the step S3141 and then carrying out ultrasonic treatment, wherein the ultrasonic treatment time is 0-15 min, preferably 5min, and the ultrasonic treatment is carried out for 0-10 min every 1-2 h in the emulsification reaction; preferably, the ultrasonic treatment is carried out for 3min every 1 h;
s3143: removing swelling medium (50% acetone aqueous solution) by rotary evaporation at 40 deg.C, performing ultrasonic treatment, diluting the rest liquid gradually, centrifuging, and observing whether the centrifuged precipitate is complete and the fluorescence intensity of the supernatant; discarding supernatant, washing the lower layer solid precipitate with pure water for 3 times, removing swelling medium solution, washing with pure water for several times until supernatant has no fluorescence after centrifugation, and removing Eu (DBM) not entering into microsphere 3 Phen, finally dissolving the obtained precipitate in 500 mu L of pure water to obtain PS bicolor microsphere solution; the time of the ultrasonic treatment in the step S3143 is 1-15 min, preferably 5min.
Specific example 1: selecting the sample extracting solution in the step S5, optimizing the sample extracting solution for optimizing the product performance, and selecting three sample extracting solutions in the following table 1 for analysis;
TABLE 1 formulation of sample extracts
The preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a membrane coating solution for detecting an antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody), preparing the concentration of a quality control antibody goat anti-chicken IgY (IgY), coating the detecting antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use; the step S1 comprises the following specific steps:
s11: preparing a detection line (T line) coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) into the detection line coating solution to enable the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) to be 1.5mg/mL;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating a detection line coating solution added with a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) and a quality control line coating solution added with a quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 16h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (prepared 1000 mL) of the treatment solution in step S2 includes: 7g of S9 surfactant (manufacturer: schhuier Biotech Co., ltd., yangzhou, cat # SPBHS 9), 10g of BSA, 970mL of LS002, 30mL of LS009, 0.5mL of blocker (the formula of the blocker is 0.02% sodium azide by mass/volume, 10mM of PBS, pH 7.4), 0.2mL of ProClin 300; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s3, marking and preparing a metal spraying bonding pad: respectively labeling the monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY with bicolor microspheres, respectively diluting the labeled monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY and bicolor microsphere conjugates into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solutions, spraying the gold spraying solutions onto a conjugate pad, placing the conjugate pad in a drying room, placing the conjugate pad in an oven at 45 ℃ for drying for 4 hours, and drying for later use; the formula of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water;
the specific steps of the step S3 are as follows:
s31: marking a monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) antibody and chicken IgY (IgY) by using 10mM PB marking buffer solution and 300nm bicolor microspherical particles, centrifuging and drying to respectively prepare a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microspherical conjugate and a chicken IgY-bicolor microspherical conjugate; wherein the preparation method of the bicolor microspherical particles adopts the preparation method of the bicolor microspherical particles in the step S31 in the embodiment; in the step S31, the microsphere marking solution is 10mM PB, and the particle size of the bicolor microsphere particles is 300nm; the mass ratio of the monoclonal antibody 2019-nCoV N protein antibody 2 labeled antibody to the bicolor microspherical particles is 15;
s32: diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate into 1.5mg/mL monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution by using a microsphere preservation solution; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere conjugate is 0.5mg/mL; spraying gold by using a film-scribing gold spraying instrument according to the spraying amount of 2.0 mu L/cm; spraying onto the bonding pad, placing in a drying room, and drying in a 45 deg.C oven for 4 hr;
s4, assembling a detection card: cutting and sticking the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection plate, and putting the stuck detection plate into a corresponding card shell for detecting card assembly to obtain a detection card;
s5, preparing a sample extract: preparing and subpackaging sample extract according to table 1; respectively adding 100 mu L of sample extract to test for 3 times (S1-S8 are N protein recombinant antigen dilution reference products), and S9-S10 are blank reference products; the obtained test results are shown in table 2.
TABLE 2 test results of three sample extracts
Wherein, the signal test value is negative G4, and is positive G4, the antigen concentration represented by the numerical value is higher, the color is more obvious, and S1-S10 are reference substances. As can be seen from the test results in Table 2, the three sample extracts have light colors and no obvious distinction when tested in S7-S10, and the sample extract-3 has good performance when distinguished from yin and yang, and meanwhile, the fluorescence signal detection result also conforms to the result trend. Thus sample extract-3 is optimal, its formulation is 10mM PBS +2% Tween80+0.02% Proclin300, pH 7.4. + -.1.
