CN111812336A - Detection kit for detecting coronavirus antibodies and preparation method thereof - Google Patents

Abstract

The invention discloses a detection kit for detecting coronavirus antibodies, comprising a first reagent and a second reagent, the first reagent is a solution containing first microspheres, and the first microspheres are coated with coronavirus Latex microspheres of viral recombinant protein; 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 of the present invention coats the novel coronavirus recombinant protein antigen and the paired monoclonal antibody on latex microspheres respectively, and uses the latex microspheres coated with the recombinant protein antigen to first combine with the novel coronavirus antibody in the sample to be tested, Then, it is combined with an indirect competition method of latex microspheres coated with anti-recombinant protein monoclonal antibodies, and the change in the absorbance (absorbance) of the solution is used to determine whether there are new coronavirus antibodies in the sample to be tested. The detection kit for detecting novel coronavirus antibodies of the present invention has strong specificity, high sensitivity, wide detection range, and can greatly reduce the cost of reagents.

Description

Detection kit for detecting coronavirus antibodies and preparation method thereof

technical field

The invention relates to the technical field of biomedicine, in particular to a detection kit for detecting coronavirus antibodies and a preparation method of the detection kit.

Background technique

Pneumonia caused by the new coronavirus is an acute infectious disease whose pathogen is a coronavirus that has not been found in humans before, namely SARS-CoV-2, and the disease it causes is named by the World Health Organization as COVID-19 , mainly through respiratory droplets and close contact transmission, is highly contagious, and the crowd is generally susceptible to infection.

Since the development of the pneumonia epidemic caused by the new coronavirus, it is necessary to screen samples quickly and accurately on a large scale to provide a basis for the clinical diagnosis of patients with new coronavirus infection, and to provide rapid and timely judgment results for enterprises to resume work and production. In order to meet market demand, many practitioners in the diagnostic industry have developed a series of new coronavirus detection kits, mainly including nucleic acid detection and antibody detection. Nucleic acid testing generally uses the PCR method to determine whether the nucleic acid sequence of the new coronavirus exists in the sample, so as to determine whether the tested person is infected with the virus. Antibody detection is based on the basic principles of immunology. When the virus is infected, the human immune system will produce corresponding antibodies. By detecting whether there are new coronavirus antibodies in the sample, it can indirectly determine whether the person under test is infected with the virus.

Nucleic acid detection based on PCR technology has high accuracy, but it takes a long time, requires special equipment and places, and has high requirements on operators. The existing antibody detection mainly includes colloidal gold and chemiluminescence. The colloidal gold method is simple to operate and fast, and is suitable for the immediate detection market such as rapid inspection. The disadvantage is that it cannot be automated, cannot perform large-scale screening, and at the same time, the sensitivity is not high, and the repeatability is not easy to control. The antibody detection scheme based on chemiluminescence method is suitable for high-throughput screening, but it requires supporting instruments. The existing instrument platforms of chemiluminescence products on the market are closed platforms, and the reagents cannot be used universally, which is very unfavorable for promotion and inconvenient to use. It is used in fast inspection scenarios such as real-time inspection.

Particles Enhanced Turbidity Immuno-Assay-PETIA (Particles Enhanced Turbidity Immuno-Assay-PETIA) uses antigens or antibodies adsorbed on latex microspheres with a diameter of tens to hundreds of nanometers to capture the antibodies or antigens in the sample to be tested, thereby forming a space between the microspheres. The cross-linking of the solution changes the scattering degree or transmission absorbance (i.e. turbidity) of the solution. By measuring the change of the scattering degree or transmittance absorbance of the solution, the concentration of the analyte can be known qualitatively or quantitatively. The conventional latex-enhanced immune turbidimetric reagent includes two parts, R1 and R2. R1 is the diluent, which is used to dilute the test sample, and R2 is the latex microsphere solution, which is used to produce absorbance changes. The above-mentioned turbidimetric method and the latex reagent designed by the conventional competitive method require a single novel coronavirus antigen to bridge the antibodies on more than two microspheres at the same time to cause turbidity changes. The efficiency of this reaction is very low, resulting in more obvious turbidity changes when there are higher concentrations of antibody in the sample, thus reducing the sensitivity of the detection.

SUMMARY OF THE INVENTION

In view of this, in order to overcome the defects of the prior art, the purpose of the present invention is to provide an improved detection kit for detecting coronavirus antibodies, which has high sensitivity, strong specificity, and reduced reagent cost.

