CN114578050B

CN114578050B - Detection reagent for novel coronavirus antibodies and preparation method thereof

Info

Publication number: CN114578050B
Application number: CN202210236548.0A
Authority: CN
Inventors: 刘晓娟; 张小锐; 蔡华雅; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2025-02-28
Anticipated expiration: 2042-03-11
Also published as: CN114578050A

Abstract

The present application relates to a detection reagent for novel coronavirus antibodies and a method for preparing the same. Specifically, the present application relates to a kit for determining the content of SARS-CoV-2 antibodies in a sample by latex-enhanced immunoturbidimetry, which comprises a first reagent, a second reagent and a calibrator. The first reagent comprises a stabilizer, an aggregation promoter and a buffer. The second reagent comprises nanoparticles coated with SARS-CoV-2 antigens and a buffer. The kit of the present application has strong specificity, repeatability, sensitivity and detection range that can meet the requirements of clinical applications.

Description

Novel coronavirus antibody detection reagent and preparation method thereof

Technical Field

The present disclosure belongs to the field of in vitro diagnosis medicine immunization, and relates to a kit for determining SARS-CoV-2 antibody content in human sample by using latex enhanced turbidimetric immunoassay.

Background

The novel coronavirus (SARS-CoV-2) is a causative agent of the novel coronavirus pneumonia (COVID-19), and is an enveloped beta-type single-stranded RNA coronavirus.

The SARS-CoV-2 latency is typically 2 to 14 days, and the virus is at high risk of transmission from person to person, primarily through respiratory secretions and aerosols. Therefore, timely diagnosis of the carrier or infected person is critical to control epidemic and disease treatment. At least 27 proteins in SARS-CoV-2 include 15 nonstructural proteins (nsp 1 to nsp10, nsp12 to nsp 16), 4 structural proteins (S, E, M and N), and 8 accessory proteins (3 a, 3b, p6, 7a, 7b, 8b, 9b, and orf 14). The 4 structural proteins are spinous process protein (S), envelope (E), membrane (M) and nucleocapsid (N).

The S protein is a transmembrane protein, and forms a unique trimeric structure on the surface of coronaviruses. Each S monomer consists of an N-terminal S1 subunit and a membrane proximal S2 subunit. The S1 subunit comprises a receptor binding domain RBD (receptor binding domain) which binds to the host cell angiotensin converting enzyme 2 (angiotensin-converting enzyme ACE 2) receptor. RBD can induce organism to produce neutralizing antibody, and is ideal antigen for diagnosis and vaccine research.

Serological testing plays an important role in understanding viral infections and vaccine studies. At present, the main methods for detecting the serum SARS-CoV-2 specific antibody include a colloidal gold method, a chemiluminescent method and the like. The colloidal gold method has simple operation, no special equipment, low cost, low requirement on hardware, low sensitivity, only qualitative detection and low flux, and can finish detection within 15-30 min. The chemiluminescence method has high sensitivity and specificity, can perform quantitative detection, can be operated in batches, but requires special instruments, and is suitable for large and medium-sized hospitals. The latex enhanced immunoturbidimetry is simple and rapid to operate, has high flux, and can quantitatively determine SARS-CoV-2 antibody in human serum or plasma. Quantification of antibody responses facilitates the determination of specific antibody concentrations and allows for longitudinal monitoring of individual antibody dynamic levels.

CN113252911B discloses a detection kit for SARS-CoV-2 neutralizing antibody, which is characterized in that the detection kit is used for simultaneously detecting a neutralizing antibody combined with RBD protein and a neutralizing antibody combined with NTD protein, the detection kit comprises a component a and a component B, the component a comprises RBD protein coated on magnetic particles and NTD protein coated on magnetic particles, the component B comprises acridinium ester marked ACE2 protein and acridinium ester marked AXL protein, and the detection kit further comprises ACE2 antibody and AXL antibody.

CN113341150a relates to a SARS-CoV-2 detection kit, said kit comprising a) RBD specific ligand coupled to solid support, b) RBD recombinant protein coupled with label of acridine fluorophore, and first buffer for dissolving a component a, ph=5.5-6.5, second buffer for dissolving b component, ph=5.5-8.5, third buffer for lysing sample to release RBD antigen in virus, ph=3.0-4.5.