Specific example 2: determination of microsphere labeling conditions (labeling buffer, antibody amount).
The preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a membrane coating solution for a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1, preparing the concentration of a membrane coating solution for a quality control antibody goat anti-chicken IgY, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use; the step S1 comprises the following specific steps:
s11: preparing a detection line (T line) coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) in the detection line coating solution to enable the final concentration of the antibody to be 1.5mg/mL;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating the detection line coating solution added with the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control line coating solution added with the quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 15h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (formulation 1000 mL) of the treatment solution in step S2 includes: 7g of S9 surfactant (manufacturer: schhuier Biotech Co., ltd., yangzhou, cat # SPBHS 9), 10g of BSA, 970mL of LS002, 30mL of LS009, 0.5mL of blocker (the formula of the blocker is 0.02% sodium azide by mass/volume, 10mM of PBS, pH 7.4), 0.2mL of ProClin 300; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s3, marking and preparing a metal spraying bonding pad: respectively mixing a monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and chicken IgY with a microsphere marking solution, then marking the mixture with bicolor microspheres, respectively diluting the marked monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and chicken IgY with a bicolor microsphere conjugate with a microsphere preservation solution to obtain a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution, spraying the gold spraying solutions onto a conjugate pad, placing the pad in a drying chamber, placing the pad in a 45 ℃ oven for drying, wherein the drying time is 4 hours, and drying for later use; the formula of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water;
the specific steps of the step S3 are as follows:
s31: labeling the monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M) antibody) and the chicken IgY according to the labeling process of the following table 3, centrifuging and drying to respectively prepare a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and a chicken IgY-bicolor microsphere conjugate;
TABLE 3 different amounts of labeling buffer and antibody for the groups
| Experimental group | Labeling buffer | Microsphere particle (particle size) | Antibody dosage (microsphere ratio) |
| 1 | 5mM MES | 100nm | 10:1 |
| 2 | 5mM MES | 200nm | 15:1 |
| 3 | 5mM MES | 300nm | 20:1 |
| 4 | 5mM HEPES | 100nm | 15:1 |
| 5 | 5mM HEPES | 200nm | 20:1 |
| 6 | 5mM HEPES | 300nm | 10:1 |
| 7 | 10mM PB | 100nm | 20:1 |
| 8 | 10mM PB | 200nm | 10:1 |
| 9 | 10mM PB | 300nm | 15:1 |
In each group, 2019-nCov (H08A 1M) antibody was respectively labeled according to the process parameters in Table 3, after preparing an antibody-microsphere conjugate by centrifugation, the antibody-microsphere conjugate was sprayed onto the treated conjugate pad by a film-cutting gold-spraying instrument at 2.0. Mu.L/cm (2019-nCov (H08A 1M) microsphere label 1.5mg/mL; chicken IgY microsphere label 0.5 mg/mL), and dried for use.
S32: diluting the 9 groups of monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugates in the step S31 into 1.5mg/mL monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere conjugate is 0.5mg/mL; spraying gold on the bonding pad by using a film-scribing gold spraying instrument according to the spraying amount of 2.0 mu L/cm; spraying onto the bonding pad, placing in a drying room, and drying in a 45 deg.C oven for 4 hr;
s4, assembling a detection card: cutting the coated film, the sprayed gold combination pad, the processed sample pad and the absorbent paper, then pasting the cut films into a detection plate, putting the pasted detection plate into a corresponding card shell for detection card assembly, and obtaining 9 groups of detection cards; the results of detection of S1-S10 (S1-S8 are N protein recombinant antigen dilution references and S9-S10 are blank references) with 9 sets of detection cards are shown in Table 4.
TABLE 4 detection results of detection cards obtained by using different amounts of labeled buffer and antibody in groups
Wherein, the signal test value is negative G4 or more, the signal test value is positive G4, the larger the numerical value is, the higher the antigen concentration represented by the numerical value is, the more obvious the color is, and S1-S10 are reference substances (S1-S8 are N protein recombinant antigen dilution reference substances, and S9-S10 are blank reference substances). The results in table 4 show that, in the tests S1 to S10, the measured values of the experimental group nine are relatively good overall, the overall color is clearly distinguished, and meanwhile, the fluorescence signal detection result also conforms to the trend of the result, so the labeling process of the experimental group nine, that is, the labeling process with 10mM PB labeling buffer, 300nm two-color microspheres, and 15 antibody usage amount, is finally selected.
