Detection reagent for novel coronavirus antibodies and preparation method thereof

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

Technical Field

[0001] The present invention belongs to the field of in vitro diagnostic medical immunology and relates to a kit for determining the content of SARS-CoV-2 antibodies in human samples by latex-enhanced immunoturbidimetry. Background Art

[0002] The new coronavirus (SARS-CoV-2) is the pathogen of the new coronavirus pneumonia (COVID-19) and is an enveloped β-type single-stranded RNA coronavirus.

[0003] The incubation period of SARS-CoV-2 is usually 2 to 14 days. The virus has a high risk of human-to-human transmission, mainly through respiratory secretions and aerosols. Therefore, timely diagnosis of carriers or infected persons is crucial for epidemic control and disease treatment. There are at least 27 proteins in SARS-CoV-2, including 15 non-structural proteins (nsp1 to nsp10, nsp12 to nsp16), 4 structural proteins (S, E, M and N) and 8 auxiliary proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14). The 4 structural proteins are: spike protein (S), envelope (E), membrane (M) and nucleocapsid (N).

[0004] The S protein is a transmembrane protein that forms a unique trimer structure on the surface of the coronavirus. Each S monomer consists of an N-terminal S1 subunit and a membrane-proximal S2 subunit. The S1 subunit contains a receptor binding domain (RBD), which can bind to the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell. RBD can induce the body to produce neutralizing antibodies and is an ideal antigen for diagnosis and vaccine research.

[0005] Serological testing plays an important role in understanding viral infections and vaccine research. At present, the main methods for detecting serum SARS-CoV-2 specific antibodies are colloidal gold method, chemiluminescence method, etc. The colloidal gold method is simple to operate, does not require special equipment, completes the test within 15-30 minutes, has a relatively low cost, and does not require high hardware. It can be used in grassroots laboratories, but has low sensitivity and can only be used for qualitative detection, and has low throughput. The chemiluminescence method has high sensitivity and specificity, can perform quantitative detection, and can be operated in batches, but requires special instruments and is suitable for large and medium-sized hospitals. The latex-enhanced immunoturbidimetry method is simple to operate, rapid, and has high throughput. It can quantitatively determine SARS-CoV-2 antibodies in human serum or plasma. Quantification of antibody response helps to determine specific antibody concentrations and can monitor the dynamic antibody levels of individuals longitudinally.

[0006] CN113252911B discloses a detection kit for SARS-CoV-2 neutralizing antibodies, characterized in that the detection kit is used to simultaneously detect neutralizing antibodies bound to RBD protein and neutralizing antibodies bound to NTD protein; the detection kit comprises component A and component B; component A comprises RBD protein coated on magnetic particles and NTD protein coated on magnetic particles; component B comprises ACE2 protein labeled with acridinium ester and AXL protein labeled with acridinium ester; the detection kit also comprises ACE2 antibody and AXL antibody.

[0007] CN113341150A relates to a SARS-CoV-2 detection kit; the kit comprises a) an RBD-specific ligand coupled to a solid support, b) an RBD recombinant protein coupled to a marker of an acridine fluorophore, and: a first buffer for dissolving component a, pH=5.5-6.5; a second buffer for dissolving component b, pH=5.5-8.5; and a third buffer for lysing a sample to release the RBD antigen in the virus, pH=3.0-4.5.

[0008] CN113495141A provides a SARS-CoV-2 IgM antibody detection kit, comprising two antigens labeled with acridinium ester and mouse anti-human IgM antibodies coated on a solid phase carrier; wherein the two antigens are SARS-CoV-2 RBD antigen and SARS-CoV-2 N antigen.

[0009] CN111537743A relates to a SARS-CoV-2 novel coronavirus antibody detection kit, comprising a solid carrier, and a specific polypeptide combination connected to the solid carrier, which also includes the RBD domain of the S protein of SARS-CoV-2SARS-CoV-2 connected to the solid carrier.

[0010] CN113607957A discloses a specific neutralizing antibody competition ELISA kit for the SARS-CoV-2 RBD domain, which contains a solid phase carrier coated with human ACE2 protein and a biotin-labeled RBD protein.