CN113495141A provides a SARS-CoV-2IgM antibody detection kit, which comprises two antigens marked by acridinium ester and a mouse anti-human IgM antibody coated on a solid phase carrier, wherein the two antigens are SARS-CoV-2RBD antigen and SARS-CoV-2N antigen.

CN111537743a relates to a novel coronavirus antibody detection kit of SARS-CoV-2, comprising a solid support, and a specific polypeptide combination attached to said solid support, further comprising an RBD domain of the S protein of SARS-CoV-2 attached to said solid support.

CN113607957a discloses a specific neutralizing antibody competition method ELISA kit for SARS-CoV-2RBD domain, which contains solid phase carrier coated with human ACE2 protein, biotin-labeled RBD protein.

CN113671184a provides a detection kit and method for detecting SARS-CoV-2 neutralizing antibody, and adopts protein tag to modify RBD and ACE2 so as to make them have better stability and reactivity.

Disclosure of Invention

With the advent of more viral variants or subtypes (e.g., alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu, omicron, etc.), single protogenic/wild-type RBDs have not met the needs of clinical testing. In view of this, the present disclosure provides a technical solution for RBD association.

Microsphere(s)

The present disclosure provides a nanoparticle having an antigen coated on a surface thereof. The nanoparticle and antigen are covalently or non-covalently bound, preferably covalently bound.

In some embodiments, the antigen is an RBD antigen selected from the group consisting of an RBD of SARS-CoV-2, a polypeptide comprising an RBD of SARS-CoV-2, or a polypeptide comprising an epitope of an RBD of SARS-CoV-2.

In the present disclosure, "RBD antigen" is to be understood broadly, as a full-length RBD, fragment or epitope thereof, so long as it is recognized and bound by an antibody to SARS-CoV-2 in the sample to be tested. Accordingly, the RBD antigen comprises RBD or an epitope thereof selected from any of the SARS-CoV-2 subtype, wild-type (also referred to herein as protogenic), alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron. When more subtypes are present in the future, the present disclosure is intended to cover these subtypes.

In some embodiments, SARS-CoV-2RBD is expressed by a host cell and is obtained by chromatographic purification. The above antigens can be prepared by art-recognized genetic recombination techniques, and commercial products can also be purchased.

In some specific embodiments, the SARS-CoV-2 antigen is an S protein, preferably the RBD of the S protein as the antigen.

In some embodiments, the nanoparticle is a core-shell structure. As an example, the core of the nanoparticle is a polystyrene polymer and the shell is composed of styrene, n-butyl acrylate, methacrylic acid copolymer. In some embodiments, the nanoparticle is a polystyrene nanoparticle particle.

In some embodiments, the nanoparticle surface bears a chemical group selected from the group consisting of a sulfate group, a sulfonate group, a carboxyl group, an amino group, a hydroxyl group, a hydrazide group, a chloromethyl group, and combinations thereof.

In some embodiments, the nanoparticle has an average particle size ranging from 200 to 450nm (inclusive), including but not limited to 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450nm, and ranges between any two of the foregoing. In some specific embodiments, the nanoparticle has a particle size of 325nm.

It should be emphasized here that "325nm" in this context does not mean that the particle size of each nanoparticle in the reagent is exactly 325nm. In practice, 325nm is a statistically significant number, and since the nanospheres are subject to errors during manufacture, the 325nm specification of nanospheres refers to a particle size range of about 325nm, e.g., 300 to 350nm, and more e.g., 310 to 340 nm.

In the present disclosure, the nanoparticle is not homogeneous, comprising a mixture of two nanoparticles, each coated with a different RBD antigen.

In some embodiments, the nanoparticle comprises a first nanoparticle and a second nanoparticle. The first nanometer microsphere is coated with SARS-CoV-2 first RBD antigen, and the second nanometer microsphere is coated with SARS-CoV-2 second RBD antigen. The first RBD antigen comprises RBDs or epitopes thereof selected from any of the SARS-CoV-2 subtypes Alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron, and the second RBD antigen comprises RBDs or epitopes thereof selected from any of the SARS-CoV-2 subtypes wild-type, alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron. The first RBD antigen and the second RBD antigen are different.