Specific example 3: and determining the concentration of the gold spraying solution of the labeled antibody-microsphere conjugate, searching the optimal concentration of the gold spraying solution for optimizing the product performance, and performing gold spraying treatment on the conjugate pad by adopting different concentrations of the gold spraying solution (the gold spraying solution of the labeled antibody-microsphere conjugate is diluted into antibody-microsphere conjugate of 1mg/mL, 1.5mg/mL, 2mg/mL and 2.5 mg/mL).
The preparation method of the neocorona antigen detection kit comprises the following steps:
s1, preparing a nitrocellulose membrane coated with an antibody: preparing the concentration of a membrane coating solution for a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1, preparing the concentration of a membrane coating solution for a quality control antibody goat anti-chicken IgY, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
the step S1 comprises the following specific steps:
s11: preparing a detection line coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) into the detection line coating solution to enable the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 to be 1.5mg/mL;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating the detection line coating solution added with the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control line coating solution added with the quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 17h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (prepared 1000 mL) of the treatment solution in step S2 includes: 7g of S9 surfactant (manufacturer: schhuier Biotech Co., ltd., yangzhou, cat # SPBHS 9), 10g of BSA, 970mL of LS002, 30mL of LS009, 0.5mL of blocker (the formula of the blocker is 0.02% sodium azide by mass/volume, 10mM of PBS, pH 7.4), 0.2mL of ProClin 300; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s3, marking and preparing a metal spraying bonding pad: respectively labeling the monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY with bicolor microspheres, respectively diluting the labeled monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M) antibody) and the chicken IgY and bicolor microsphere conjugate into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solutions, spraying the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution onto a conjugate pad, placing the conjugate pad into a drying room, drying the conjugate pad in an oven at 45 ℃, wherein the drying time is 4 hours, and drying the conjugate pad for later use; the formula of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water;
the specific steps of the step S3 are as follows:
s31: according to the optimized marking process, 10mM PB marking buffer solution and 300nm bicolor microsphere particles are used, the 2019-nCov (I09C 3M) antibody and the chicken IgY are respectively marked by the antibody dosage of 15; wherein the two-color microsphere particle is fluorescent dye and color dye (Eu (DBM) 3 Phen fluorescent dye and oil-soluble yellow R color dye), the preparation method of which adopts the preparation method of the two-color microsphere in step S31 in the example;
s32: diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution by using a microsphere preserving fluid according to the table 5; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere conjugate is 0.5mg/mL; spraying gold on the bonding pad by using a film-scribing gold spraying instrument according to the spraying amount of 2.0 mu L/cm; spraying onto the bonding pad, placing in a drying room, and drying in a 45 deg.C oven for 4 hr;
s4, assembling a detection card: cutting the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper, then pasting the cut films, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection board, and putting the pasted detection board into a corresponding card shell for detection card assembly to obtain 4 groups of detection cards; the test strips were used to test S1-S10 (S1-S8 are N protein recombinant antigen dilution references and S9-S10 are blank references), and the test results are shown in Table 5 below.
TABLE 5 detection results of different concentrations of gold spraying solution for preparing detection card
| Detection object | 1mg/mL | 1.5mg/mL | 2mg/mL | 2.5mg/mL |
| S1 | ≥G8 | ≥G10 | ≥G10 | ≥G10 |
| S2 | ≥G6 | ≥G8 | ≥G9 | ≥G8 |
| S3 | ≥G5 | ≥G7 | ≥G7 | ≥G7 |
| S4 | ≥G4 | ≥G5 | ≥G5 | ≥G5 |
| S5 | ≥G4 | ≥G4 | ≥G4 | ≥G4 |
| S6 | ≥G4 | ≥G4 | ≥G4 | ≥G4 |
| S7 | ≥G3 | ≥G4 | ≥G4 | ≥G4 |
| S8 | ≥G3 | ≥G3 | ≥G3 | ≥G3 |
| S9 | <G1 | <G1 | <G1 | <G1 |
| S10 | <G1 | <G1 | <G1 | <G1 |
Wherein, the signal test value is negative G4, positive G4 or more, the larger the value is, the higher the antigen concentration represented by the larger the value is, the more obvious the color is, S1-S10 is the reference (S1-S8 are N protein recombinant antigen dilution reference, S9-S10 are blank reference). As shown in Table 5, the test results show that, when the gold spraying concentration is 1mg/mL in the tests S1-S10, the whole color is lighter, the influence of the gold spraying concentration of 1.5 mg/mL-2.5 mg/mL on the color is smaller, and meanwhile, the fluorescence signal detection result also conforms to the result trend. Considering the cost comprehensively, the concentration of the gold spraying (labeling) of the 2019-nCov (H08A 1M) microsphere conjugate is 1.5mg/mL.