In order to achieve the above object, the present invention adopts the following technical scheme:

A detection kit for detecting coronavirus antibodies, comprising a first reagent and a second reagent, the first reagent is a solution containing the first microspheres, and the first microspheres are coated with a coronavirus recombinant protein. Latex microspheres; the second reagent is a solution containing second microspheres, and the second microspheres are latex microspheres coated with an anti-coronavirus recombinant protein antibody; the antibody is a monoclonal antibody.

In this application, coating refers to the binding of antigens or antibodies to the surface of a solid support (latex microspheres). In the actual product of the detection kit of the present application, the amount of the second reagent can be much smaller than the amount 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 reagents in the detection kit of the present invention and the conventional latex reagents using the competition method is that the components in the conventional reagents used to compete with the target to be tested (in this application, the coronavirus recombinant protein antigen) are Present in the reagent in a free state, and the recombinant protein antigen used for competition in the reagent of the present application has been coated on latex microspheres.

The method of coating the antigen for competition on latex microspheres has the following advantages: 1. The sensitivity of the reagent can be greatly improved. The latex reagent designed by the conventional competition method requires a single coronavirus antigen to bridge the antibodies on more than two microspheres at the same time to cause absorbance changes. The efficiency of this reaction is very low, resulting in a relatively obvious change in absorbance only when there is a higher concentration of antibody in the sample, thereby reducing the sensitivity of the reagent. In the present invention, the antigen used for competition has been coated on the latex microspheres, and it only needs to react with an antibody on the latex microspheres in the second reagent to bridge the latex microspheres and cause a significant change in the absorbance of the solution, thus greatly improving the Sensitivity of reagents. 2. The cost of reagents can be greatly reduced. Because the antigen in the first reagent is coated on the latex microspheres, the amount of antigen used can be greatly reduced, correspondingly, the amount of the antibody coated on the latex microspheres in the second reagent can be greatly reduced, thereby greatly reducing the amount of the antigen used in the second reagent. Reduce reagent costs.

According to some preferred implementation aspects of the present invention, in terms of mass ratio, the amount of recombinant coronavirus protein in the first microspheres is 0.01-0.025 times the amount of the original latex microspheres; if the amount is less than 0.01 times, the kit will be reduced The detection sensitivity of 0.025 times is the amount of coronavirus recombinant protein covering the surface of latex microspheres, that is, the upper limit. The positive impact on the results above the upper limit will not be significantly improved, and the cost of reagents will increase. The amount of the anti-coronavirus recombinant protein antibody in the second microspheres is 0.04-0.08 times the amount of the original latex microspheres; if it is less than 0.04 times, the detection sensitivity of the kit is greatly reduced, and the amount of 0.08 times is the antibody spread. The amount of one layer on the surface of the full latex microsphere is the upper limit. The antibody used in the second reagent in the detection kit is a monoclonal antibody, which has high specificity and further improves the detection sensitivity.

In this application, 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 implementation aspects of the present invention, the average particle size of the original latex microspheres is 120-210 nm; the latex microspheres are carboxyl polystyrene microspheres. When the average particle size of latex microspheres is less than 120nm, the detection sensitivity of the kit decreases and the amount of raw materials increases, that is, the cost increases; when the average particle size of latex microspheres is less than 210nm, the cost of raw materials decreases, but the particle size of latex particles becomes larger, and its specific surface area will become smaller. The amount of carboxyl groups available for antibody linking on the surface of the unit sphere decreases, so when the amount of latex microspheres remains the same, the amount of antibody that can be coated will decrease if the particle size of the latex microspheres increases, and the signal value will increase on the basis of the increase in noise. It will not increase significantly, resulting in a decrease in the detection signal-to-noise ratio of the kit, that is, low repeatability.

According to some preferred implementation aspects of the present invention, the first reagent includes first microspheres and a buffer system solution, and the concentration of the first microspheres in the first reagent is 0.2g/L-0.35g/L; The second reagent includes a second microsphere and a buffer system solution, and the concentration of the second microsphere in the second reagent is 0.5g/L-1.0g/L.

According to some preferred implementation aspects of the present invention, the buffer system solution is a morpholine buffer system, specifically a MOPSO buffer system. In some embodiments, its components and the concentrations of each component in the buffer system solution are as follows:

The first reagent using the above buffer system solution is the first microsphere, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative (PC- 300) and a mixed solution of solvent water; the second reagent is the second microsphere, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, A mixed solution of preservative (PC-300) and solvent water.

According to some preferred implementation aspects of the present invention, the detection kit further includes calibrators of anti-coronavirus recombinant protein antibodies at different concentrations (0-10ug/mL), and the calibrators include anti-coronavirus recombinant protein antibodies and calibrators Preservation solution.