In some embodiments, the RBD antigen can be bound to the nanoparticle surface by physical adsorption or chemical coupling. As an example, a single protogenic RBD antigen, or a single mutant RBD antigen, or a combination of both are bound to the surface of the nanoparticle by a chemical coupling and crosslinking method. In some specific embodiments, the native RBD antigen and the mutant RBD antigen can be crosslinked to the surface of the nanoparticle, respectively, and then mixed according to a specific ratio, so that a better use effect can be achieved.

Reagent/kit

In some embodiments, there is provided a SARS-CoV-2 antibody detection kit comprising:

a first reagent comprising a stabilizer, a polymerization promoter, and a buffer;

A second reagent comprising:

The aforementioned first nanoparticle according to the present disclosure,

The aforementioned second nanoparticle according to the present disclosure,

Optionally, a stabilizer, and

And (3) a buffer solution.

In some embodiments, the first nanoparticle is coated with a SARS-CoV-2 first RBD antigen and the second nanoparticle is coated with a SARS-CoV-2 second RBD antigen.

In some embodiments, the first RBD antigen comprises RBD or an epitope thereof selected from any of SARS-CoV-2 subtype Alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron, and the second RBD antigen comprises RBD or an epitope thereof selected from any of SARS-CoV-2 subtype wild-type, alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron.

In some embodiments, the first RBD antigen and the second RBD antigen are different.

In some embodiments, the stabilizer is selected from any one of fatty alcohol polyoxyethylene ether, BSA, tween 20, brijL23, sucrose, or a combination thereof.

In some embodiments, by way of example, the stabilizing agent is selected from the group consisting of 0.1% to 5% bovine serum albumin by weight/volume (preferably 0.1% to 0.5%), sucrose in a concentration range of 1% to 10% by weight/volume, brijL23 in a concentration range of 0.1% to 1% by weight/volume (preferably 0.1% to 1%; more preferably 0.1% to 0.5%), and combinations thereof.

In some embodiments, the polymerization promoter is selected from any one of PEG6000, PEG8000, PEG20000, PVP, dextran, or a combination thereof.

In some embodiments, the coagulant is selected from the group consisting of PEG4000, PEG6000, PEG8000, PEG10000, PEG20000, dextran T-40, dextran T-70, dextran T-500, PVP, and combinations thereof. The accelerator concentration ranges from 0.1% to 10% by weight/volume, e.g., 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and ranges between any two of the foregoing.

In some embodiments, the buffer in the first and second reagents is independently selected from any one of phosphate buffer, glycine buffer, HEPES buffer, MES buffer, boric acid buffer, acetate buffer, ammonium chloride buffer, or a combination thereof.

In some embodiments, the concentration of buffer in the first reagent and the second reagent is independently 10mM to 500mM. In some embodiments, the concentration of the buffer includes, but is not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500mM, and ranges between any two of the foregoing.

In some embodiments, the pH of the buffer in the first and second reagents is independently from 6 to 8, preferably from 7.0 to 7.5, e.g., 7.2, 7.3, 7.4, 7.5.

In some embodiments, the buffers in the first reagent and the second reagent are the same or different.

In some embodiments, the concentration of the nanoparticle is 0.025% to 0.25% (weight/volume). For example, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25% and ranges between any two of the foregoing values. In some specific embodiments, the concentration of RBD coated polystyrene nanospheres is 0.125% (total of both microspheres) by weight/volume.

In some embodiments, the first agent further comprises any one or combination selected from 1% to 10% nacl by mass/volume, 0.05% to 0.2% preservative by mass/volume.

In some embodiments, the preservative is selected from the group consisting of sodium azide, thimerosal, phenol, sodium ethylmercuric thiosulfate, and combinations thereof. In some embodiments, the preservative concentration is from 0.02% to 0.1% by weight/volume, e.g., from 0.05% to 0.09%. The type/concentration of preservative in each agent may be the same or different.

In some embodiments, the kit further comprises a calibrator and/or a quality control comprising a known concentration of SARS-CoV-2 antibody.

