CN111812336A - Detection kit for detecting coronavirus antibody and preparation method thereof - Google Patents
Detection kit for detecting coronavirus antibody and preparation method thereof Download PDFInfo
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
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- 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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- G01N33/50—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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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention discloses a detection kit for detecting coronavirus antibodies, which comprises a first reagent and a second reagent, wherein the first reagent is a solution containing first microspheres, and the first microspheres are latex microspheres coated with coronavirus recombinant proteins; the second reagent is a solution containing second microspheres, and the second microspheres are latex microspheres coated with anti-coronavirus recombinant protein antibodies. The detection kit disclosed by the invention is characterized in that a novel coronavirus recombinant protein antigen and a paired monoclonal antibody are respectively coated on latex microspheres, and whether the novel coronavirus antibody exists in a sample to be detected is judged through the change of solution absorbance (absorbance) by utilizing an indirect competition method that the latex microspheres coated with the recombinant protein antigen are firstly combined with the novel coronavirus antibody in the sample to be detected and then combined with the latex microspheres coated with an anti-recombinant protein monoclonal antibody. The detection kit for detecting the novel coronavirus antibody has the advantages of strong specificity, high sensitivity and wide detection range, and can greatly reduce the cost of the reagent.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a detection kit for detecting coronavirus antibodies and a preparation method of the detection kit.
Background
The pneumonia caused by the novel coronavirus is an acute infectious disease, the pathogen of the pneumonia is a coronavirus which is not found in human bodies before, namely SARS-CoV-2, the disease caused by the pneumonia is named as COVID-19 by the world health organization, the disease is mainly transmitted through respiratory droplets and close contact infection, the pneumonia has strong infectivity, and people are generally easy to infect.
The development of pneumonia epidemic caused by the novel coronavirus needs to be carried out on a large scale, rapidly and accurately screening samples, providing basis for clinical diagnosis of novel coronavirus infectors and providing rapid and timely judgment results for enterprise repeated production. In order to meet the market demand, practitioners in the diagnostic industry develop a series of novel coronavirus detection kits, which mainly comprise two categories of nucleic acid detection and antibody detection. In general, nucleic acid detection is performed by determining whether or not a nucleic acid sequence of a new coronavirus is present in a sample by a PCR method, thereby determining whether or not a subject is infected with a virus. The antibody detection is based on the basic principle of immunology, the human immune system can generate corresponding antibodies when the virus is infected, and whether the detected person is infected with the virus can be indirectly judged by detecting whether a new coronavirus antibody exists in a sample.
The nucleic acid detection based on the PCR technology has high accuracy, but the time consumption is long, special instruments and equipment and places are needed, the requirement on operators is high, and the nucleic acid detection is difficult to popularize in the basic level for mass screening. The existing antibody detection mainly comprises two schemes of colloidal gold and chemiluminescence. The colloidal gold method is simple to operate, high in speed and suitable for the markets of instant detection such as quick detection. Its disadvantages are not automation, not being able to carry out large-scale screening, low sensitivity and not easy to control the repeatability. The antibody detection scheme based on the chemiluminescence method is suitable for high-throughput screening, but a matched instrument is needed, instrument platforms of the existing chemiluminescence products in the market are all closed platforms, reagents cannot be universal, popularization is not facilitated, and the method is also not convenient to be used in quick detection scenes such as instant detection and the like.
Latex Enhanced turbidimetric immunoassay (particle Enhanced turbidimetric immunoassay-Assay-PETIA) captures antibodies or antigens in a sample to be detected by using antigens or antibodies adsorbed on latex microspheres with diameters of dozens to hundreds of nanometers, so that crosslinking among the microspheres is formed, and the scattering degree or transmission absorbance (namely Turbidity) of a solution is changed. By measuring the change in the degree of scattering or in the absorbance of transmission of the solution, the concentration of the analyte can be known qualitatively or quantitatively. Conventional latex-enhanced turbidimetric immunoassay reagents include two moieties R1 and R2. R1 is a diluent used to dilute the test sample, and R2 is a latex microsphere solution used to generate absorbance changes. The turbidimetric method and the latex reagent designed by the conventional competition method need a single new coronavirus antigen to bridge antibodies on more than two microspheres simultaneously to cause turbidity change. The efficiency of this reaction is low, resulting in a relatively significant turbidity change only at higher concentrations of antibody in the sample, and thus a decrease in the sensitivity of the assay.