In the calibrator, the concentration of the anti-coronavirus recombinant protein antibody is selected from two or more concentrations in the range of 0-10ug/mL. Specifically, in some embodiments of the present invention, the concentration of the anti-coronavirus recombinant protein antibody in the calibrator is: 0ug/mL, 1ug/mL and 5ug/mL. The selection of the three concentrations is based on the clinical negative-positive reference interval, where 0ug/mL is negative, 1ug/mL is the median value, and 5ug/mL is the high value.

According to some preferred implementation aspects of the present invention, the components in the calibrator preservation solution and the concentrations of each component in the calibrator preservation solution are as follows: 9g/L sodium chloride, 25mM HEPES buffer with pH 7.6, 2g/L of laureth (Thesit), 200mL/L of calf negative serum and 1g/L of preservatives. The preservative is selected from one or more of sodium azide, PC-300 and PC-950.

According to some preferred implementation aspects of the present 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 (new coronavirus).

The present application also provides a method for preparing the above-mentioned detection kit for detecting coronavirus antibodies, comprising the following steps:

A coronavirus recombinant protein or an anti-coronavirus recombinant protein antibody is added to the activated latex microspheres, and a first microsphere or a second microsphere is prepared correspondingly;

Mix the first microspheres or the second microspheres with the buffer system solution to prepare the first reagent and the second reagent correspondingly.

According to some preferred implementation aspects of the present invention, the preparation method of the first reagent includes the following steps: adding an activation reagent to a buffer solution containing latex microspheres, mixing, centrifuging, and discarding the supernatant; adding the buffer solution again, and performing Disperse after resuspension, add coronavirus recombinant protein, mix evenly, centrifuge, and discard the supernatant; add buffer system solution, resuspend, disperse, and mix to obtain the first reagent.

In some embodiments of the present invention, the first reagent preparation method specifically includes the following steps:

1) Activated latex microspheres: Add activation reagent EDC to the MES buffer containing 5g/L-15g/L latex microspheres, stir or shake to mix for 1.0-2.0h, centrifuge the solution after the time is up, discard the supernatant, Obtain latex microsphere precipitation;

2) Use MES buffer to resuspend the latex microspheres obtained in step 1) and precipitate until the latex microsphere concentration in the resuspension is 5g/L-10g/L. After dispersion, add coronavirus recombinant protein, mix for 1.5-3h, and time After the solution is centrifuged, the supernatant is discarded, and the latex microspheres are precipitated; the unstable first microspheres are obtained at this time;

3) Use the buffer system solution to resuspend the latex microspheres obtained in step 2) and precipitate the latex microspheres into the resuspended liquid with a concentration of 0.2g/L-0.35g/L. After ultrasonic or homogenous dispersion, stir or shake and mix for 1.5- 3h, after the timer is over, place it at 37°C for 14-20h to obtain the first reagent.

According to some preferred implementation aspects of the present invention, the preparation method of the second reagent includes the following steps: adding an activation reagent to the buffer solution containing latex microspheres, mixing, centrifuging, and discarding the supernatant; adding the buffer solution again, and performing After resuspending, disperse, add anti-coronavirus recombinant protein antibody, mix evenly, centrifuge, and discard the supernatant; add buffer system solution, resuspend, disperse, and mix to obtain the second reagent.

In some embodiments of the present invention, the preparation method of the second reagent specifically includes the following steps:

1) Activated latex microspheres: Add activation reagent EDC to the MES buffer containing 5g/L-15g/L latex microspheres, stir or shake to mix for 1.0-2.0h, centrifuge the solution after the time is up, discard the supernatant, Obtain latex microsphere precipitation;

2) Use MES buffer to resuspend the latex microspheres obtained in step 1) and precipitate the latex microspheres into the resuspension. , stir or shake and mix for 1.5-3h, centrifuge the solution at the end of time, discard the supernatant, and get latex microsphere precipitation; at this time, the unstable second microsphere is obtained;

3) Use the buffer system solution to resuspend the latex microspheres obtained in step 2) to precipitate the latex microspheres in the resuspended liquid with a concentration of 0.5g/L-1.0g/L. After ultrasonic or homogeneous dispersion, stir or shake and mix for 1.5- 3h, after the timer is over, place it at 37°C for 14-20h to obtain the second reagent.

In some preferred embodiments of the present invention, the centrifugation conditions in the preparation of the above two latex microspheres are 11,000 g, and the centrifugation time is 15 minutes; the dispersion conditions are ultrasonic dispersion, 30% power, and the ultrasonic time is 5 minutes.