In some embodiments, the SARS-CoV-2 antibody in the calibrator or quality control is a monoclonal or polyclonal antibody and is derived from any of human, rabbit, sheep, avian, equine, bovine, monkey, camel, murine, recombinant antibody, the concentration of the SARS-CoV-2 antibody is any one or a combination selected from the group consisting of 0, 15, 50, 100, 150, 200, 250BAU/ml.

In some embodiments, the calibrator or quality control comprises at least two concentrations of SARS-CoV-2RBD antibody.

In some specific embodiments, the first agent comprises:

50mM pH7.0 HEPES buffer,

5% Weight/volume sodium chloride,

0.45% W/v polyvinylpyrrolidone,

0.5% Weight/volume BSA,

BrijL23 at 0.5% w/v,

0.09% W/v sodium azide.

In some specific embodiments, the second agent comprises:

0.125% w/v total of the first and second nanomicrospheres,

8% Sucrose by weight/volume,

0.09% Sodium azide by weight/volume,

50MM glycine buffer pH 7.5.

Method for preparing microsphere

The present disclosure provides a method for preparing a nanoparticle coated with SARS-CoV-2RBD antigen, comprising the steps of:

A first step comprising:

1.1 Activating the nano-microsphere to obtain an activated nano-microsphere;

1.2 Coupling the first RBD antigen to the activated nanoparticle to obtain a nanoparticle coated with the first RBD antigen;

1.3 Closing the nano-microsphere obtained in the step 1.2) to obtain a first nano-microsphere;

A second step, comprising:

2.1 Activating the nano-microsphere to obtain an activated nano-microsphere;

2.2 Coupling a second RBD antigen to the activated nanoparticle to obtain a nanoparticle coated with the second RBD antigen;

2.3 Closing the nano-microsphere obtained in the step 2.2) to obtain a second nano-microsphere;

And the third step is to mix the first nanometer microsphere and the second nanometer microsphere.

In some embodiments, the order of the first step and the second step is parallel, or interchangeable.

In some embodiments, the first RBD antigen comprises RBD or an epitope thereof selected from any one of SARS-CoV-2 subtype Alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron, and the second RBD antigen comprises RBD or an epitope thereof selected from any one of SARS-CoV-2 subtype wild-type, alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron;

The first RBD antigen and the second RBD antigen are different.

In some embodiments, the first and second nanomicrospheres are mixed in a mass ratio of 1:2 to 2:1, preferably 1:1.

In some embodiments, the first and second nanospheres are independently polymerized from one or more selected from the group consisting of polystyrene, acrylic acid, acrylate, and preferably the nanospheres are polystyrene latex microspheres.

In some embodiments, the average particle size of the first and second nanoparticles is independently from 200nm to 450nm, preferably from 300nm to 350nm.

In some embodiments, the first nanoparticle and the second nanoparticle are the same or different.

In some embodiments, the nanoparticle is a carboxyl-modified nanoparticle.

In some embodiments, the polystyrene nanoparticle is provided in a buffer. As one example, the buffer is Hepes buffer at pH 7 to 8, at a concentration of 0.02M to 0.5M, e.g., 0.05M to 0.1M, e.g., 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35M.

In some embodiments, 0.1% to 2.0% polystyrene nanoparticle by weight/volume is provided in buffer, e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, preferably 0.5% to 1.5%, more preferably 0.75% to 1.25%.

In some embodiments, SARS-CoV-2 protogenic RBD and mutant RBD are separately cross-linked to polystyrene nanomicrospheres. The concentration of SARS-CoV-2RBD in the nanoparticle buffer is 0.05 to 0.5mg/ml, such as 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.13, 0.14, 0.15, 0.2, 0.25, 0.30, 0.35mg/ml, preferably 0.05 to 0.2mg/ml.

In some embodiments, the SARS-CoV-2RBD and polystyrene nanobeads are cross-linked at 28 to 40℃for 1 to 4 hours, preferably at 37℃for 2 to 3.5 hours.

Optionally, the crosslinking reaction is terminated, preferably by 0.1M glycine buffer pH8.4, preferably for 4 to 16 hours.