[0011] CN113671184A provides a detection kit and method for detecting SARS-CoV-2 neutralizing antibodies, which uses protein tags to modify RBD and ACE2 so that they have better stability and reactivity. Summary of the invention

[0012] With the emergence of more virus variants or subtypes (such as Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu and Omicron, etc.), a single native / wild-type RBD cannot meet the needs of clinical testing. In view of this, the present disclosure provides a technical solution for RBD combination.

[0013] Microspheres

[0014] The present disclosure provides a nano-microsphere, on the surface of which an antigen is coated. The nano-microsphere and the antigen are covalently or non-covalently bound, preferably covalently bound.

[0015] In some embodiments, the antigen is an RBD antigen selected from: the RBD of SARS-CoV-2, a polypeptide comprising the SARS-CoV-2 RBD, or a polypeptide comprising an epitope of the SARS-CoV-2 RBD.

[0016] In the present disclosure, "RBD antigen" should be understood broadly, and can be a full-length RBD, a fragment or an epitope thereof, as long as it can be recognized and bound by the antibodies of SARS-CoV-2 in the sample to be tested. Therefore, the RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: wild type (also referred to herein as native type), Alpha, Beta, Gamma, Delta, DeltaPlus, Epsilon, Lambda, Mu and Omicron. When more subtypes appear in the future, the present disclosure intends to cover these subtypes.

[0017] In some embodiments, SARS-CoV-2 RBD is expressed by host cells and obtained by chromatography purification. The above antigens can be prepared by genetic recombination techniques recognized in the art, or commercial products can be purchased.

[0018] In some specific embodiments, the SARS-CoV-2 antigen is the S protein, and the RBD of the S protein is preferably used as the antigen.

[0019] In some embodiments, the nanosphere is a core-shell structure. As an example, the core of the nanosphere is a polystyrene polymer, and the shell is composed of styrene, n-butyl acrylate, and methacrylic acid copolymer. In some embodiments, the nanosphere is a polystyrene nanosphere particle.

[0020] In some embodiments, the surface of the nanospheres carries chemical groups selected from the group consisting of sulfate, sulfonic acid, carboxyl, amino, hydroxyl, hydrazide, chloromethyl, and combinations thereof.

[0021] In some embodiments, the average particle size of the nanospheres ranges from 200 to 450 nm (including endpoints); including but not limited to 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450 nm, and the range between any two of the above values. In some specific embodiments, the particle size of the nanospheres is 325 nm.

[0022] It should be emphasized here that "325nm" in this context does not mean that the particle size of each nanosphere in the reagent is exactly 325nm. In fact, 325nm is a statistically significant value; since there are errors in the manufacturing process of nanospheres, the 325nm specification of nanospheres refers to the particle size range of about 325nm, such as 300 to 350nm, and another example is 310 to 340nm.

[0023] In the present disclosure, the nanospheres are not homogeneous, and include a mixture of two nanospheres, each coated with a different RBD antigen.

[0024] In some embodiments, the nanosphere comprises a first nanosphere and a second nanosphere. The first nanosphere is coated with a SARS-CoV-2 first RBD antigen; the second nanosphere is coated with a SARS-CoV-2 second RBD antigen. The first RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron; the second RBD antigen comprises an RBD or an epitope thereof selected from any of the following 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.

[0025] In some embodiments, the RBD antigen can be bound to the surface of the nanosphere by physical adsorption or chemical coupling. As an example, a single native RBD antigen, or a single mutant RBD antigen, or a combination of the two is bound to the surface of the nanosphere by a chemical coupling cross-linking method. In some specific embodiments, the native RBD antigen and the mutant RBD antigen can also be cross-linked to the surface of the nanosphere separately, and then mixed in a specific ratio to achieve a better use effect.

[0026] Reagents / Kits

[0027] In some embodiments, a SARS-CoV-2 antibody detection kit is provided, comprising:

[0028] A first reagent comprising a stabilizer, an aggregation promoter, and a buffer;

[0029] A second reagent comprising:

[0030] According to the aforementioned first nanosphere of the present disclosure,

[0031] According to the aforementioned second nanosphere of the present disclosure,

[0032] Optionally, a stabilizer, and

[0033] Buffer.