Disclosure of Invention
In view of the above, in order to overcome the defects of the prior art, the present invention aims to provide an improved detection kit for detecting coronavirus antibodies, which has the advantages of high sensitivity, strong specificity and reduced reagent cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection kit for detecting coronavirus antibodies comprises a first reagent and a second reagent, wherein the first reagent is a solution containing first microspheres, and the first microspheres are latex microspheres coated with coronavirus recombinant proteins; the second reagent is a solution containing second microspheres, and the second microspheres are latex microspheres coated with anti-coronavirus recombinant protein antibodies; the antibody is a monoclonal antibody.
In this application, coating refers to binding of an antigen or antibody to the surface of a solid support (latex microspheres). In the actual product of the detection kit, the amount of the second reagent can be much smaller than that of the first reagent, and the volume ratio of the first reagent to the second reagent is preferably 3-5: 1.
The most obvious difference between the reagent in the detection kit of the present invention and the conventional latex reagent using the competition method is that the component (in this case, the coronavirus recombinant protein antigen) in the conventional reagent for competing with the target to be detected is present in the reagent in a free state, and the recombinant protein antigen for competing in the reagent of the present invention is coated on the latex microsphere.
The use of the method of coating the latex microspheres with the antigen for competition has the following advantages: 1. the sensitivity of the reagent can be greatly improved. Latex reagents designed by the conventional competition method need a single coronavirus antigen to simultaneously bridge antibodies on more than two microspheres so as to cause the absorbance change. The efficiency of this reaction is low, resulting in a relatively significant change in absorbance at higher concentrations of antibody in the sample, and thus a decrease in sensitivity of the reagent. The competing antigen is coated on the latex microsphere, and the latex microsphere can be bridged only by reacting with an antibody on the latex microsphere in the second reagent, so that the absorbance of the solution is obviously changed, and the sensitivity of the reagent is greatly improved. 2. The reagent cost can be greatly reduced. Because the antigen in the first reagent is coated on the latex microsphere, the use amount of the antigen can be greatly reduced, and correspondingly, the use amount of the antibody coated on the latex microsphere in the second reagent can be greatly reduced, thereby greatly reducing the reagent cost.
According to some preferred embodiments of the invention, the amount of coronavirus recombinant protein in the first microspheres is 0.01-0.025 times the amount of original latex microspheres by mass ratio; if the amount is less than 0.01 time, the detection sensitivity of the kit is reduced, and 0.025 time is the amount of the coronavirus recombinant protein which is paved on one layer of the surface of the latex microsphere, namely the upper limit value, the positive influence on the result is not obviously improved and the reagent cost is increased. The amount of the anti-coronavirus recombinant protein antibody in the second microspheres is 0.04-0.08 times of that of the original latex microspheres; if the amount is less than 0.04 times, the detection sensitivity of the kit is greatly reduced, and the amount of 0.08 times is the amount of the antibody which is spread on the surface of the latex microsphere and is one layer, namely the upper limit value. The antibody adopted by the second reagent in the detection kit is a monoclonal antibody, so that the detection kit has high specificity and further improves the detection sensitivity.
In the present application, the original latex microspheres refer to latex microspheres that are not coated with coronavirus recombinant protein or anti-coronavirus recombinant protein antibody. According to some preferred embodiments of the present invention, the primary latex microspheres have an average particle size of 120-210 nm; the latex microspheres are carboxyl polystyrene microspheres. When the average particle size of the latex microspheres is less than 120nm, the detection sensitivity of the kit is reduced, and the consumption of raw materials is increased, namely the cost is increased; when the particle size of the latex particles is larger than 210nm, the specific surface area of the latex particles is reduced, the quantity of carboxyl groups available for antibody linkage on the surface of the unit sphere is reduced, so that when the quantity of the latex microspheres is unchanged, the quantity of the antibody capable of being coated is reduced when the particle size of the latex microspheres is larger, the signal value is not obviously increased on the basis of noise increase, the detection signal-to-noise ratio of the kit is reduced, and the repeatability is low.
According to some preferred embodiments of the present invention, the first reagent comprises first microspheres and a buffer system solution, wherein the concentration of the first microspheres in the first reagent is 0.2g/L to 0.35 g/L; the second reagent comprises second microspheres and a buffer system solution, and the concentration of the second microspheres in the second reagent is 0.5g/L-1.0 g/L.
According to some preferred embodiments of the invention, the buffer system solution is a morpholine buffer system, in particular a MOPSO buffer system. In some embodiments, the components and the concentrations of the components in the buffer system solution are as follows:
the first reagent adopting the buffer system solution is a mixed solution consisting of first microspheres, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water; the second reagent is a mixed solution consisting of second microspheres, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), an MOPSO buffer solution, a preservative (PC-300) and solvent water.