According to some preferred implementation aspects of the present invention, in terms of mass ratio, the amount of the activating reagent is 10-20 times the amount of carboxyl groups on the latex microspheres. The dosage of the activating reagent is based on the stirring or shaking time in the above step 1). Under the premise of the stirring time of 1.0-2.0h, the amount of the activating reagent with 10-20 times the amount of carboxyl group is the optimal dosage, that is, it can achieve better The activation effect can also control the amount of activation reagent.

The buffer used in the preparation of the above two microspheres is MES buffer, pH value is 6.5, and the concentration of MES is 25mM.

The present invention also provides a detection method for the detection kit for detecting coronavirus antibodies, comprising the following steps:

Mix and incubate the sample to be tested with the first reagent, and the recombinant coronavirus protein coated on the first microspheres in the first reagent is combined with the coronavirus antibody in the sample to be tested;

Add the second reagent to the system, mix and incubate, the recombinant coronavirus protein on the first microspheres in the first reagent that is not bound to the coronavirus antibody in the sample to be tested is coated with the second microspheres in the second reagent The anti-coronavirus recombinant protein antibody binds and produces absorbance change, and the absorbance change value is recorded and analyzed.

According to some preferred implementation aspects of the present invention, the mixing ratio of the first reagent and the calibrator or the sample to be tested is 8:1 by volume.

According to some preferred implementation aspects of the present invention, the mixing ratio of the system after the reaction of the first reagent with the calibrator or the sample to be tested and the second reagent is 9:2 by volume.

According to some preferred implementation aspects of the present invention, the wavelength used by the analytical instrument to record the absorbance change value is 546 nm.

Different from conventional competition method reagents, the first reagent and the second reagent in the detection kit of the present invention are latex solutions, wherein the first reagent is a solution containing latex microspheres coated with new coronavirus recombinant protein, and the second reagent is a latex solution. The reagent is a solution containing latex microspheres coated with anti-2019-nCoV recombinant protein monoclonal antibody. The detection method is designed according to the competition method. During the detection, the sample to be tested is first reacted with the first reagent, and the coronavirus antibody in the sample is combined with the antigen on the latex microspheres in the first reagent. Then, the second reagent is added to the reaction system, and the antibody on the latex microspheres in the second reagent reacts with the remaining antigens on the latex microspheres in the first reagent, so that the absorbance (turbidity) of the reaction system changes.

When there is no novel coronavirus antibody in the sample to be tested, the first reagent is fully combined with the latex microspheres in the second reagent, and the absorbance changes the most. If the sample to be tested contains coronavirus antibodies, the antibodies in the sample to be tested will consume part of the antigens on the latex microspheres in the first reagent, resulting in insufficient binding of the latex microspheres of the first reagent and the second reagent when they are mixed sufficient, the resulting absorbance change value decreases. By comparing the change in absorbance, it can be known whether the test sample contains new coronavirus antibodies and the content level of new coronavirus antibodies in the sample.

Due to the adoption of the above technical solution, compared with the prior art, the present invention has the advantages that: the detection kit for detecting novel coronavirus antibodies of the present invention comprises a latex microbe coated with a novel coronavirus recombinant protein. The first reagent of the ball and the second reagent containing the latex microspheres coated with the anti-new coronavirus recombinant protein antibody are used to coat the new coronavirus recombinant protein antigen on the latex microspheres, and the first reagent is coated with the latex of the antigen. The microspheres only need to react with an antibody on the latex microspheres coated with the antibody in the second reagent to bridge the latex microspheres and cause a significant change in the absorbance of the solution, thus greatly improving the sensitivity of the reagent; by using in the first reagent The antigen is coated on latex microspheres, which can greatly reduce the amount of antigen and the antibody coated on latex microspheres used in the second reagent, thereby greatly reducing the cost of reagents.

Detailed ways

In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

The detection kit for detecting novel coronavirus antibodies in this embodiment includes a first reagent, a second reagent and a calibrator, and the volume ratio of the first reagent and the second reagent in the detection kit is 4:1.

1. The first reagent

The first reagent is latex microspheres coated with new coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative (PC- 300) and a mixed solution of solvent water.