Optionally, after terminating the reaction step, the nanomicrospheres are rinsed. Preferably, the nanoparticle is washed with 50mM glycine buffer pH7.5 to obtain RBD coated polystyrene nanoparticle.

There is provided a method for preparing RBD-coated polystyrene nanoparticle, comprising the steps of:

1. providing a first microsphere:

1) Providing 0.5% to 1.5% by weight/volume of the nanoparticle in 0.02M to 0.1M HEPES buffer at pH 6.5 to 8.0;

2) Contacting the nanomicrospheres with 0.5% to 5% carbodiimide by weight/volume at 28 to 40 ℃ (preferably 37 ℃) for 20 to 30 minutes;

3) Adding 0.05mg/ml to 0.2mg/ml of a first RBD to the solution obtained in the step 2);

4) At 28 ℃ to 40 ℃ (preferably 37 ℃) the first RBD and the nanomicrospheres are contacted for 1 to 4 hours to effect crosslinking;

5) Optionally, terminating the crosslinking reaction;

6) Optionally, rinsing the particles obtained after crosslinking;

7) Obtaining a first RBD coated nanoparticle;

2. providing a second microsphere, preparing a second RBD coated nanoparticle according to the same steps 1) to 7);

3. the first microsphere is mixed with the second microsphere in a 1:1 ratio.

The present disclosure also provides the SARS-CoV-2RBD antigen coated nanoparticle obtained by this method.

Use of the same

The disclosure also provides the use of the foregoing nanoparticle in the preparation of a detection reagent. The nano microsphere is used for preparing a latex enhanced immunoturbidimetry kit for measuring the content of SARS-CoV-2 antibody.

Without being limited to a particular theory, the present disclosure provides an optimized crosslinking method that reduces the false negative rate by mixing the nanoparticle coated with one RBD of SARS-CoV-2 with the nanoparticle 1:1 coated with the other RBD of SARS-CoV-2, and thus improves the sensitivity and specificity of the prepared reagent by utilizing the complementarity of the mutant antigen and the native antigen.

The ordinal terms "first," "second," and the like as used herein are not intended to be limited to a particular order or hierarchy and are used solely to distinguish between different features, steps, products, and active ingredients.

Drawings

FIG. 1 comparison of scaled absorbance for the disclosed kit and control kit.

Detailed Description

Examples

EXAMPLE 1 preparation of SARS-CoV-2 antibody detection kit

1. Preparing a first reagent comprising:

50mM HEPES buffer pH7.0,

5% Weight/volume sodium chloride,

0.45% Weight/volume polyvinylpyrrolidone (e.g., K90),

0.5% Weight/volume BSA,

BrijL23 at 0.5% w/v,

0.09% W/v sodium azide.

2. Preparation of the second reagent

2.1 First microspheres

Polystyrene nanoparticle solution (concentration 10% by weight/volume) with 325nm diameter is 0.1mL, 1.2mL of 0.05M HEPES buffer (pH 8.0) is added, and then 0.18mg/mL of cross-linking agent EDAC is added for activation at 37 ℃ for 30 minutes;

crosslinking 0.12mg SARS-CoV-2 protogenic RBD antigen onto the polystyrene nano-microsphere, reacting for 2 hours at 37 ℃ to make the antigen concentration be 0.16mg/ml;

Terminating the reaction by adding 0.6ml of 0.1M glycine buffer solution with pH of 8.4, shaking overnight at 37 ℃, and centrifuging to remove the supernatant;

The nanoparticle coated with SARS-CoV-2 protogenic RBD antigen was harvested by washing 3 times with 6ml of 50mM glycine buffer pH 7.5.

2.2 Second microspheres

The nanoparticle coated with SARS-CoV-2Omicron mutant RBD antigen was harvested by the same method.

2.3 The first and second microspheres were dispersed as milky suspensions in 8ml of 50mM glycine buffer pH7.5 containing 8% sucrose by weight/volume, 0.09% sodium azide by weight/volume, respectively, and mixed in equal volumes to prepare a second reagent. The total concentration of the first microspheres and the second microspheres in the second reagent was 0.125% by weight/volume.