According to some preferred embodiments of the invention, the test kit further comprises calibrators of anti-coronavirus recombinant protein antibodies at different concentrations (0-10ug/mL), the calibrators comprising anti-coronavirus recombinant protein antibodies and a calibrator stock.
In the calibrator, the concentration of the anti-coronavirus recombinant protein antibody is selected from two or more of 0-10 ug/mL. In particular, in some embodiments of the invention, the concentration of anti-coronavirus recombinant protein antibody in the calibrator is: 0ug/mL, 1ug/mL, and 5 ug/mL. The selection of three concentrations was based on the clinical negative-positive reference interval setting, 0ug/mL was negative, 1ug/mL was median, 5ug/mL was high.
According to some preferred embodiments of the invention, the components and the concentrations of the components in the calibrator preservation solution are as follows: 9g/L of sodium chloride, 25mM HEPES buffer solution with the pH value of 7.6, 2g/L of polyoxyethylene lauryl ether (Thesit), 200mL/L of calf negative serum and 1g/L of preservative. The preservative is selected from one or more of sodium azide, PC-300 and PC-950.
According to some preferred embodiments of the invention, the coronavirus is SARS-CoV-2. That is, the detection kit of the present application can be used in the detection of coronaviruses such as SARS-CoV-2 (novel coronavirus).
The application also provides a preparation method of the detection kit for detecting the coronavirus antibody, which comprises the following steps:
adding coronavirus recombinant protein or an anti-coronavirus recombinant protein antibody into the activated latex microspheres, and correspondingly preparing to obtain first microspheres or second microspheres;
and mixing the first microspheres or the second microspheres with a buffer system solution to correspondingly prepare a first reagent and a second reagent.
According to some preferred aspects of the invention, the method of preparing the first reagent comprises the steps of: adding an activating reagent into a buffer solution containing latex microspheres, uniformly mixing, centrifuging, and removing a supernatant; adding buffer solution again, dispersing after resuspending, adding coronavirus recombinant protein, mixing uniformly, centrifuging, and removing supernatant; adding a buffer system solution, and carrying out resuspension, dispersion and uniform mixing to obtain a first reagent.
In some embodiments of the invention, the first reagent preparation method specifically comprises the steps of:
1) activating the latex microspheres: adding an activating reagent EDC into MES buffer solution containing 5g/L-15g/L latex microspheres, stirring or shaking for uniformly mixing for 1.0-2.0h, centrifuging the solution after timing is finished, and removing supernatant to obtain latex microsphere precipitate;
2) re-suspending the latex microsphere precipitate obtained in the step 1) by using MES buffer solution until the concentration of the latex microsphere in the re-suspension solution is 5g/L-10g/L, adding coronavirus recombinant protein after dispersion, uniformly mixing for 1.5-3h, centrifuging the solution after timing is finished, and removing supernatant to obtain the latex microsphere precipitate; this time, the first unstable microspheres are obtained;
3) and (3) resuspending the latex microspheres obtained in the step 2) by using a buffer system solution until the concentration of the latex microspheres in the resuspension solution is 0.2-0.35 g/L, carrying out ultrasonic or homogeneous dispersion, stirring or uniformly mixing for 1.5-3h, and placing at 37 ℃ for 14-20h after timing to obtain the first reagent.
According to some preferred aspects of the invention, the method of preparing the second reagent comprises the steps of: adding an activating reagent into a buffer solution containing latex microspheres, uniformly mixing, centrifuging, and removing a supernatant; adding buffer solution again, dispersing after resuspending, adding anti-coronavirus recombinant protein antibody, mixing uniformly, centrifuging, and removing supernatant; adding a buffer system solution, and carrying out resuspension, dispersion and uniform mixing to obtain a second reagent.
In some embodiments of the invention, the method of preparing the second reagent specifically comprises the steps of:
1) activating the latex microspheres: adding an activating reagent EDC into MES buffer solution containing 5g/L-15g/L latex microspheres, stirring or shaking for uniformly mixing for 1.0-2.0h, centrifuging the solution after timing is finished, and removing supernatant to obtain latex microsphere precipitate;
2) re-suspending the latex microsphere precipitate obtained in the step 1) by using MES buffer solution until the concentration of the latex microsphere in the re-suspension solution is 5g/L-10g/L, adding an anti-coronavirus recombinant protein monoclonal antibody after ultrasonic or homogeneous dispersion, stirring or shaking and uniformly mixing for 1.5-3h, finishing timing, centrifuging the solution, and removing the supernatant to obtain the latex microsphere precipitate; this time, the second unstable microsphere is obtained;
3) and (3) resuspending the latex microspheres obtained in the step 2) by using a buffer system solution until the concentration of the latex microspheres in the resuspension solution is 0.5-1.0 g/L, carrying out ultrasonic or homogeneous dispersion, stirring or uniformly mixing for 1.5-3h, and placing at 37 ℃ for 14-20h after timing to obtain a second reagent.