The preparation method of the first reagent in this embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) The MOPSO solution with a concentration of 50 mM is prepared, Arg, NaCl, trehalose, BSA, PC-300 are added successively to the MOPSO solution, stirred and dissolved, and the pH of the solution is adjusted to 7.0 with 6 mol/L HCl to obtain a MOPSO buffer system ; The concentration of each substance in the MOPSO buffer system is as follows: Arg 40g/L, NaCl 9g/L, trehalose 40g/L, BSA 10g/L, PC-3001g/L;

3) Take carboxylated polystyrene microspheres with a particle size of 123 nm, dilute with MES buffer to a concentration of 10 g/L of microspheres, add EDC powder with 10 times the mass of the carboxyl groups of the microspheres taken by mass ratio, and stir and mix at 300 rpm. evenly 1.0h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 5 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the recombinant protein of the novel coronavirus with 0.025 times the mass of the microspheres taken in ) was stirred and mixed at 300 rpm for 1.5 h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres in step 6) and precipitate to a latex solution with a microsphere concentration of 0.35g/L, then use a cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300rpm for 1.5 minutes. h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 14 hours to obtain the first reagent.

2. Second reagent

The second reagent is latex microspheres coated with anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative ( PC-300) and solvent water.

The preparation method of the second reagent in the present embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) prepare the MOPSO solution with a concentration of 50 mM, add Arg, NaCl, trehalose, BSA, PC-300 to the solution successively, stir and dissolve, and adjust the pH of the solution to 7.0 with 6 mol/L HCl, to obtain a MOPSO buffer system;

3) Take carboxylated polystyrene microspheres with a particle size of 123 nm, dilute with MES buffer to a concentration of 10 g/L of microspheres, add EDC powder with 10 times the mass of the carboxyl groups of the microspheres taken by mass ratio, and stir and mix at 300 rpm. evenly 1.0h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 10 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the anti-novel coronavirus recombinant protein monoclonal antibody with 0.08 times the mass of the microspheres used in ) was stirred and mixed at 300 rpm for 1.5 h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres of step 6) and precipitate to a latex solution with a microsphere concentration of 1.0 g/L, then use a cell crusher to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 1.5 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 14 hours to obtain the second reagent.

3. Calibrator

The calibrators in this example include calibrators of anti-novel coronavirus recombinant protein antibody solutions with different concentrations of 0ug/mL, 1ug/mL and 5ug/mL.

Each concentration of anti-2019-nCoV recombinant protein antibody solution includes anti-2019-nCoV recombinant protein antibody and calibrator preservation solution. Among them, the calibrator preservation solution includes sodium chloride, HEPES buffer, lauryl alcohol polyoxyethylene ether (Thesit), calf negative serum and PC-300.

The preparation method of the anti-novel coronavirus SARS-CoV-2 recombinant protein monoclonal antibody calibrator in this embodiment specifically includes the following steps:

1) Prepare a HEPES solution with a concentration of 25 mM, and use 6 mol/L NaOH to adjust the pH of the solution to 7.6;

2) Add NaCl, laureth (Thesit), calf negative serum, PC-300 to the solution in step 1) in turn, stir and mix to obtain a calibrator preservation solution; The concentrations are: NaCl 9g/L, laureth 2g/L, calf negative serum 200mL/L, PC-3001g/L.

3) Take the anti-novel coronavirus SARS-CoV-2 recombinant protein monoclonal antibody, and use the calibrator preservation solution in step 2) to dilute it into calibrators with concentrations of 1ug/mL and 5ug/mL, that is, the calibrator.

Embodiment 2

The detection kit for detecting novel coronavirus antibodies in this embodiment includes a first reagent, a second reagent and a calibrator, and the volume ratio of the first reagent and the second reagent in the detection kit is 5:1.

1. The first reagent

The first reagent is latex microspheres coated with new coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative (PC- 300) and a mixed solution of solvent water.

The preparation method of the first reagent in this embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) prepare the MOPSO solution with a concentration of 50 mM, add Arg, NaCl, trehalose, BSA, PC-300 to the solution successively, stir and dissolve, and adjust the pH of the solution to 7.0 with 6 mol/L HCl, to obtain a MOPSO buffer system;

3) Take the carboxylated polystyrene microspheres with a particle size of 198nm, dilute to a microsphere concentration of 5g/L with MES buffer and add the EDC powder of 20 times the carboxyl group mass of the microspheres taken by mass ratio, and stir and mix at 300rpm. evenly 2.0h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 5 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the recombinant protein of the novel coronavirus with 0.01 times the mass of the microspheres taken in ) was stirred and mixed at 300 rpm for 3.0 h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres in step 6) and precipitate to a latex solution with a microsphere concentration of 0.2 g/L, then use a cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 3.0 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 20 hours to obtain the first reagent.

2. Second reagent

The second reagent is latex microspheres coated with anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative ( PC-300) and solvent water.