Specific example 4: the selection of the coating concentration of the detection antibody on the detection line (T line) is carried out, namely the optimal coating concentration of the 2019-nCov (I09C 3M) antibody is groped.
The preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 by using a film coating solution, preparing the concentration of a quality control antibody goat anti-chicken IgY by using a film coating solution, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
the step S1 comprises the following specific steps:
s11: preparing a detection line (T line) coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) into the detection line coating solution, namely diluting the antibody into 1.0mg/mL, 1.5mg/mL, 2mg/mL and 2.5mg/mL through the detection line coating solution;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating the detection line coating solution added with the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control line coating solution added with the quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 20h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (prepared 1000 mL) of the treatment solution in step S2 includes: 7g of S9 surfactant (manufacturer: schhuier Biotech Co., ltd., yangzhou, cat # SPBHS 9), 10g of BSA, 970mL of LS002, 30mL of LS009, 0.5mL of blocker (the formula of the blocker is 0.02% sodium azide by mass/volume, 10mM of PBS, pH 7.4), 0.2mL of ProClin 300; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s3, marking and preparing a metal spraying bonding pad: respectively labeling the monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY with bicolor microspheres, respectively diluting the labeled monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY and bicolor microsphere conjugates into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solutions, spraying the gold spraying solutions onto a conjugate pad, placing the conjugate pad in a drying room, placing the conjugate pad in an oven at 45 ℃ for drying for 4 hours, and drying for later use; the formula of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water;
the specific steps of the step S3 are as follows:
s31: marking a 2019-nCov (H08A 1M) antibody and chicken IgY by using 10mM PB marking buffer solution, 300nm double-color microsphere particles and 15 nm antibody dosage, centrifuging and drying to respectively prepare a monoclonal antibody 2019-nCoV N protein antibody 2-double-color microsphere conjugate and a chicken IgY-double-color microsphere conjugate; wherein the preparation method of the bicolor microspherical particles adopts the preparation method of the bicolor microspherical particles in the step S31 in the embodiment;
s32: diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate into an antibody-microsphere conjugate gold spraying solution of 1.5mg/mL; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere marker is 0.5mg/mL; spraying gold by using a film-scribing gold spraying instrument according to the spraying amount of 2.0 mu L/cm; spraying onto the bonding pad, placing in a drying room, and drying in a 45 deg.C oven for 4 hr;
s4, assembling a detection card: cutting the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper, then pasting the cut films, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection board, and putting the pasted detection board into a corresponding card shell for detection card assembly to obtain 4 groups of detection cards; respectively detecting S1-S10 (S1-S8 are N protein recombinant antigen dilution reference substances, and S9-S10 are blank reference substances), and the detection results are shown in the following table 6.
TABLE 6 detection results of 4 detection cards prepared by antibody coating concentration on different detection lines (T lines)
| Detection object | 1mg/mL | 1.5mg/mL | 2mg/mL | 2.5mg/mL |
| S1 | ≥G7 | ≥G10 | ≥G10 | ≥G10 |
| S2 | ≥G7 | ≥G9 | ≥G9 | ≥G9 |
| S3 | ≥G6 | ≥G6 | ≥G7 | ≥G6 |
| S4 | ≥G4 | ≥G5 | ≥G5 | ≥G5 |
| S5 | ≥G4 | ≥G4 | ≥G4 | ≥G4 |
| S6 | ≥G4 | ≥G4 | ≥G4 | ≥G2 |
| S7 | ≥G4 | ≥G4 | ≥G4 | ≥G2 |
| S8 | ≥G2 | ≥G3 | ≥G3 | ≥G2 |
| S9 | <G1 | <G1 | <G1 | ≥G2 |
| S10 | <G1 | <G1 | <G1 | <G1 |
Wherein, the signal test value is negative G4 or more, the signal test value is positive G4, the larger the numerical value is, the higher the antigen concentration represented by the numerical value is, the more obvious the color is, and S1-S10 are reference substances (S1-S8 are N protein recombinant antigen dilution reference substances, and S9-S10 are blank reference substances). As can be seen from the test results in Table 6, in the test of S1-S10, the test color is lighter when the streaking concentration is 1.0mg/mL, the color distinction is obvious when the streaking concentration is 1.5mg/mL and 2mg/mL, and when the streaking concentration is 2.5mg/mL, the color distinction of S6-S10 is impossible, and meanwhile, the fluorescence signal detection result also conforms to the result trend. For cost reasons, the streaking concentration of 2019-nCov (I09C 3M) was 1.5mg/mL.