[0034] In some embodiments, the first nanosphere is coated with the first RBD antigen of SARS-CoV-2; and the second nanosphere is coated with the second RBD antigen of SARS-CoV-2.

[0035] In some embodiments, the first RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron; the second RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: wild type, Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron.

[0036] In some embodiments, the first RBD antigen and the second RBD antigen are different.

[0037] In some embodiments, the stabilizer is selected from any one of the following: fatty alcohol polyoxyethylene ether, BSA, Tween 20, Brij L23, sucrose, or a combination thereof.

[0038] In some embodiments, as an example, the stabilizer is selected from: bovine serum albumin in a range of 0.1% to 5% 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.

[0039] In some embodiments, the aggregation promoter is selected from any one of the following: PEG6000, PEG8000, PEG20000, PVP, dextran or a combination thereof.

[0040] In some embodiments, the coagulant is selected from PEG4000, PEG6000, PEG8000, PEG10000, PEG20000, dextran T-40, dextran T-70, dextran T-500, PVP and combinations thereof. The coagulant concentration range is 0.1% to 10% by weight / volume; for example, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and the range between any two of the above values.

[0041] In some embodiments, the buffers in the first reagent and the second reagent are independently selected from any one of the following: phosphate buffer, glycine buffer, HEPES buffer, MES buffer, borate buffer, acetate buffer, ammonium chloride buffer, or a combination thereof.

[0042] In some embodiments, the concentration of the 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 the range between any two of the above values.

[0043] In some embodiments, the pH of the buffer in the first reagent and the second reagent is independently 6 to 8, preferably 7.0 to 7.5; for example 7.2, 7.3, 7.4, 7.5.

[0044] In some embodiments, the buffer in the first reagent and the buffer in the second reagent are the same or different.

[0045] In some embodiments, the concentration of the nanospheres is 0.025% to 0.25% (by weight / volume). For example, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25% and ranges between any two of the above values. In some specific embodiments, the concentration of the RBD-coated polystyrene nanospheres is 0.125% by weight / volume (total of the two microspheres).

[0046] In some embodiments, the first reagent further comprises any one or a combination selected from the following: 1% to 10% NaCl by mass / volume, 0.05% to 0.2% preservative by mass / volume.

[0047] In some embodiments, the preservative is selected from the group consisting of sodium azide, thimerosal, phenol, ethylmercuric sodium thiosulfate, and combinations thereof. In some embodiments, the preservative concentration is 0.02% to 0.1% by weight / volume; e.g., 0.05% to 0.09%. The preservative type / concentration in each reagent may be the same or different.

[0048] In some embodiments, the kit further comprises: a calibrator and / or a quality control material, wherein the calibrator or the quality control material comprises a known concentration of a SARS-CoV-2 antibody.

[0049] In some embodiments, the SARS-CoV-2 antibody in the calibrator or control is a monoclonal antibody or a polyclonal antibody and is derived from any of the following: human, rabbit, sheep, poultry, horse, cattle, monkey, camel, mouse, or recombinant antibody; the concentration of the SARS-CoV-2 antibody is selected from any one of the following or a combination thereof: 0, 15, 50, 100, 150, 200, 250 BAU / ml.

[0050] In some embodiments, the calibrator or control comprises at least two concentrations of SARS-CoV-2 RBD antibodies.

[0051] In some specific embodiments, the first reagent comprises:

[0052] 50mM pH7.0 HEPES buffer,

[0053] 5% weight / volume sodium chloride,

[0054] 0.45% weight / volume polyvinyl pyrrolidone,

[0055] 0.5% weight / volume BSA,

[0056] 0.5% weight / volume BrijL23,

[0057] 0.09% w / v sodium azide.

[0058] In some specific embodiments, the second reagent comprises:

[0059] A total of 0.125% w / v of the first nanospheres and the second nanospheres,

[0060] 8% sucrose by weight / volume,

[0061] 0.09% sodium azide by weight / volume,

[0062] 50 mM pH 7.5 glycine buffer.