The preparation method of the second reagent in the present embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) prepare the MOPSO solution with a concentration of 50 mM, add Arg, NaCl, trehalose, BSA, PC-300 to the solution successively, stir and dissolve, and adjust the pH of the solution to 7.0 with 6 mol/L HCl, to obtain a MOPSO buffer system;

3) Take the carboxylated polystyrene microspheres with a particle size of 198nm, dilute to a microsphere concentration of 5g/L with MES buffer and add the EDC powder of 20 times the carboxyl group mass of the microspheres taken by mass ratio, and stir and mix at 300rpm. evenly 2.0h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 5 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the anti-novel coronavirus recombinant protein monoclonal antibody with 0.04 times the mass of the microspheres was used, and stirred and mixed at 300 rpm for 3.0 h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres in step 6) and precipitate the microspheres to a latex solution with a microsphere concentration of 0.5 g/L, then use a cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 3.0 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 20 hours to obtain the second reagent.

3. Calibrator

The calibrator and its preparation method in this example are the same as those in Example 1.

Embodiment 3

The detection kit for detecting novel coronavirus antibodies in this embodiment includes a first reagent, a second reagent and a calibrator, and the volume ratio of the first reagent and the second reagent in the detection kit is 5:1.

1. The first reagent

The first reagent and the preparation method thereof in this embodiment are the same as those in the second embodiment.

2. Second reagent

The second reagent is latex microspheres coated with anti-novel coronavirus recombinant protein antibody, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative ( PC-300) and solvent water.

The preparation method of the second reagent in the present embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) prepare the MOPSO solution with a concentration of 50 mM, add Arg, NaCl, trehalose, BSA, PC-300 to the solution successively, stir and dissolve, and adjust the pH of the solution to 7.0 with 6 mol/L HCl, to obtain a MOPSO buffer system;

3) Take the carboxylated polystyrene microspheres with a particle size of 198nm, dilute to a microsphere concentration of 8g/L with MES buffer and add the EDC powder of 15 times the carboxyl group mass of the microspheres taken by mass ratio, and stir and mix at 300rpm. uniform for 1.5h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 8 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the anti-novel coronavirus recombinant protein monoclonal antibody of 0.06 times the mass of the microspheres used in ) was stirred and mixed at 300rpm for 2.5h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres in step 6) and precipitate to a latex solution with a microsphere concentration of 0.8 g/L, then use a cell crusher to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 2.5 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 16 hours to obtain the second reagent.

3. Calibrator

The calibrator and its preparation method in this example are the same as those in Example 1.

Embodiment 4

The detection kit for detecting novel coronavirus antibodies in this embodiment includes a first reagent, a second reagent and a calibrator, and the volume ratio of the first reagent and the second reagent in the detection kit is 3:1.

1. The first reagent

The first reagent is latex microspheres coated with new coronavirus recombinant protein, arginine (Arg), sodium chloride (NaCl), trehalose, bovine serum albumin (BSA), MOPSO buffer, preservative (PC- 300) and a mixed solution of solvent water.

The preparation method of the first reagent in this embodiment specifically includes the following steps:

1) Prepare an MES solution with a concentration of 25 mM, and adjust the pH of the solution to 6.5 with 6 mol/L NaOH to obtain MES buffer;

2) prepare the MOPSO solution with a concentration of 50 mM, add Arg, NaCl, trehalose, BSA, PC-300 to the solution successively, stir and dissolve, and adjust the pH of the solution to 7.0 with 6 mol/L HCl, to obtain a MOPSO buffer system;

3) Take carboxylated polystyrene microspheres with a particle size of 123 nm, dilute with MES buffer to a concentration of 8 g/L of microspheres, add EDC powder with 15 times the carboxyl mass of the microspheres taken by mass ratio, and stir and mix at 300 rpm. evenly 1.0h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 8 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the recombinant protein of the novel coronavirus with 0.02 times the mass of the microspheres used in ) was stirred and mixed at 300rpm for 2.5h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres of step 6) and precipitate to a latex solution with a microsphere concentration of 0.3 g/L, then use a cell crusher to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 2.5 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 16 hours to obtain the first reagent.

2. Second reagent

The second reagent in this embodiment and the preparation method thereof are the same as those in Embodiment 3.

3. Calibrator

The calibrator and its preparation method in this example are the same as those in Example 1.