In some preferred embodiments of the present invention, the centrifugation conditions in the preparation of the two latex microspheres are 11000g, and the centrifugation time is 15 minutes; the dispersing conditions are ultrasonic dispersing, 30% power and 5 minutes of ultrasonic time.
According to some preferred embodiments of the present invention, the activating agent is used in an amount of 10 to 20 times by mass of the amount of the carboxyl groups on the latex microspheres. The dosage of the activating reagent is considered based on the stirring or oscillating time in the step 1), and under the premise of stirring for 1.0-2.0h, the dosage of the activating reagent with 10-20 times of the carboxyl quantity is the optimal dosage, so that a better activating effect can be achieved, and the dosage of the activating reagent can be controlled.
The buffer used in the preparation of the two microspheres was MES buffer with pH 6.5, wherein the MES concentration was 25 mM.
The invention also provides a detection method for detecting the coronavirus antibody by using the detection kit, which comprises the following steps:
mixing and incubating a sample to be detected with a first reagent, wherein coronavirus recombinant protein coated on a first microsphere in the first reagent is combined with a coronavirus antibody in the sample to be detected;
and adding a second reagent into the system, mixing and incubating, wherein the coronavirus recombinant protein which is not combined with the coronavirus antibody in the sample to be detected on the first microsphere in the first reagent is combined with the anti-coronavirus recombinant protein antibody coated on the second microsphere in the second reagent, and absorbance change is generated, and the absorbance change value is recorded and analyzed.
According to some preferred embodiments of the present invention, the mixing ratio of the first reagent and the calibrator or the sample to be measured is 8: 1.
according to some preferred embodiments of the present invention, the mixing ratio of the system after the reaction of the first reagent and the calibrator or the sample to be tested to the second reagent is 9: 2.
according to some preferred embodiments of the invention, the analytical instrument records the absorbance change at a wavelength of 546 nm.
Different from the conventional competition method reagent, the first reagent and the second reagent in the detection kit are latex solutions, wherein the first reagent is a solution containing latex microspheres coated with the novel coronavirus recombinant protein, and the second reagent is a solution containing latex microspheres coated with the monoclonal antibody against the novel coronavirus recombinant protein. The detection method is designed according to a competition method, when in detection, a sample to be detected firstly reacts with a first reagent, and coronavirus antibodies in the sample are combined with antigens on latex microspheres in the first reagent. And then adding a second reagent into the reaction system, wherein the antibody on the latex microsphere in the second reagent reacts with the residual antigen on the latex microsphere in the first reagent, so that the absorbance (turbidity) of the reaction system is changed.
When the novel coronavirus antibody does not exist in the sample to be detected, the latex microspheres in the first reagent and the second reagent are fully combined, and the absorbance change is the largest. If the sample to be detected contains coronavirus antibodies, the antibodies in the sample to be detected consume part of the antigens on the latex microspheres in the first reagent, so that the latex microspheres in the first reagent and the second reagent are not sufficiently combined when the first reagent and the second reagent are mixed, and the generated absorbance change value is reduced. By comparing the change of the absorbance, the detection sample can be known to contain the new coronavirus antibody and the content level of the new coronavirus antibody in the sample.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the beneficial effects that: the detection kit for detecting the novel coronavirus antibody comprises a first reagent containing latex microspheres coated with the novel coronavirus recombinant protein and a second reagent containing latex microspheres coated with an anti-novel coronavirus recombinant protein antibody, wherein the novel coronavirus recombinant protein antigen is coated on the latex microspheres, and the latex microspheres coated with the antigen in the first reagent can be bridged with the latex microspheres to cause obvious change of solution absorbance only by reacting with an antibody on the latex microspheres coated with the antibody in the second reagent, so that the sensitivity of the reagent is greatly improved; by coating the latex microspheres with the antigen used in the first reagent, the amount of the antigen used and the antibody coated on the latex microspheres used in the second reagent can be greatly reduced, thereby greatly reducing the reagent cost.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The detection kit for detecting the novel coronavirus antibody in the embodiment comprises a first reagent, a second reagent and a calibrator, wherein the volume ratio of the first reagent to the second reagent in the detection kit is 4: 1.