Specific example 5: selecting the gold spraying concentration of the chicken IgY-microsphere conjugate, and searching the optimal gold spraying solution concentration for optimizing the product performance, wherein the chicken IgY antibody is marked by using bicolor microsphere particles in the specific embodiment;
the preparation method of the neocorona antigen detection kit comprises the following steps:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a membrane coating solution for a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1, preparing the concentration of a membrane coating solution for a quality control antibody goat anti-chicken IgY, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
the step S1 comprises the following specific steps:
s11: preparing a detection line (T line) coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 (2019-nCov (I09C 3M) antibody) in the detection line coating solution to enable the final concentration of the antibody to be 1.5mg/mL;
s12: preparing a quality control line (C line) coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating a detection line coating solution added with a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and a quality control line coating solution added with a quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm, and coating the coating solution by 30cm;
s14: drying in a 45 ℃ oven for 12h for later use;
s2 treatment of the sample pad: treating a sample pad by using a treatment solution, placing the treated sample pad on a clean and dry net rack, and drying the sample pad in a 45 ℃ drying oven for 4 hours for later use; the recipe (prepared 1000 mL) of the treatment solution in step S2 includes: 7g of S9 surfactant (manufacturer: schhuier Biotech Co., ltd., yangzhou, cat # SPBHS 9), 10g of BSA, 970mL of LS002, 30mL of LS009, 0.5mL of blocker (the formula of the blocker is 0.02% sodium azide by mass/volume, 10mM of PBS, pH 7.4), 0.2mL of ProClin 300; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL of Tween20, 0.02mL of ProClin300 and the balance of purified water;
s3, marking and preparing a metal spraying bonding pad: respectively labeling the monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY with bicolor microspheres, respectively diluting the labeled monoclonal antibody 2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and the chicken IgY and bicolor microsphere conjugate into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solutions, spraying the gold spraying solutions onto a conjugate pad, placing the conjugate pad into a drying room, placing the drying room into a 45 ℃ oven for drying for 4 hours, and drying for later use; the formula of the microsphere preservation solution in the step S3 is as follows: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water;
the specific steps of the step S3 are as follows:
s31: marking a monoclonal anti-2019-nCoV N protein antibody 2 (2019-nCov (H08A 1M)) and chicken IgY by using 10mM PB marking buffer solution, 300nm bicolor microsphere particles and the antibody dosage of 15; wherein the preparation method of the bicolor microspherical particles adopts the preparation method of the bicolor microspherical particles in the step S31 in the embodiment;
s32: diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere marker into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution of 1.5mg/mL; diluting the chicken IgY-bicolor microsphere conjugate into a gold spraying solution of chicken IgY-bicolor microsphere markers with the concentrations of 0.3mg/mL, 0.5mg/mL, 0.8mg/mL and 1mg/mL; spraying gold by using a film-scribing gold spraying instrument according to the spraying amount of 2.0 mu L/cm; spraying onto the bonding pad, placing in a drying room, drying in a 45 deg.C oven for 4 hr, and drying;
s4, assembling a detection card: cutting the coated film, the sprayed gold bonding pad, the processed sample pad and the absorbent paper, then pasting the cut films, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection board, and putting the pasted detection board into a corresponding card shell for detection card assembly to obtain 4 groups of detection cards; respectively detecting S1-S10 (S1-S8 are N protein recombinant antigen dilution reference substances, and S9-S10 are blank reference substances), and the detection results are shown in the following table 7.