Detection method:

During detection, first perform calibration: mix and incubate the first reagent and calibrator, then add the second reagent to the system, mix and incubate, the first microspheres in the first reagent and the second microspheres in the second reagent Due to the combination of immune responses and the amplification of the dual latex microspheres, the immune response produces obvious changes in absorbance in the reaction system. This reaction process has both the high specificity of monoclonal antibodies and the dual latex microspheres. High sensitivity due to amplification. The instrument records the absorbance change value of the above reaction system due to the addition of the second reagent, and a calibration curve for the concentration of the novel coronavirus antibody can be made from a set of calibrators and the corresponding absorbance change value.

Detection:

Mix and incubate the first reagent and the calibrator, then add the second reagent into the reaction system, mix and incubate, the instrument records the absorbance change value of the reaction system caused by the addition of the second reagent, and brings this absorbance change value into the aforementioned calibration The calibration curve obtained in the step can calculate the concentration of the novel coronavirus antibody in the test sample and then judge whether the sample contains novel coronavirus antibody.

Embodiments 5 and 6 respectively take semi-automatic and fully automatic biochemical analyzers as examples, and use the detection kits prepared in the above embodiments to detect novel coronavirus antibodies.

Embodiment 5

In this example, the specific protein analyzer EZ-400 produced by Nanjing Laura Electronics Co., Ltd. is used as an example.

First, add 160uL of the first reagent to the reaction cup of the analyzer, add 20uL of the sample to be tested, put the reaction cup into the test hole of the analyzer, stir for 30 seconds, add 40uL of the second reagent to the reaction cup, and stir for 10 seconds After 3 minutes of reaction, the analyzer records the change in absorbance of the solution in the reaction cup after adding the second reagent.

Use the absorbance change values of the calibrators tested multiple times to make a calibration curve, and then substitute the absorbance change values of the test samples into the calibration curve to determine whether the samples contain novel coronavirus antibodies.

Embodiment 6

This embodiment takes the automatic biochemical analyzer BS-400 produced by Shenzhen Mindray Biomedical Electronics Co., Ltd. as an example.

Use the calibrator in Example 1 to calibrate on Mindray BS-400, an automatic biochemical analyzer, and then test its accuracy and repeatability. The analysis method is the endpoint method, the calibration rule is Spline, the reaction direction is rising, the dominant wavelength is 546 nm, the blank correction is 43, the reaction time is 80, the first reagent volume is 160uL, the sample volume to be tested is 20uL, and the second reagent volume It is 40uL, and the concentration unit is ug/mL.

First take 160uL of the first reagent, incubate at 37°C for 112 seconds; add 20uL of the sample to be tested, incubate at 37°C for 270 seconds, add 40uL of the second reagent, and react at 37°C for 342 seconds; the instrument records at 546nm From the absorbance in the above reaction process, the absorbance change value after adding the second reagent in a single test process is obtained.

Set the instrument to use the calibrator as the sample to make a calibration curve, and then substitute the absorbance change value when testing the sample to be tested into the calibration curve to obtain the concentration of the new coronavirus antibody in the sample, and then determine whether the sample contains the new coronavirus.

Comparative Example 1

In this comparative example, the reagent combination of the latex-enhanced immune turbidimetric method commonly used in the industry is used as an example for comparison, that is, the first reagent is a buffer solution or a diluent, in this example, it is a MOPSO buffer system, and the second reagent is a buffer system containing The latex solution of the new coronavirus recombinant protein latex microspheres is used to detect the concentration of the new coronavirus antibody in the sample.

The preparation method of the second reagent in this comparative example is as follows:

1) prepare MES buffer, same as Example 1;

2) prepare MOPSO buffer system, same as Example 1;

3) Take the carboxylated polystyrene microspheres with a particle size of 198 nm, dilute to a microsphere concentration of 10 g/L with MES buffer, and add the EDC powder of 20 times the carboxyl mass of the microspheres taken by mass ratio, and stir and mix at 300 rpm. uniform for 1.5h;

4) After the timing is over, centrifuge the solution obtained in the above step 3) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

5) Use MES buffer to resuspend the microspheres of step 4) and precipitate to a latex solution with a microsphere concentration of 10 g/L, use an ultrasonic cell pulverizer to ultrasonically disperse the latex solution at 30% power for 5 minutes, and add step 3 by mass ratio ), the recombinant protein of the new coronavirus with 0.01 times the mass of the microspheres taken in ) was stirred and mixed at 300 rpm for 2.0 h;

6) After the timing is over, centrifuge the solution obtained in the above step 5) at 11000g for 15 minutes, discard the supernatant, and obtain microsphere precipitation;

7) Use the MOPSO buffer system to resuspend the microspheres in step 6) and precipitate to a latex solution with a microsphere concentration of 0.8 g/L, then use a cell crusher to ultrasonically disperse the latex solution at 30% power for 5 minutes, stir and mix at 300 rpm for 2.5 h;

8) After the timing is over, place the latex solution obtained in step 7) in a 37° C. oven for 16 hours to obtain the second reagent.