[0063] Preparation method of microspheres

[0064] The present disclosure provides a method for preparing nanoparticles coated with SARS-CoV-2 RBD antigen, comprising the steps of:

[0065] The first step comprises:

[0066] 1.1) activating the nano-microspheres to obtain activated nano-microspheres;

[0067] 1.2) coupling the first RBD antigen to the activated nanoparticles to obtain nanoparticles coated with the first RBD antigen;

[0068] 1.3) sealing the nanospheres obtained in step 1.2) to obtain first nanospheres;

[0069] The second step comprises:

[0070] 2.1) activating the nano-microspheres to obtain activated nano-microspheres;

[0071] 2.2) coupling the second RBD antigen to the activated nanoparticles to obtain nanoparticles coated with the second RBD antigen;

[0072] 2.3) sealing the nanospheres obtained in step 2.2) to obtain second nanospheres;

[0073] The third step: mixing the first nanospheres and the second nanospheres.

[0074] In some embodiments, the order of the first step and the second step is parallel or interchangeable.

[0075] In some embodiments, the first RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron; the second RBD antigen comprises an RBD or an epitope thereof selected from any of the following SARS-CoV-2 subtypes: wild type, Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron;

[0076] The first RBD antigen and the second RBD antigen are different.

[0077] In some embodiments, the first nanospheres and the second nanospheres are mixed at a mass ratio of 1:2 to 2:1, preferably 1:1.

[0078] In some embodiments, the first nanosphere and the second nanosphere are independently polymerized from one or more selected from the group consisting of polystyrene, acrylic acid, and acrylate; preferably, the nanosphere is a polystyrene latex microsphere.

[0079] In some embodiments, the average particle size of the first nanosphere and the second nanosphere is independently 200 nm to 450 nm, preferably 300 nm to 350 nm.

[0080] In some embodiments, the first nanosphere and the second nanosphere are the same or different.

[0081] In some embodiments, the nanospheres are carboxyl-modified nanospheres.

[0082] In some embodiments, the polystyrene nanospheres are provided in a buffer. As an example, the buffer is a Hepes buffer at pH 7 to 8; a concentration of 0.02M to 0.5M, such as 0.05M to 0.1M; such as 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 buffer.

[0083] In some embodiments, 0.1% to 2.0% polystyrene nanospheres are provided in the buffer by weight / volume; for example, 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%.

[0084] In some embodiments, the SARS-CoV-2 native RBD and mutant RBD are cross-linked to polystyrene nanospheres, respectively. The SARS-CoV-2 RBD has a concentration of 0.05 to 0.5 mg / ml in the nanosphere buffer, 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, 0.35 mg / ml, preferably 0.05 to 0.2 mg / ml.

[0085] In some embodiments, the SARS-CoV-2 RBD and polystyrene nanospheres are cross-linked at 28 to 40° C. for 1 to 4 hours, preferably at 37° C. for 2 to 3.5 hours.

[0086] Optionally, the cross-linking reaction is terminated; preferably, the cross-linking reaction is terminated by 0.1 M glycine buffer at pH 8.4, and the reaction is preferably carried out for 4 to 16 hours.

[0087] Optionally, after the step of terminating the reaction, the nanospheres are washed. Preferably, the nanospheres are washed with 50 mM glycine buffer at pH 7.5 to obtain RBD-coated polystyrene nanospheres.

[0088] A method for preparing RBD-coated polystyrene nanospheres is provided, comprising the steps of:

[0089] 1. Provide the first microsphere:

[0090] 1) providing 0.5% to 1.5% by weight / volume of nanospheres in 0.02M to 0.1M HEPES buffer at pH 6.5 to 8.0;

[0091] 2) contacting the nanospheres with 0.5% to 5% carbodiimide by weight / volume at 28 to 40° C. (preferably 37° C.) for 20 to 30 minutes;

[0092] 3) adding 0.05 mg / ml to 0.2 mg / ml of the first RBD to the solution obtained in step 2);

[0093] 4) contacting the first RBD and the nanospheres at 28° C. to 40° C. (preferably 37° C.) for 1 to 4 hours to perform cross-linking;

[0094] 5) optionally, terminating the cross-linking reaction;

[0095] 6) optionally, washing the cross-linked particles;

[0096] 7) obtaining a first RBD-coated nanoparticle;

[0097] 2. Providing a second microsphere: preparing a second RBD-coated nanosphere according to the same steps 1) to 7);

[0098] 3. Mix the first microspheres with the second microspheres in a 1:1 ratio.