TABLE 7 detection results of detection cards prepared from concentrations of different chicken IgY-bicolor microsphere conjugates in gold spraying solution
| Detection object | 0.3mg/mL | 0.5mg/mL | 0.8mg/mL | 1mg/mL |
| S1 | ≥G8 | ≥G10 | ≥G10 | ≥G10 |
| S2 | ≥G6 | ≥G8 | ≥G9 | ≥G8 |
| S3 | ≥G5 | ≥G6 | ≥G6 | ≥G6 |
| S4 | ≥G4 | ≥G5 | ≥G5 | ≥G4 |
| S5 | ≥G4 | ≥G4 | ≥G4 | ≥G4 |
| S6 | ≥G4 | ≥G4 | ≥G4 | ≥G4 |
| S7 | ≥G3 | ≥G4 | ≥G4 | ≥G4 |
| S8 | ≥G3 | ≥G3 | ≥G3 | ≥G3 |
| S9 | <G1 | <G1 | <G1 | <G1 |
| S10 | <G1 | <G1 | <G1 | <G1 |
Wherein, the signal test value is negative G4, positive G4 or more, the larger the value is, the higher the antigen concentration represented by the larger the value is, the more obvious the color is, S1-S10 is the reference (S1-S8 are N protein recombinant antigen dilution reference, S9-S10 are blank reference). As can be seen from the test results in Table 7, in the tests S1-S10, when the concentration of the metal spraying is 0.3mg/mL, the whole color is lighter, the influence of the concentration of the metal spraying of 0.5mg/mL-1mg/mL on the color is smaller, and meanwhile, the detection result of the fluorescence signal also conforms to the trend of the result. Comprehensively considering the cost, the concentration of the gold spraying (marking) of the chicken IgY-bicolor microsphere conjugate is 0.5mg/mL.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present invention, and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements and the like, such as changes in shape or material of some components, are made within the spirit and principle of the present invention; are intended to be included within the scope of the present invention.
Claims (19)
1. The preparation method of the neocorona antigen detection kit is characterized by comprising the following steps of:
s1 preparation of an antibody-coated nitrocellulose membrane: preparing the concentration of a membrane coating solution for a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1, preparing the concentration of a membrane coating solution for a quality control antibody goat anti-chicken IgY, coating the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and the quality control antibody goat anti-chicken IgY on a nitrocellulose membrane, and drying for later use;
s2 treatment of the sample pad: treating the sample pad by using the treatment solution, and drying the treated sample pad in an oven for later use;
s3, marking and preparing a metal spraying bonding pad: respectively marking the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microsphere particles, respectively diluting the marked monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY with bicolor microsphere conjugates into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution by using microsphere preservation solution, spraying the gold spraying solutions onto a conjugate pad, placing the conjugate pad in a drying room, drying the conjugate pad in an oven, and drying the conjugate pad for later use;
s4, assembling a detection card: cutting and sticking the coated nitrocellulose membrane, the sprayed gold bonding pad, the processed sample pad and the absorbent paper into a detection plate, and putting the stuck detection plate into a corresponding card shell for detection card assembly to obtain the detection card.
2. The method for preparing a neocorona antigen detection kit according to claim 1, further comprising a step S5 of preparing a sample extract: preparing and subpackaging a sample extract; the extract is used for extracting a sample and is used as a component of a neocorona antigen detection kit for sample detection.
3. The method for preparing the kit for detecting the neocorona antigen according to claim 2, wherein the step S1 comprises the following steps:
s11: preparing a detection line coating solution, and adding a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 into the detection line coating solution to ensure that the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 is 1.0-2.5 mg/mL;
s12: preparing a quality control line coating solution, and adding a quality control antibody goat anti-chicken IgY into the quality control line coating solution to ensure that the final concentration of the quality control antibody goat anti-chicken IgY is 1.0mg/mL;
s13: coating a detection line coating solution added with a detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 and a quality control line coating solution added with a quality control antibody goat anti-chicken IgY on a test area and a quality control area of a nitrocellulose membrane by a film cutting machine according to the amount of 1 mu L/cm;
s14: drying in a 45 ℃ oven for 12-20 h for later use.
4. The method for preparing the reagent kit for detecting neocoronary antigen according to claim 3, wherein the step S3 comprises the following steps:
s31: mixing the microsphere marking solution and the bicolor microsphere particles, respectively carrying out marking reaction on the monoclonal antibody 2019-nCoV N protein antibody 2 and the chicken IgY, and respectively preparing a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and a chicken IgY-bicolor microsphere marker after centrifugation;
s32: and then diluting the monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate and the chicken IgY-bicolor microsphere marker into a monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution and a chicken IgY-bicolor microsphere conjugate gold spraying solution respectively by using microsphere preservation solutions, spraying the gold spraying solutions onto the conjugate pad treated in the step S2 through a film-scribing gold spraying instrument, putting the conjugate pad into a 45-DEG C oven for drying, drying for 4 hours, and drying for later use.