Take the MOPSO buffer system prepared in step 2) as the first reagent, use the calibrator in Example 1 to calibrate on the automatic biochemical analyzer Mindray BS-400, and then test its accuracy and repeatability, and the test method is the same as the implementation Example six.

Comparative Example 2

In this comparative example, the reagent combination of the competitive method commonly used in the industry is used as an example for comparison, that is, the free state new coronavirus recombinant protein is directly added to the buffer as the first reagent, and the recombinant protein containing the anti-new coronavirus is added to the buffer as the first reagent. The latex solution of the cloned antibody latex microspheres is the second reagent to detect the concentration of the novel coronavirus antibody in the sample.

The MOPSO buffer system was prepared with reference to the methods of Examples 1 to 4 and the novel coronavirus recombinant protein was added as the first reagent, wherein the concentration of the novel coronavirus recombinant protein was 2ug/mL;

With reference to the preparation steps of the second reagent in Example 3 or 4, the latex solution is prepared as the second reagent;

Use the calibrator in Example 1 for calibration on an automatic biochemical analyzer Mindray BS-400, and then test its accuracy and repeatability. The test method is the same as Example 6.

Experimental results and analysis

Using the detection instrument and detection method in Example 6, the detection kits prepared in Examples 1 to 4, as well as Comparative Examples 1 and 2, were tested for three concentrations of calibrators, and the detection results are shown in the following table. The detection results in the table are the reactivity values, which are calculated by the detection instrument according to the absorbance.

Table 1 Example and Comparative Example Comparison of Responsiveness in Automatic Biochemical Analyzer

The data in Table 1 shows that when using the automatic biochemical analyzer to detect the calibrator of the same concentration, the reactivity measured in Examples 1 to 4 is significantly improved compared to Comparative Examples 1 and 2; and when detecting calibrators of different concentrations, the embodiment One to four can obtain a higher difference in reactivity, that is, a higher unit antibody concentration reactivity, which shows that the sensitivity of the novel coronavirus antibody detection using Examples 1 to 4 is significantly higher than that of the methods in the prior art. promote.

In order to meet the detection needs of the new coronavirus in the market, and at the same time to overcome the shortcomings of the existing products, the present invention provides a high-sensitivity, strong specificity, which can not only meet the needs of real-time detection at the grassroots level, but also meet the requirements of automatic rapid high-throughput screening Required detection kits and detection methods for novel coronavirus antibodies. The detection kit of the present invention is based on the basic principle of immune detection, by using latex microspheres coated with a novel coronavirus monoclonal antibody and the novel coronavirus antibody in the sample to compete and combine with another type of novel coronavirus coated with a novel coronavirus recombinant recombinant Antigen latex microsphere method to determine whether there are antibodies to the new coronavirus in the sample. The detection kit of the present invention uses a latex homogeneous reaction system, and a single detection can be completed in a few minutes, and can be applied to a fully automatic biochemical analyzer commonly used in clinics to achieve rapid and high-throughput detection, and can also shoulder the heavy responsibility of large-scale testing. The bedside rapid detection is carried out on small POCT equipment such as specific protein analyzers for instant detection to meet the identification needs of grassroots units, and the detection is fast. It can simultaneously detect IgM, IgG, IgA and other total antibodies of the new coronavirus in human serum and plasma samples , high sensitivity and wide detection range. The principle basis of the competition method can effectively avoid the HOOK effect of the detection kit; the use of high-specificity monoclonal antibodies can enhance the immune response through double latex to achieve high sensitivity.

The method of the invention can be applied on a general large-scale automatic biochemical analyzer. General-purpose large-scale automatic biochemical analyzers have been popularized in most hospitals, and they are all open platforms, which can adapt to latex reagents from various manufacturers. The method of the present invention can also be used in small POCT (point-of-care detection) equipment such as specific protein analyzers, which have a large number of applications in primary hospitals. Therefore, compared with the existing chemiluminescence or colloidal gold methods on the market, the method of the present invention has significantly stronger applicability, and can well meet the needs of use in different places from large tertiary hospitals to community primary hospitals in my country.

The detection kit for detecting coronavirus antibodies of the present application can be used in the detection of new coronaviruses, and is mainly used in the detection of coronavirus antibodies for non-diagnostic purposes, but can also be used for detection purposes for diagnostic purposes.

The above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement them accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included 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.