3. Preparation of a calibration Material

Mixing SARS-CoV-2 protogenic RBD antibody and Omicron mutant antibody at a ratio of 1:1, adding into matrix (such as commercially available normal human serum) at concentrations of 0, 15, 50, 100, 150, 250BAU/ml (binding antibody unit BAU), and stirring to obtain multi-point calibrator.

EXAMPLE 2 control preparation method 1

1. Preparation of the first reagent

The first reagent was prepared in the same manner as in example 1.

2. Preparation of the second reagent

The only difference compared to example 1 is that only the native RBD antigen is coated.

3. Preparation of a calibration Material

The calibrator was prepared as in example 1.

EXAMPLE 3 control preparation method 2

1. Preparation of the first reagent

The first reagent was prepared in the same manner as in example 1.

2. Preparation of the second reagent

The only difference compared to example 1 is that only mutant RBD antigens are coated.

3. Preparation of a calibration Material

The calibrator was prepared as in example 1.

EXAMPLE 4 control preparation method 3

1. Preparation of the first reagent

The first reagent was prepared in the same manner as in example 1.

2. Preparation of the second reagent

Mixing 0.06mg of SARS-CoV-2 protogenic RBD antigen with 0.06mg of SARS-CoV-2Omicron mutant RBD antigen to obtain mixed antigen, reacting at 37deg.C for 2 hr to crosslink the mixed antigen to the polystyrene nanometer microsphere;

washing 3 times with 6ml 50mM glycine buffer solution of pH7.5, and harvesting the nano-microsphere coated with SARS-CoV-2RBD mixed antigen;

dispersing the antigen-coated nanoparticle into milky suspension with 8ml of 50mM glycine buffer (pH 7.5) containing 8% sucrose by weight/volume and 0.09% sodium azide by weight/volume to prepare a second reagent;

The final concentration of the nanomicrospheres in the second reagent was 0.125% by weight/volume.

3. Preparation of a calibration Material

The calibrator was prepared as in example 1.

Test example 1 determination procedure for SARS-CoV-2 antibody detection kit

Taking AU5800 biochemical analyzer as an example, measuring wavelength of 570nm, adding 144 μl of the first reagent, reacting at 37deg.C for 18 seconds, adding 8 μl of calibrator, reacting for 180 seconds, adding 48 μl of the second reagent, measuring absorbance values (A1 and A2) at 18 and 180 seconds, and calculating absorbance difference DeltaA=A2-A1 (Table 1). And repeatedly measuring for 2 times in each tube, taking the absorbance difference delta A measured for 2 times in each calibrator tube as an ordinate, and the corresponding concentration as an abscissa, preparing a concentration-absorbance difference calibration curve, and taking a sample to be measured. The absorbance difference of the sample is measured by the same method and is substituted into a calibration curve, and the content of SARS-CoV-2 antibody in the sample to be measured can be calculated according to the figure 1.

TABLE 1

Test example 2 sensitivity and specificity of the kits of the present disclosure versus control kits

38 Cases of SARS-CoV-2 antibody positive samples and 12 cases of SARS-CoV-2 antibody negative samples in a certain hospital are respectively detected by using the kit and the control kit.

Examples of 50 cases include vaccinators and SARS-CoV-2 infected individuals. The results of the measurements are shown in tables 2 to 3. As can be seen from the results of the following table, the sensitivity and specificity of the kit of the present disclosure are higher than those of the kit of the control preparation method. The kit prepared by the control method has higher false positive and false negative rates.

TABLE 2 clinical sample results with the disclosed kits and control kits

TABLE 3 sensitivity to specificity of the kits of the present disclosure versus control kits

Project	The disclosed kit	Control preparation method 1	Control preparation method 2	Control preparation method 3
					Sensitivity of	97.4%	84.2%	60.5%	92.1%
Specificity of the sample	100.0%	75.0%	66.7%	91.7%

Test example 3 comparison of the scaled absorbance of the kits of the disclosure with control kits

Absorbance was detected by simultaneous calibration with the kits of the present disclosure and control kits. As can be seen from the data below, the kits of the present disclosure scale absorbance higher than the control kits 1to 3.