[0099] The present disclosure also provides nanoparticles coated with SARS-CoV-2 RBD antigen obtained by this method.

[0100] use

[0101] The present disclosure also provides the use of the aforementioned nanoparticles in preparing a detection reagent. The nanoparticles according to the present disclosure are used to prepare a latex enhanced immunoturbidimetric assay kit for determining the content of SARS-CoV-2 antibodies.

[0102] Without being limited to a specific theory, the present disclosure provides an optimized cross-linking method by mixing nanoparticles coated with one RBD of SARS-CoV-2 with nanoparticles coated with another RBD of SARS-CoV-2 in a 1:1 ratio, and utilizing the complementarity between the mutant antigen and the native antigen to reduce the false negative rate, thereby improving the sensitivity and specificity of the prepared reagent.

[0103] The ordinal numbers "first", "second", etc. used in this application are not intended to limit a specific order or level, but are only used to distinguish different features, steps, products, and active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 : Comparison of the calibrated absorbance of the disclosed kit and the control kit. DETAILED DESCRIPTION

[0105] Example

[0106] Example 1. Preparation of SARS-CoV-2 Antibody Detection Kit

[0107] 1. Prepare a first reagent, which comprises:

[0108] 50mM HEPES buffer, pH 7.0,

[0109] 5% weight / volume sodium chloride,

[0110] 0.45% weight / volume polyvinyl pyrrolidone (such as K90),

[0111] 0.5% weight / volume BSA,

[0112] 0.5% weight / volume BrijL23,

[0113] 0.09% w / v sodium azide.

[0114] 2. Preparation of the Second Reagent

[0115] 2.1 The first microsphere

[0116] 0.1 mL of a polystyrene nanosphere solution (concentration 10% by weight / volume) with a diameter of 325 nm was added with 1.2 mL of 0.05 M HEPES buffer (pH 8.0), and then 0.18 mg / ml of the cross-linking agent EDAC was added, and activated at 37°C for 30 minutes;

[0117] 0.12 mg of SARS-CoV-2 native RBD antigen was cross-linked to the polystyrene nanospheres and reacted at 37°C for 2 hours to make the antigen concentration 0.16 mg / ml;

[0118] The reaction was terminated by adding 0.6 ml of 0.1 M glycine buffer (pH 8.4), shaking at 37 °C overnight, and centrifuging to remove the supernatant;

[0119] Wash three times with 6 ml of 50 mM glycine buffer, pH 7.5; harvest the nanoparticles coated with SARS-CoV-2 native RBD antigen.

[0120] 2.2 Second microsphere

[0121] Nanoparticles coated with SARS-CoV-2Omicron mutant RBD antigen were harvested using the same method.

[0122] 2.3 The first microsphere and the second microsphere were dispersed into a milky white suspension using 8 ml of 50 mM pH 7.5 glycine buffer containing 8% sucrose by weight / volume and 0.09% sodium azide by weight / volume, and then mixed in equal volumes to prepare a second reagent. The total concentration of the first microsphere and the second microsphere in the second reagent was 0.125% by weight / volume.

[0123] 3. Preparation of Calibrators

[0124] SARS-CoV-2 native RBD antibody and Omicron mutant antibody were mixed in a 1:1 ratio and added to a matrix (such as commercially available normal human serum) at concentrations of 0, 15, 50, 100, 150, and 250 BAU / ml (binding antibody unit BAU), respectively. The mixture was stirred evenly to prepare a multi-point calibrator.

[0125] Example 2. Comparative preparation method 1

[0126] 1. Preparation of the first reagent

[0127] The preparation method of the first reagent is the same as that of Example 1.

[0128] 2. Preparation of the Second Reagent

[0129] Compared with Example 1, the only difference is that only the native RBD antigen is coated.

[0130] 3. Preparation of Calibrators

[0131] The preparation of the calibrator is the same as in Example 1.

[0132] Example 3. Comparative preparation method 2

[0133] 1. Preparation of the first reagent

[0134] The preparation method of the first reagent is the same as that of Example 1.

[0135] 2. Preparation of the Second Reagent

[0136] Compared with Example 1, the only difference is that only the mutant RBD antigen is coated.