1. A first reagent
The first reagent is a mixed solution consisting of latex microspheres coated with novel coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the first reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the MOPSO solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system; the concentrations of the various substances in the MOPSO buffer system were as follows: arg 40g/L, NaCl 9g/L, trehalose 40g/L, BSA 10g/L, PC-3001 g/L;
3) taking a carboxylated polystyrene microsphere with the particle size of 123nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 10g/L, adding EDC powder which is 10 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 1.0 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 5g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the novel coronavirus recombinant protein which is 0.025 times of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 1.5 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.35g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing at 300rpm for 1.5 hours;
8) and after timing is finished, placing the latex solution obtained in the step 7) in an oven at 37 ℃ for 14h to obtain a first reagent.
2. Second reagent
The second reagent is a mixed solution consisting of latex microspheres coated with the anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the second reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system;
3) taking a carboxylated polystyrene microsphere with the particle size of 123nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 10g/L, adding EDC powder which is 10 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 1.0 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 10g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the anti-novel coronavirus recombinant protein monoclonal antibody which is 0.08 times of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 1.5 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 1.0g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing at 300rpm for 1.5 hours;
8) and after timing is finished, placing the latex solution obtained in the step 7) in an oven at 37 ℃ for 14h to obtain a second reagent.
3. Calibration article
The calibrator in the embodiment comprises calibrators of anti-novel coronavirus recombinant protein antibody solutions with different concentrations of 0ug/mL, 1ug/mL and 5 ug/mL.
The anti-novel coronavirus recombinant protein antibody solution at each concentration comprises an anti-novel coronavirus recombinant protein antibody and a calibrator stock solution. Wherein the calibrator stock solution comprises sodium chloride, HEPES buffer solution, polyoxyethylene lauryl ether (Thesit), calf negative serum and PC-300.
The preparation method of the monoclonal antibody calibrator for resisting the novel coronavirus SARS-CoV-2 recombinant protein in the embodiment specifically comprises the following steps:
1) preparing a HEPES solution with the concentration of 25mM, and adjusting the pH value of the solution to be 7.6 by using 6mol/L NaOH;
2) sequentially adding NaCl, polyoxyethylene lauryl ether (Thesit), calf negative serum and PC-300 into the solution obtained in the step 1), and uniformly stirring to obtain a calibrator preservation solution; the concentration of each substance in the calibrator preservation solution is as follows: 9g/L of NaCl, 2g/L of polyoxyethylene lauryl ether and 200mL/L, PC-3001g/L of calf negative serum.
3) And (3) taking the monoclonal antibody of the anti-new coronavirus SARS-CoV-2 recombinant protein, and diluting the monoclonal antibody into the calibrator with the concentration of 1ug/mL and 5ug/mL by using the calibrator preservation solution in the step 2) to obtain the calibrator.
Example two
The detection kit for detecting the novel coronavirus antibody in the embodiment comprises a first reagent, a second reagent and a calibrator, wherein the volume ratio of the first reagent to the second reagent in the detection kit is 5: 1.
1. A first reagent
The first reagent is a mixed solution consisting of latex microspheres coated with novel coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the first reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system;
3) taking a carboxylated polystyrene microsphere with the particle size of 198nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 5g/L, adding EDC powder which is 20 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 2.0 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 5g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the novel coronavirus recombinant protein which is 0.01 time of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 3.0 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.2g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing the latex solution at 300rpm for 3.0 hours;
8) and after timing is finished, putting the latex solution obtained in the step 7) into an oven at 37 ℃ for 20 hours to obtain the first reagent.
2. Second reagent
The second reagent is a mixed solution consisting of latex microspheres coated with the anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the second reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system;
3) taking a carboxylated polystyrene microsphere with the particle size of 198nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 5g/L, adding EDC powder which is 20 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 2.0 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 5g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the anti-novel coronavirus recombinant protein monoclonal antibody which is 0.04 times of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 3.0 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.5g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing the latex solution at 300rpm for 3.0 hours;
8) and after timing is finished, putting the latex solution obtained in the step 7) into a 37 ℃ oven for 20 hours to obtain a second reagent.
3. Calibration article
The calibrator and the preparation method thereof in this embodiment are the same as those in the first embodiment.
EXAMPLE III
The detection kit for detecting the novel coronavirus antibody in the embodiment comprises a first reagent, a second reagent and a calibrator, wherein the volume ratio of the first reagent to the second reagent in the detection kit is 5: 1.
1. A first reagent
The first reagent and the preparation method thereof in this example are the same as in example two.