5. The method for preparing the neocoronary antigen detection kit according to claim 4, wherein the microsphere labeling solution in step S31 comprises 5mM MES, 5mM HEPES or 10mM PB, and the particle size of the bi-color microsphere particles is 100-300 nm; the mass ratio of the monoclonal antibody 2019-nCoV N protein antibody 2 labeled antibody to the bicolor microspherical particles is (10-20): 1.
6. The method for preparing the reagent kit for detecting the neocorona antigen according to claim 5, wherein in the step S32, the 2019-nCoV N protein antibody 2 labeled antibody-bi-color microsphere conjugate is diluted into 1-2.5 mg/mL of gold spraying solution of the antibody-microsphere conjugate; the concentration of the chicken IgY-bicolor microsphere conjugate is 0.3-1 mg/mL; and the film-scribing metal spraying instrument sprays metal according to the spraying amount of 2.0 mu L/cm.
7. The method for preparing the neocorolla antigen detection kit according to claim 4, wherein the formulation of the extraction solution in the step S5 comprises, by mass or volume percent: PBS buffer solution 10nM, emulsifier 1-2% and Proclin300 0.01-0.03%, pH value is 7.4-7.8 + -1.
8. The method for preparing the neocoronary antigen detection kit according to claim 6, wherein the microsphere labeling solution in step S31 is 10mM PB labeling buffer solution, the bi-color microspherical particles are polystyrene microspheres with coexisting fluorochromes and colorized dyes, the particle size is 300nm, and the ratio of the 2019-nCoV N protein antibody 2 labeled antibody to the bi-color microspherical particles is 15 by mass; in the step S32, the 2019-nCoV N protein antibody 2 labeled antibody-bicolor microsphere conjugate is diluted into a 1.5mg/mL monoclonal antibody 2019-nCoV N protein antibody 2-bicolor microsphere conjugate gold spraying solution; the concentration of the gold spraying solution of the chicken IgY-bicolor microsphere marker is 0.5mg/mL.
9. The method for preparing the neocorona antigen detection kit according to claim 7, wherein the emulsifier in the extract in the step S5 is Tween20 or/and Tween80; in the step S11, the final concentration of the detection antibody monoclonal antibody 2019-nCoV N protein antibody 1 added into the detection line coating solution is 1.5mg/mL.
10. The method for preparing a reagent kit for detecting neocorona antigen according to claim 2, further comprising a step S6 of packing in a test card: putting the detection card and the drying agent into an aluminum foil bag for packaging, and marking; s7, detection card outer packaging: the detection card, the instruction book and the extraction tube are put into an external packing box, and the self-adhesive sticker of the packing box is pasted.
11. The method for preparing the reagent kit for detecting neocoronary antigen in claim 2, wherein the preparation of 1000mL of the treating solution in step S2 comprises: 6-9 g of S9 surfactant, 9-11 g of BSA, 25-35 mL of LS009 intermediate solution, 0.5-0.6 mL of blocking agent, 0.15-0.25 mL of ProClin, and the balance of LS002 intermediate solution.
12. The method for preparing a neocoronary antigen detection kit according to claim 11, wherein the 100mL formulation of the microsphere preservation solution prepared in step S3 is: 45-55 mL of LS002 intermediate solution, 0.1-0.8 mL of LS009 intermediate solution, 0.2-0.8 g of BSA, 0.01-0.03 mL of ProClin, and the balance of purified water.
13. The method for preparing the kit for detecting the neocoronary antigen according to claim 12, wherein the formula of the blocking agent is as follows: sodium azide at 0.02% mass to volume and 10mM PBS, pH 7.4; the LS002 intermediate solution is Tris buffer solution, and the prepared 100mL formula is as follows: 1.58g Tris, 1.7g sodium chloride, 0.02mL ProClin300, and the balance purified water; the formula for preparing 100mL of LS009 intermediate solution is as follows: 10mL Tween20, 0.02mL ProClin300, and the balance purified water.
14. The method for preparing the neocoronary antigen detection kit according to claim 4, wherein the two-color microsphere in step S31 is a polystyrene microsphere with Eu (DBM) 3Phen fluorochrome and oil-soluble yellow R color dye coexisting, and the method for preparing the kit comprises the following steps:
s311: preparation of fluorescent dye: respectively preparing a DBM and ethanol mixed solution, a Phen and ethanol mixed solution and a rare earth ion and ethanol mixed solution according to a ratio, reacting the DBM and ethanol mixed solution and the Phen and ethanol mixed solution with the rare earth ion and ethanol mixed solution, and filtering and drying to obtain fluorescent dye powder;
s312 preparation of polystyrene microspheres: preparing polystyrene microspheres by a soap-free emulsion polymerization method to obtain white emulsion, namely polystyrene microsphere liquid;
s313 preparation of polystyrene color microspheres: mixing and emulsifying the polystyrene microsphere liquid obtained in the step S312 by using a color dye, and centrifuging to remove supernatant liquid to obtain polystyrene color microspheres;
s314 preparation of polystyrene PS bicolor microspheres: and (2) mixing the fluorescent dye powder prepared in the step (S1) with the polystyrene colorful microspheres prepared in the step (S313), emulsifying, and centrifuging to remove supernatant to obtain the polystyrene PS double-color microspheres.