[0137] 3. Preparation of Calibrators

[0138] The preparation of the calibrator is the same as in Example 1.

[0139] Example 4. Comparative preparation method 3

[0140] 1. Preparation of the first reagent

[0141] The preparation method of the first reagent is the same as that of Example 1.

[0142] 2. Preparation of the Second Reagent

[0143] 0.1 mL of a polystyrene nanosphere solution (concentration 10% by weight / volume) with a diameter of 325 nm was added with 1.2 mL of 0.05 M HEPES buffer (pH 8.0), and then 0.18 mg / ml of the cross-linking agent EDAC was added, and activated at 37°C for 30 minutes;

[0144] Mix 0.06 mg of SARS-CoV-2 native RBD antigen and 0.06 mg of SARS-CoV-2Omicron mutant RBD antigen to obtain a mixed antigen; react at 37° C. for 2 hours to cross-link the mixed antigen to the polystyrene nanoparticles;

[0145] The reaction was terminated by adding 0.6 ml of 0.1 M glycine buffer (pH 8.4), shaking at 37 °C overnight, and centrifuging to remove the supernatant;

[0146] Wash three times with 6 ml of 50 mM pH 7.5 glycine buffer; harvest the nanoparticles coated with SARS-CoV-2 RBD mixed antigens;

[0147] Using 8 ml of 50 mM glycine buffer at pH 7.5 containing 8% sucrose by weight / volume and 0.09% sodium azide by weight / volume, the nanoparticles coated with the antigen are dispersed into a milky white suspension to prepare a second reagent;

[0148] The final concentration of the nanospheres in the second reagent was 0.125% by weight / volume.

[0149] 3. Preparation of Calibrators

[0150] The preparation of the calibrator is the same as in Example 1.

[0151] Test Example 1. Assay steps for SARS-CoV-2 antibody detection kit

[0152] Take the AU5800 biochemical analyzer as an example: the measurement wavelength is 570nm, first add 144μl of the first reagent, react at 37℃ for 18 seconds, then add 8μl of the calibration substance, and then react for 180 seconds before adding 48μl of the second reagent. Measure the absorbance values ​​at 18 seconds and 180 seconds of the reaction (A1, A2), and calculate the absorbance difference △A=A2-A1 (Table 1). Repeat the measurement twice for each tube, and use the absorbance difference △A measured twice for each calibration tube as the vertical coordinate, and the corresponding concentration as the horizontal coordinate to make a "concentration-absorbance difference" calibration curve, and take the sample to be tested. Measure the absorbance difference of the sample in the same way, and substitute it into the calibration curve, see Figure 1 The content of SARS-CoV-2 antibodies in the sample to be tested can be calculated.

[0153] Table 1

[0154]

[0155] Test Example 2. Comparison of sensitivity and specificity between the disclosed kit and the control kit

[0156] The kit disclosed herein and the control kit were used to test 38 SARS-CoV-2 antibody-positive samples and 12 SARS-CoV-2 antibody-negative samples in a hospital.

[0157] The 50 samples included: vaccine recipients, SARS-CoV-2 infected persons. The test results are shown in Tables 2 to 3. As can be seen from the results in the following table, the sensitivity and specificity of the disclosed kit are higher than those of the control preparation method kit. The kit prepared by the control method has a higher false positive and false negative rate.

[0158] Table 2. Results of clinical samples tested by the disclosed kit and the control kit

[0159]

[0160] Table 3. Comparison of sensitivity and specificity of the disclosed kit and the control kit

[0161] project The disclosed kit Control preparation method 1 Control preparation method 2 Control preparation method 3 Sensitivity 97.4％ 84.2％ 60.5％ 92.1％ Specificity 100.0％ 75.0％ 66.7％ 91.7％

[0162] Test Example 3. Comparison of the calibrated absorbance of the disclosed kit and the control kit

[0163] The absorbance was detected by calibrating the kit of the present invention and the control kit at the same time. As can be seen from the following data, the absorbance of the calibrated kit of the present invention is higher than that of the control kits 1 to 3.