2. Second reagent
The second reagent is a mixed solution consisting of latex microspheres coated with the anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the second reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system;
3) taking a carboxylated polystyrene microsphere with the particle size of 198nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 8g/L, adding EDC powder which is 15 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 1.5 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 8g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the anti-novel coronavirus recombinant protein monoclonal antibody which is 0.06 times of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 2.5 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.8g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing the latex solution at 300rpm for 2.5 hours;
8) and after timing is finished, placing the latex solution obtained in the step 7) in an oven at 37 ℃ for 16h to obtain a second reagent.
3. Calibration article
The calibrator and the preparation method thereof in this embodiment are the same as those in the first embodiment.
Example four
The detection kit for detecting the novel coronavirus antibody in the embodiment comprises a first reagent, a second reagent and a calibrator, wherein the volume ratio of the first reagent to the second reagent in the detection kit is 3: 1.
1. A first reagent
The first reagent is a mixed solution consisting of latex microspheres coated with novel coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, Bovine Serum Albumin (BSA), MOPSO buffer solution, preservative (PC-300) and solvent water.
The preparation method of the first reagent in this embodiment specifically includes the following steps:
1) preparing 25mM MES solution, and adjusting the pH of the solution to 6.5 by using 6mol/L NaOH to obtain MES buffer solution;
2) preparing an MOPSO solution with the concentration of 50mM, sequentially adding Arg, NaCl, trehalose, BSA and PC-300 into the solution, stirring for dissolving, and adjusting the pH of the solution to 7.0 by using 6mol/L HCl to obtain an MOPSO buffer system;
3) taking a carboxylated polystyrene microsphere with the particle size of 123nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 8g/L, adding EDC powder which is 15 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 1.0 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 8g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the novel coronavirus recombinant protein which is 0.02 times of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 2.5 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.3g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing at 300rpm for 2.5 hours;
8) and after timing is finished, placing the latex solution obtained in the step 7) in an oven at 37 ℃ for 16h to obtain a first reagent.
2. Second reagent
The second reagent and the method for preparing the same as in the third embodiment.
3. Calibration article
The calibrator and the preparation method thereof in this embodiment are the same as those in the first embodiment.
The detection method comprises the following steps:
during the detection, firstly, calibration is carried out: the method comprises the steps of taking a first reagent and a calibrator, mixing and incubating, adding a second reagent into a system, mixing and incubating, wherein a first microsphere in the first reagent and a second microsphere in the second reagent are combined due to immunoreaction, and the immunoreaction generates obvious absorbance change in the reaction system through the amplification effect of double latex microspheres. The instrument records the absorbance change value of the reaction system caused by the addition of the second reagent, and a calibration curve of the novel coronavirus antibody concentration can be prepared by a group of calibrators and the corresponding absorbance change values.
And (3) detection:
and (3) mixing and incubating the first reagent and the calibrator, adding the second reagent into the reaction system, mixing and incubating, recording the absorbance change value of the reaction system caused by the addition of the second reagent by using an instrument, and substituting the absorbance change value into the calibration curve obtained in the calibration step to calculate the concentration of the novel coronavirus antibody in the detection sample so as to judge whether the sample contains the novel coronavirus antibody.
The fifth example and the sixth example are respectively a semi-automatic biochemical analyzer and a full-automatic biochemical analyzer, and the detection kit prepared in the above examples is used for detecting the novel coronavirus antibody.
EXAMPLE five
In this embodiment, taking specific protein analyzer EZ-400 produced by luo jing laola electronics ltd as an example, the detection method of the detection kit for detecting the novel coronavirus antibody is as follows:
firstly, 160uL of first reagent is added into a reaction cup matched with an analyzer, 20uL of sample to be detected is added, the reaction cup is placed into a test hole of the analyzer, 40uL of second reagent is added into the reaction cup after stirring for 30 seconds, the reaction cup is stirred for 10 seconds and then reacts for 3 minutes, and the analyzer records the absorbance change value of the solution in the reaction cup after the second reagent is added.
And (3) preparing a calibration curve by using the absorbance change values of the calibrator in multiple tests, and substituting the absorbance change values in the test of the sample into the calibration curve to judge whether the sample contains the novel coronavirus antibody.
EXAMPLE six
In this embodiment, taking the fully automatic biochemical analyzer BS-400 produced by shenzhen mirui biomedical electronics limited company as an example, the detection method of the detection kit for detecting the novel coronavirus antibody is as follows,
the calibrator of example one was used for calibration on a fully automated biochemical analyzer miyer BS-400 and then tested for accuracy and reproducibility. The analysis method is an endpoint method, the calibration rule is Spline, the reaction direction is ascending, the main wavelength is 546nm, the blank correction is 43, the reaction time is 80, the first reagent amount is 160uL, the sample amount to be detected is 20uL, the second reagent amount is 40uL, and the concentration unit ug/mL.