TABLE 4 comparison of scaled absorbance for the presently disclosed kits and control kits

Test example 4 comparative reproducibility of the presently disclosed kit control kit

Serum sample reproducibility was detected simultaneously with the control kit using the kits of the present disclosure. Sample reproducibility variation coefficient less than 5% is used as a qualification standard.

As can be seen from the following data, the coefficient of variation of the kit of the present disclosure is 3.6%, less than 8.40% of the coefficient of variation of control kit 1, less than 18.8% of the coefficient of variation of control kit 2, and less than 13.3% of the coefficient of variation of control kit 3.

TABLE 5 reproducibility comparison of the kits of the present disclosure with control kits

Comparative example 1

Kits were prepared according to the methods of examples 1 to 4, except that the native and/or omacron mutant RBD antigen was replaced with other mutant corresponding antigen (the first and second microspheres may be of different subtypes), and test comparisons were performed using the methods of test examples 1 to 4. Similar results were obtained, and the preparation strategy of mixing the first and second microspheres was superior to other preparation strategies ("single antigen" strategy, and "pre-antigen mix-post-coat" strategy) in terms of improving false negative/positive, improving reproducibility (data not shown).

Comparative example 2

PVP was optimal for the sensitivity and reproducibility of the kit, PEG times (PEG 8000> PEG6000> PEG 20000) (data not shown).

Comparative example 3

With reference to the preparation method of example 1, the effect of the SARS-CoV-2RBD antigen coated on latex microspheres at various concentrations (0.05 mg/ml, 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml; this concentration refers to the concentration in the latex buffer prior to addition of the blocking solution) on the second reagent was compared.

The thermal stability of the prepared kit was optimal at SARS-CoV-2RBD antigen concentrations ranging from 0.1mg/ml to 0.2mg/ml in latex buffer (data not shown).

Comparative example 4

The inventors compared the effect of sucrose, trehalose, mannitol, glucose at different concentrations (1%, 5%, 8%, 10%) on the second reagent with reference to the preparation method of example 1.

Sucrose and trehalose were observed to have a better viscosity protecting effect than equal concentrations of mannitol and glucose (data not shown). Without being limited to a particular theory, it may be explained that sucrose and trehalose have higher glass transition temperatures.

Theoretically, trehalose has a higher glass transition temperature than sucrose, and should provide a better stabilizing effect. However, it was unexpectedly noted that sucrose, at a specific concentration of 8% in combination with 50mM glycine buffer pH7.5, gave a compromise between the stability of the second reagent (absorbance change over time) and the batch-to-batch reproducibility of the kit (data not shown).

Claims

1. A SARS-CoV-2 antibody detection kit comprising:

A first reagent comprising:

0.1 to 10% by weight/volume of a stabilizer,

0.1 To 10% by weight/volume of a polymerization promoter, and

10MM to 500mM buffer;

A second reagent comprising:

0.025% to 0.25% by weight/volume of first nanomicrospheres,

From 0.025% to 0.25% by weight/volume of second nanomicrospheres,

0.1 To 10% by weight/volume of a stabilizer,

10MM to 500mM buffer;

wherein:

The first nano microsphere is coated with SARS-CoV-2 first RBD antigen;

the second nano microsphere is coated with SARS-CoV-2 second RBD antigen;

The first RBD antigen is the RBD of wild type SARS-CoV-2;

The second RBD antigen is selected from the group consisting of RBD of any of SARS-CoV-2 subtype Alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron;

The first RBD antigen and the second RBD antigen are different;

The stabilizer is independently selected from any one of fatty alcohol polyoxyethylene ether, BSA, tween 20, brijL23, sucrose, or a combination thereof;

the polymerization promoter is selected from any one of PEG6000, PEG8000, PEG20000, PVP, dextran or a combination thereof;

The buffers in the first and second reagents are independently selected from any one of a phosphate buffer, glycine buffer, HEPES buffer, MES buffer, boric acid buffer, acetate buffer, ammonium chloride buffer, or a combination thereof;

the pH of the buffer in the first and second reagents is independently 6 to 8;

The buffers in the first reagent and the second reagent are the same or different.