[0164] Table 4. Comparison of the calibration absorbance of the disclosed kit and the control kit

[0165]

[0166] Test Example 4. Comparison of the Repeatability of the Kit of the Present Disclosure and the Control Kit

[0167] The kit disclosed herein and the control kit are used to simultaneously test the repeatability of serum samples. The sample repeatability coefficient of variation of less than 5% is used as a qualified standard.

[0168] It can be seen from the following data that the coefficient of variation of the disclosed kit is 3.6%, which is smaller than the coefficient of variation of control kit 1 (8.40%), smaller than the coefficient of variation of control kit 2 (18.8%), and smaller than the coefficient of variation of control kit 3 (13.3%).

[0169] Table 5. Comparison of the repeatability of the disclosed kit and the control kit

[0170]

[0171]

[0172] Comparative Example 1

[0173] The kit was prepared by referring to the methods of Examples 1 to 4, except that the native and / or Omicron mutant RBD antigens were replaced with corresponding antigens of other mutants (the subtypes of the first microsphere and the second microsphere were different), and the test comparison was performed using the methods of Test Examples 1 to 4. The results obtained were similar, and the preparation strategy of mixing the first microsphere and the second microsphere was superior to other preparation strategies (the "single antigen" strategy and the "antigen mixing-then coating" strategy) in terms of improving false negative / positive and improving repeatability (data not shown).

[0174] Comparative Example 2

[0175] Regarding the sensitivity and repeatability of the kit: PVP was the best, followed by PEG (PEG8000>PEG6000>PEG20000) (data not shown).

[0176] Comparative Example 3

[0177] Referring to the preparation method of Example 1, the effects of different concentrations (0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml; the concentration refers to the concentration in the latex buffer before the blocking solution is added) of SARS-CoV-2 RBD antigen coated on the latex microspheres on the second reagent were compared.

[0178] When the concentration of SARS-CoV-2RBD antigen in latex buffer was 0.1 mg / ml to 0.2 mg / ml, the thermal stability of the prepared kit was optimal (data not shown).

[0179] Comparative Example 4

[0180] The inventors referred to the preparation method of Example 1 and compared the effects of different concentrations (1%, 5%, 8%, 10%) of sucrose, trehalose, mannitol and glucose on the second reagent.

[0181] Sucrose and trehalose were observed to have a better viscosity protection effect than mannitol and glucose at equal concentrations (data not shown). Without being bound by a particular theory, a possible explanation is that sucrose and trehalose have higher glass transition temperatures.

[0182] Theoretically, trehalose has a higher glass transition temperature than sucrose, and should provide a better stabilization effect. However, it was unexpectedly noted that at a specific concentration of 8% and in combination with 50 mM pH 7.5 glycine buffer, sucrose better balanced the stability of the second reagent (absorbance change over time) and the inter-batch and intra-batch repeatability of the kit than trehalose (data not shown).

Claims

1. A SARS-CoV-2 antibody detection kit comprising: The first reagent comprises: 0.1% to 10% by weight / volume stabilizer, 0.1% to 10% by weight / volume of polymerization promoter, and 10mM to 500mM buffer; A second reagent comprising: 0.025% to 0.25% by weight / volume of the first nanospheres, 0.025% to 0.25% by weight / volume of a second nanosphere, 0.1% to 10% by weight / volume stabilizer, 10mM to 500mM buffer; in: The first nanoparticle is coated with the first RBD antigen of SARS-CoV-2; The second nanoparticle is coated with a second RBD antigen of SARS-CoV-2; The first RBD antigen is the RBD of wild-type SARS-CoV-2; The second RBD antigen is selected from any of the following SARS-CoV-2 subtypes of RBD: 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 the following: fatty alcohol polyoxyethylene ether, BSA, Tween 20, BrijL23, sucrose, or a combination thereof; The polymerization promoter is selected from any one of the following: PEG6000, PEG8000, PEG20000, PVP, dextran or a combination thereof; The buffer in the first reagent and the second reagent are independently selected from any one of the following: phosphate buffer, glycine buffer, HEPES buffer, MES buffer, borate buffer, acetate buffer, ammonium chloride buffer, or a combination thereof; The pH of the buffer in the first reagent and the second reagent are independently 6 to 8; The buffer in the first reagent and the buffer in the second reagent are the same or different.