Firstly, 160uL of first reagent is taken and incubated for 112 seconds at the temperature of 37 ℃; adding 20uL of sample to be detected, incubating for 270 seconds at 37 ℃, adding 40uL of second reagent, and reacting for 342 seconds at 37 ℃; and recording the absorbance of the reaction process by an instrument at 546nm to obtain the absorbance change value after the second reagent is added in the single test process.
And setting an instrument to prepare a calibration curve by taking the calibrator as a sample, substituting the absorbance change value during testing of the sample to be tested into the calibration curve to obtain the concentration of the novel coronavirus antibody in the sample, and further judging whether the sample contains the novel coronavirus.
Comparative example 1
In the comparison example, the reagent combination of the latex enhanced immunoturbidimetry commonly used in the industry is taken as an example for comparison, namely the first reagent is a buffer solution or a diluent, the first reagent is a MOPSO buffer system in the example, the second reagent is a latex solution containing latex microspheres coated with the novel coronavirus recombinant protein, and the concentration of the novel coronavirus antibody in the sample is detected.
The second reagent of this comparative example was prepared as follows:
1) preparing MES buffer solution as in the first example;
2) preparing a MOPSO buffer system as in the first embodiment;
3) taking a carboxylated polystyrene microsphere with the particle size of 198nm, diluting the carboxylated polystyrene microsphere with MES buffer solution until the microsphere concentration is 10g/L, adding EDC powder which is 20 times of the mass of the carboxyl of the taken microsphere according to the mass ratio, and uniformly stirring at 300rpm for 1.5 h;
4) after timing is finished, centrifuging the solution obtained in the step 3) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
5) re-suspending the microspheres obtained in the step 4) by using MES buffer solution until the concentration of the microspheres is 10g/L, ultrasonically dispersing the latex solution for 5 minutes by using an ultrasonic cell crusher according to 30% power, adding the novel coronavirus recombinant protein which is 0.01 time of the mass of the microspheres obtained in the step 3) according to the mass ratio, and uniformly stirring at 300rpm for 2.0 hours;
6) after timing is finished, centrifuging the solution obtained in the step 5) for 15 minutes under the condition of 11000g, and removing supernatant to obtain microsphere precipitate;
7) resuspending the microspheres obtained in step 6) by using an MOPSO buffer system to obtain a latex solution with the microsphere concentration of 0.8g/L, ultrasonically dispersing the latex solution by using a cell crusher according to the power of 30% for 5 minutes, and stirring and uniformly mixing the latex solution at 300rpm for 2.5 hours;
8) and after timing is finished, placing the latex solution obtained in the step 7) in an oven at 37 ℃ for 16h to obtain a second reagent.
The MOPSO buffer system prepared in the step 2) is used as a first reagent, the calibrator in the first embodiment is used for calibration on a full-automatic biochemical analyzer Mirui BS-400, and then the accuracy and the repeatability of the calibration are tested, wherein the test method is the same as that in the sixth embodiment.
Comparative example No. two
In the comparison example, the reagent combination of the competition method commonly used in the industry is taken as an example for comparison, namely, the free novel coronavirus recombinant protein is directly added into the buffer solution to be used as a first reagent, the latex solution containing the latex microspheres coated with the monoclonal antibody against the novel coronavirus recombinant protein is used as a second reagent, and the concentration of the novel coronavirus antibody in the sample is detected.
Preparing a MOPSO buffer system according to the method of the first to the fourth embodiments and adding a novel coronavirus recombinant protein as a first reagent, wherein the concentration of the novel coronavirus recombinant protein is 2 ug/mL;
preparing a latex solution as a second reagent according to the preparation steps of the second reagent in the third or fourth example;
the calibrator of example one was used for calibration on a fully automated biochemical analyzer Mirey BS-400 and then tested for accuracy and repeatability, as in example six.
Results and analysis of the experiments
The detection instrument and the detection method in the sixth embodiment are adopted to detect the detection kits prepared in the first to fourth embodiments and the 3 concentration calibrators in the first and second comparative examples, and the detection results are shown in the following table. The results in the table are the values of the reactivity, which are calculated by the detection instrument based on the absorbance.