15. The method for preparing a neocorona antigen detection kit according to claim 14, wherein the specific step of step S311 includes:
s3111: weighing raw materials including Eu 3+ Salt, DBM and Phen for standby;
s3112: respectively dissolving DBM and Phen in absolute ethyl alcohol to obtain a mixed solution of DBM and ethyl alcohol and a mixed solution of Phen and ethyl alcohol; eu is added 3+ Dissolving salt in absolute ethyl alcohol to obtain an absolute ethyl alcohol mixed solution of rare earth ions;
s3113: adding DBM and ethanol mixed solution into anhydrous ethanol solution of rare earth ions, placing the mixture into a three-neck flask, heating the mixture at 60-70 ℃, carrying out reflux stirring reaction, dropwise adding the Phen and ethanol mixed solution into the three-neck flask by using a constant-pressure dropping funnel, continuously reacting, adjusting the pH value of the reaction solution, and then continuously carrying out reflux stirring reaction until the reaction is finished;
s3114: transferring the reaction solution into a clean open container, standing at room temperature, cooling with ice water bath, vacuum filtering, and drying to obtain Eu (DBM) 3 Phen fluorescent dye powder.
16. The method for preparing the kit for detecting neocorona antigen according to claim 14, wherein the step S312 includes:
s3121: adding 200mL of water and 10mL of refined styrene into a three-neck flask, introducing nitrogen, heating in a water bath, and stirring;
s3122: when the temperature of the water bath is raised to 70-80 ℃, adding initiator ammonium persulfate, and continuously stirring for reaction to obtain uniform white emulsion, namely the polystyrene microsphere liquid.
17. The method for preparing a neocorona antigen detection kit according to claim 16, wherein the step S313 specifically comprises:
s3131: dispersing the polystyrene microsphere solution obtained in the step S312 in a swelling medium; dissolving an oil-soluble yellow R dye in a swelling medium to obtain a dye swelling medium mixed solution;
s3132: mixing the microsphere solution and the dye swelling medium mixed solution in the step S3131, and emulsifying, namely carrying out oscillation reaction for 1-3 h at room temperature;
s3133: and (3) removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate for multiple times by using pure water, and finally dissolving the obtained precipitate in the pure water to obtain the PS colored microsphere liquid.
18. The method for preparing a kit for detecting neocoronary antigen according to claim 17, wherein the step S314 comprises the following steps:
s3141: dispersing the PS colored microsphere liquid obtained in the step S3133 in a swelling medium; eu (DBM) 3 Dissolving Phen fluorescent dye powder in a swelling medium to obtain a fluorescent dye swelling medium mixed solution;
s3142: mixing the microsphere solution obtained in the step S3141 with the mixed solution of the fluorescent dye swelling medium, and emulsifying, namely carrying out oscillation reaction at room temperature; after reacting for a period of time, adding methacrylic acid (MAA) and continuing the reaction;
s3143: removing the swelling medium by rotary evaporation, performing ultrasonic treatment, gradually diluting the residual liquid, centrifuging, removing the supernatant, washing the lower-layer solid precipitate with pure water for multiple times, and dissolving the obtained precipitate in the pure water to obtain the carboxyl PS double-color microsphere solution.
19. The neocorona antigen detection kit obtained by the preparation method of the neocorona antigen detection kit according to any one of claims 1 to 18, wherein the detection card of the neocorona antigen detection kit sequentially comprises a PVC bottom plate, a nitrocellulose membrane, a combination pad, a water absorption pad and a sample pad from bottom to top, wherein the combination pad and the water absorption pad are respectively arranged at two ends of the nitrocellulose membrane and are positioned on the same horizontal plane, the sample pad is arranged on the combination pad, the nitrocellulose membrane is coated with a detection line and a quality control line through an antibody, and the widths of the nitrocellulose membrane, the sample pad, the water absorption paper and the PVC bottom plate are all 3mm.
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