A SARS-CoV-2 neutralizing antibody detection kit

Abstract

The present invention relates to a SARS-CoV-2 neutralization detection kit, including a solid phase carrier, a screening protein, a competitive binding protein, an alkaline phosphatase, and an alkaline phosphatase luminescent substrate, wherein the screening protein includes an RBD recombinant protein having an amino acid sequence as shown in SEQ ID NO.1 or having at least 85% homology thereto, and / or, an S protein and a N protein fusion protein having an amino acid sequence as shown in SEQ ID NO.2 or having at least 85% homology thereto. The kit developed by the present invention can efficiently, safely, quantitatively and accurately detect SARS-CoV-2 neutralizing antibodies, and can realize large-scale rapid detection. And the RBD protein recombined by a eukaryotic expression system or a fusion protein of the S protein and the N protein is selected as the screening protein of the competitive reaction system, which can further improve the sensitivity and specificity of the detection kit.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody engineering, and in particular relates to a SARS-CoV-2 neutralizing antibody detection kit. Background Art

[0002] The development of a preventive vaccine for SARS-CoV-2 is the most fundamental means of preventing infection with the new coronavirus SARS-CoV-2. The key to judging the success of a preventive vaccine is whether it can induce an effective humoral immune response. Therefore, establishing a stable and effective neutralizing antibody (NAbs) detection method is an important means to evaluate the immunogenicity of a SARS-CoV-2 vaccine.

[0003] SARS-CoV-2 is a type of single-stranded positive-strand RNA coronavirus, whose main structural proteins are spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). For a virus to enter a cell, the cell must have its corresponding receptor. The S protein can bind to the viral receptor of the host cell and is the key protein that determines whether the virus invades susceptible cells. SARS-CoV-2 enters the cell by binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through its surface spike protein (S protein). Therefore, neutralizing antibodies (NAbs) in recovered patients and vaccine recipients can prevent SARS-CoV-2 from infecting healthy cells again by binding to the S protein on the surface of SARS-CoV-2.

[0004] At present, the main method for determining neutralizing antibodies is the traditional virus neutralization test (cVNT), which requires the handling of live viruses and cells. It must be performed by professionals in a biosafety level 3 (BSL3) laboratory, and it usually takes 2-4 days to get the results, which cannot achieve large-scale screening and testing. In addition, due to the certain diversity of the performance of live viruses in experiments, and the test results need to be visually evaluated, the results from different facilities are also lacking in comparability, so the traditional test is inefficient, cumbersome and difficult to standardize. Wang Linfa's team developed an enzyme-linked immunosorbent assay (ELISA) kit for detecting SARS-CoV-2 neutralizing antibodies. However, this method does not have supporting fully automated instruments and equipment, and requires manual sampling and other operations. It takes 2 hours to complete a test, and it still cannot achieve the purpose of large-scale screening. Moreover, ELISA has a narrow linear range and low sensitivity, and cannot accurately detect SARS-CoV-2 neutralizing antibodies. Summary of the invention

[0005] The purpose of the present invention is to provide a rapid, safe, highly accurate, fully quantitative and highly stable neutralizing antibody detection kit.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a SARS-CoV-2 neutralization detection kit, comprising a solid phase carrier, a screening protein, a competitive binding protein, alkaline phosphatase, and an alkaline phosphatase luminescent substrate, wherein the screening protein comprises an RBD recombinant protein having an amino acid sequence as shown in SEQ ID NO.1 or having at least 85% homology thereto, and / or a fusion protein of a spike protein S protein and a nucleocapsid protein N protein having an amino acid sequence as shown in SEQ ID NO.2 or having at least 85% homology thereto.

[0008] A safer and more effective way is adopted to detect SARS-CoV-2 neutralizing antibodies, by simulating the binding process of the receptor binding domain (RBD) of the new coronavirus and angiotensin converting enzyme 2 (ACE2) on the surface of the host cell in vitro, and the introduction of neutralizing antibodies (NAbs) will block this binding process. The kit of the present invention can efficiently, safely, quantitatively and accurately detect SARS-CoV-2 neutralizing antibodies to achieve large-scale rapid detection of SARS-CoV-2 and antibodies. And the RBD protein recombined by the eukaryotic expression system or the fusion protein of the spike protein S protein and the nucleocapsid protein N protein is selected as the screening protein of the competitive reaction system, which can further improve the sensitivity and specificity of the detection kit.

[0009] The preparation process of RBD recombinant protein: (1) constructing the recombinant bacmid with the RBD recombinant protein gene sequence; (2) transforming the recombinant bacmid into insect cells for expression; (3) cell culture, screening and purification.

[0010] The preparation process of fusion protein: (1) synthesize the spike protein S protein gene and the nucleocapsid protein N protein gene in their entirety; (2) construct the recombinant bacmid with the entire gene synthesis sequence; (2) transform the recombinant bacmid into insect cells for expression; (3) culture cells, screen and purify.