TABLE 1 comparison of reactivity of examples and comparative examples in fully automated Biochemical Analyzer
The data in table 1 show that when a full-automatic biochemical analyzer is used for detecting a calibrator with the same concentration, the reactivity measured in the first to fourth examples is remarkably improved compared with that measured in the first and second comparative examples; and when the calibrators with different concentrations are detected, higher reactivity difference values can be obtained in the first to fourth embodiments, namely higher unit antibody concentration reactivity, which shows that the sensitivity of the first to fourth embodiments for detecting the novel coronavirus antibody is obviously improved compared with the method in the prior art.
In order to meet the detection requirement of the new coronavirus in the market and overcome the defects of the existing product, the invention provides the detection kit and the detection method of the novel coronavirus antibody, which have high sensitivity and strong specificity, and can meet the requirements of basic-level instant detection and full-automatic rapid high-throughput screening. The detection kit of the invention judges whether the novel coronavirus antibody exists in the sample by adopting a method that the latex microsphere coated with the novel coronavirus monoclonal antibody and the novel coronavirus antibody in the sample compete with each other and are combined with another latex microsphere coated with the novel coronavirus recombinant antigen according to the basic principle of immunoassay. The detection kit provided by the invention uses a latex homogeneous reaction system, can complete single detection within minutes, can be applied to a clinical common full-automatic biochemical analyzer to realize rapid high-flux detection, can bear the burden of large-batch tests, can be used for performing bedside rapid detection on small POCT equipment such as a specific protein analyzer for instant detection and the like, realizes the identification requirement of basic units, is rapid in detection, can simultaneously detect total antibodies such as IgM, IgG, IgA and the like of new coronavirus in human serum and plasma samples, and has the advantages of high sensitivity and wide detection range. The principle basis of the competition method can ensure that the detection kit can effectively avoid the HOOK effect; high sensitivity is achieved by using a monoclonal antibody with high specificity and enhancing immunoreaction through double latex.
The method can be applied to a general large-scale automatic biochemical analyzer. The universal large-scale full-automatic biochemical analyzer is popularized in most hospitals, is an open platform and can adapt to latex reagents of various manufacturers. The method of the present invention can also be used in small POCT (point of care testing) devices such as specific protein analyzers, which have a large number of applications in primary hospitals. Compared with the existing chemiluminescence or colloidal gold method in the market, the method of the invention has obviously stronger applicability and can well meet the use requirements of different places from large-scale three hospitals to community primary hospitals in China.
The detection kit for detecting coronavirus antibodies of the present application can be used for the detection of novel coronaviruses, and is mainly applied to the detection of non-diagnostic coronavirus antibodies, but can also be applied to detection for diagnosis.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A detection kit for detecting coronavirus antibodies comprises a first reagent and a second reagent, and is characterized in that the first reagent is a solution containing first microspheres, and the first microspheres are latex microspheres coated with coronavirus recombinant proteins; the second reagent is a solution containing second microspheres, and the second microspheres are latex microspheres coated with anti-coronavirus recombinant protein antibodies.
2. The test kit of claim 1, wherein the mass of coronavirus recombinant protein in the first microspheres is 0.01-0.025 times the mass of the original latex microspheres.
3. The test kit according to claim 1, wherein the mass of the anti-coronavirus recombinant protein antibody in the second latex microsphere is 0.04-0.08 times the mass of the original latex microsphere.
4. The detection kit as claimed in claim 2 or 3, wherein the primary latex microspheres have an average particle size of 120-210 nm.
5. The detection kit of claim 1, wherein the first reagent comprises a first microsphere and a buffer system solution, and the concentration of the first microsphere in the first reagent is 0.2g/L to 0.35 g/L.
6. The detection kit of claim 1, wherein the second reagent comprises a second microsphere and a buffer system solution, and the concentration of the second microsphere in the second reagent is 0.5g/L to 1.0 g/L.
7. The detection kit according to claim 5 or 6, wherein the buffer system solution comprises the following components: buffer solution, amino acid, sodium chloride, trehalose, bovine serum albumin and preservative.
8. The test kit of claim 1, further comprising a calibrator comprising the following components: anti-coronavirus recombinant protein antibody and calibrator stock solutions.
9. The detection kit according to claim 1, wherein the coronavirus is SARS-CoV-2.
10. A method for preparing a detection kit for detecting coronavirus antibodies according to any one of claims 1-9, comprising the steps of:
adding coronavirus recombinant protein or an anti-coronavirus recombinant protein antibody into the activated latex microspheres, and correspondingly preparing to obtain first microspheres or second microspheres;
and mixing the first microspheres or the second microspheres with a buffer system solution to correspondingly prepare a first reagent and a second reagent.
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