2. The SARS-CoV-2 antibody detection kit according to claim 1, further comprising:

a calibrator and/or a quality control material,

The calibrator comprises SARS-CoV-2 antibody at a known concentration and a buffer;

the quality control product comprises SARS-CoV-2 antibody with known concentration and buffer solution;

the buffer is selected from any one of phosphate buffer, glycine buffer, HEPES buffer, MES buffer, boric acid buffer, acetate buffer, ammonium chloride buffer, or a combination thereof;

The SARS-CoV-2 antibody in the calibrator or quality control is a monoclonal or polyclonal antibody and is derived from any of human, rabbit, sheep, fowl, horse, cow, monkey, camel, mouse, recombinant antibody.

3. The SARS-CoV-2 antibody detection kit according to claim 2, wherein:

The concentration of SARS-CoV-2 antibody is any one or combination of 0, 15, 50, 100, 150, 250 BAU/ml.

4. The SARS-CoV-2 antibody detection kit according to claim 1, wherein the pH of the buffer in the first reagent and the buffer in the second reagent are independently 7.0 to 7.5.

5. The SARS-CoV-2 antibody detection kit of claim 1, wherein the first and second reagents further comprise any one or a combination of the following selected from the group consisting of:

0.1 to 10% NaCl by weight/volume,

0.05% To 0.2% preservative by mass/volume.

6. The SARS-CoV-2 antibody detection kit according to claim 1, wherein:

The nanometer microsphere is polymerized by one or more selected from polystyrene, acrylic acid and acrylic ester;

the average particle diameter of the nano microsphere is 200nm to 450nm;

the first nanoparticle and the second nanoparticle are the same or different.

7. The SARS-CoV-2 antibody detection kit according to claim 6, wherein:

The nano-microsphere is a polystyrene latex microsphere.

8. The SARS-CoV-2 antibody detection kit according to claim 6, wherein:

the average particle size of the nano microsphere is 300 nm to 350nm.

9. The SARS-CoV-2 antibody detection kit according to claim 1, wherein:

A first reagent comprising:

40 mM to 50 mM pH 7.0 HEPES buffer,

0.4 To 0.5% by weight/volume of polyvinylpyrrolidone,

0.4 To 0.5% weight/volume BSA,

From 0.4% to 0.5% weight/volume BrijL23,

4% To 5% weight/volume sodium chloride,

0.05% To 0.1% w/v sodium azide;

A second reagent comprising:

0.10 to 0.15% w/v total of the first and second nanomicrospheres,

8% Sucrose by weight/volume,

0.05 To 0.1% by weight/volume of sodium azide,

40MM to 50mM glycine buffer pH 7.5.

10. A method for preparing a SARS-CoV-2 antibody detection kit as in claim 1, wherein the preparation of said nanoparticle comprises the steps of:

A first step comprising:

1.1 Activating the nano-microsphere to obtain an activated nano-microsphere;

A second step, comprising:

2.1 Activating the nano-microsphere to obtain an activated nano-microsphere;

the third step is to mix the first nano-microsphere and the second nano-microsphere,

Wherein:

the order of the first step and the second step is parallel or interchangeable;

The first RBD antigen is the RBD of wild type SARS-CoV-2;

the second RBD antigen is an RBD selected from any of SARS-CoV-2 subtype Alpha, beta, gamma, delta, delta Plus, epsilon, lambda, mu and Omicron;

The first RBD antigen and the second RBD antigen are different;

The first nano-microsphere and the second nano-microsphere are mixed according to the mass ratio of 1:2 to 2:1.

11. The method of claim 10, wherein the first and second nanomicrospheres are mixed in a mass ratio of 1:1.

12. The method according to claim 10, wherein:

The first nano-microsphere and the second nano-microsphere are independently polymerized by one or more selected from polystyrene, acrylic acid and acrylic ester;

the average particle size of the first and second nanoparticles is independently 200nm to 450nm;

the first and second nanomicrospheres are the same or different.

13. The method according to claim 12, wherein:

the first and second nanospheres are polystyrene latex microspheres.

14. The method according to claim 12, wherein:

the average particle size of the first and second nanoparticles is independently 300 nm to 350nm.

15. The method according to claim 12, wherein:

The first and second nanospheres are carboxyl-modified nanospheres.