2. The SARS-CoV-2 antibody detection kit according to claim 1, further comprising: Calibrators and / or controls, The calibrator comprises a known concentration of SARS-CoV-2 antibody and a buffer; The quality control product contains a known concentration of SARS-CoV-2 antibody and a buffer; The buffer is selected from any one of the following: phosphate buffer, glycine buffer, HEPES buffer, MES buffer, borate buffer, acetate buffer, ammonium chloride buffer, or a combination thereof; The SARS-CoV-2 antibody in the calibrator or quality control is a monoclonal antibody or a polyclonal antibody and is derived from any of the following: human, rabbit, sheep, poultry, horse, cattle, monkey, camel, mouse, or recombinant antibody.

3. The SARS-CoV-2 antibody detection kit according to claim 2, wherein: The concentration of the SARS-CoV-2 antibody is selected from any one of the following or a combination thereof: 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 according to claim 1, wherein the first reagent and the second reagent further comprise any one or a combination thereof selected from the following: 0.1% to 10% NaCl by weight / volume, 0.05% to 0.2% by mass / volume preservatives.

6. The SARS-CoV-2 antibody detection kit according to claim 1, wherein: The nano-microspheres are polymerized by one or more selected from the following: polystyrene, acrylic acid, acrylate; The average particle size of the nanospheres is 200nm to 450nm; The first nanosphere and the second nanosphere are the same or different.

7. The SARS-CoV-2 antibody detection kit according to claim 6, wherein: The nano-microspheres are polystyrene latex microspheres.

8. The SARS-CoV-2 antibody detection kit according to claim 6, wherein: The average particle size of the nanospheres is 300 nm to 350 nm.

9. The SARS-CoV-2 antibody detection kit according to claim 1, wherein: The first reagent comprises: 40 mM to 50 mM pH 7.0 HEPES buffer, 0.4% to 0.5% weight / volume polyvinylpyrrolidone, 0.4% to 0.5% weight / volume BSA, 0.4% to 0.5% weight / volume Brij L23, 4% to 5% weight / volume sodium chloride, 0.05% to 0.1% weight / volume sodium azide; A second reagent comprising: A total of 0.10% to 0.15% w / v of the first nanospheres and the second nanospheres, 8% sucrose by weight / volume, 0.05% to 0.1% sodium azide by weight / volume, 40 mM to 50 mM pH 7.5 glycine buffer.

10. A method for preparing the SARS-CoV-2 antibody detection kit according to claim 1, wherein the preparation of the nanoparticles comprises the steps of: The first step comprises: 1.1) Activating the nano-microspheres to obtain activated nano-microspheres; 1.2) coupling the first RBD antigen to the activated nanoparticles to obtain nanoparticles coated with the first RBD antigen; 1.3) sealing the nanospheres obtained in step 1.2) to obtain first nanospheres; The second step comprises: 2.1) Activating the nano-microspheres to obtain activated nano-microspheres; 2.2) coupling the second RBD antigen to the activated nanoparticles to obtain nanoparticles coated with the second RBD antigen; 2.3) sealing the nanospheres obtained in step 2.2) to obtain second nanospheres; The third step: mixing the first nanosphere and the second nanosphere, in: 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 the following SARS-CoV-2 subtypes: Alpha, Beta, Gamma, Delta, Delta Plus, Epsilon, Lambda, Mu, and Omicron; The first RBD antigen and the second RBD antigen are different; The first nanospheres and the second nanospheres are mixed in a mass ratio of 1:2 to 2:

1. The method according to claim 10 , wherein the first nanospheres and the second nanospheres are mixed at a mass ratio of 1:

1.

12. The method according to claim 10, wherein: The first nanosphere and the second nanosphere are independently polymerized from one or more selected from the following: polystyrene, acrylic acid, acrylate; The average particle sizes of the first nanosphere and the second nanosphere are independently 200 nm to 450 nm; The first nanosphere and the second nanosphere are the same or different.

13. The method according to claim 12, wherein: The first nanospheres and the second nanospheres are polystyrene latex microspheres.

14. The method of claim 12, wherein: The average particle diameters of the first nanosphere and the second nanosphere are independently 300 nm to 350 nm.

15. The method of claim 12, wherein: The first nanosphere and the second nanosphere are carboxyl-modified nanospheres.

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