[0011] Preferably, the competitive binding protein is selected from one or more of host cell receptor angiotensin converting enzyme 2 or screening protein antibodies.

[0012] Preferably, the solid phase carrier is one or more of streptavidin magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads.

[0013] Further preferably, the cross-linking agent used for the alkaline phosphatase labeled protein is one or more of Sulfo-SMCC, 2-IT, DMP, DCC, streptavidin, and biotin.

[0014] Preferably, the kit further comprises a calibrator, and the raw material of the calibrator comprises anti-screening protein human IgG monoclonal antibody.

[0015] Further preferably, the kit contains matching calibrators and quality control products, and the preparation method is: high-affinity IgG monoclonal antibodies are screened out using the screening protein as an immunogen, and humanized so that they can simulate the combination of neutralizing IgG antibodies in patient serum and RBD protein or S protein and N protein fusion protein in the kit; the obtained RBD protein human IgG monoclonal antibody or S protein and N protein fusion protein human IgG monoclonal antibody is then diluted with diluent at a certain ratio to prepare calibrators and quality control products.

[0016] According to a specific and preferred embodiment, the diluent is a citric acid buffer, and the RBD protein human IgG monoclonal antibody or the S protein and N protein fusion protein human IgG monoclonal antibody is diluted with the citric acid buffer in proportion to 0.5 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, 1000 ng / mL, and 5000 ng / mL as a calibrator for calibrating the kit and calculating the concentration value of the sample to be tested.

[0017] Preferably, the surface of the solid phase carrier is coated with one of the screening protein or the competitive binding protein, and the surface of the other of the screening protein or the competitive binding protein is labeled with the alkaline phosphatase.

[0018] The first design scheme: the screening protein is coated on the surface of the solid phase carrier, and the alkaline phosphatase (AP enzyme) is labeled on the surface of the competitive binding protein. The principle is: the screening protein is coated on the M solid phase carrier, and the competitive binding protein is labeled with AP enzyme. NAbs and competitive binding protein compete for the binding site of the screening protein. The alkaline phosphatase-labeled complex will catalyze the alkaline phosphatase luminescent substrate to produce photons, and the final release of photons is inversely proportional to the NAbs to be detected.

[0019] The second design scheme: the competitive binding protein is coated on the surface of the solid phase carrier, and the alkaline phosphatase is labeled on the surface of the screening protein. The principle is: the competitive binding protein is coated on the M solid phase carrier, and the screening protein is labeled with the AP enzyme that catalyzes the alkaline phosphatase luminescent substrate. The neutralizing antibody in the sample to be tested will compete with the competitive binding protein for the binding site of the screening protein, and a corresponding complex will be formed. After incubation and washing, the alkaline phosphatase-labeled complex will catalyze the alkaline phosphatase luminescent substrate to produce photons, and the amount of photons released is inversely proportional to the NAbs to be tested.

[0020] The kit of the second design is preferably used, which has higher accuracy, precision, sensitivity and specificity than the kit of the first design.

[0021] The kit includes reagent M, reagent R2 and reagent R1, wherein the reagent M is a first buffer containing a solid phase carrier with surface-coated protein; the reagent R2 is a second buffer containing an alkaline phosphatase marker; and the reagent R1 is a third buffer containing a stabilizer for protecting the protein.

[0022] Further preferably, the stabilizer is one or more of aminoacetic acid, bovine serum albumin, casein, trehalose, mannitol, polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol octylphenyl ether (TritonX100), and polyethylene glycol (PEG).

[0023] Further preferably, the first buffer, the second buffer and the third buffer are independently one or more of PBS buffer, Tris buffer or MES buffer.

[0024] More preferably, the working concentration of the solid phase carrier coated with protein on the surface in the M reagent is greater than 0.4ug / mL, more preferably 0.4ug / mL to 5ug / mL, and more preferably 0.4ug / mL to 1ug / mL. The working concentration of the M reagent is calculated based on protein concentration.

[0025] More preferably, the working concentration of the alkaline phosphatase marker in the R2 reagent is greater than 0.8ug / mL, more preferably 0.8ug / mL to 5ug / mL, and more preferably 0.8ug / mL to 1.5ug / mL. The working concentration of the R2 reagent is the concentration of the alkaline phosphatase marker.

[0026] More preferably, the mass percentage of the stabilizer in the R1 reagent is 0.5-5%, and more preferably 0.5-3%.

[0027] According to a specific and preferred embodiment, when the screening protein is an RBD recombinant protein having an amino acid sequence as shown in SEQ ID NO.1, the solid phase carrier is tosyl magnetic beads, the first buffer and the third buffer are independently PBS buffer, the second buffer is MES buffer, and the stabilizer is aminoacetic acid added in an amount of 1% of the total mass of the R1 reagent and polyoxyethylene sorbitan monolaurate added in an amount of 0.5% of the total mass of the R1 reagent.

[0028] According to another specific and preferred embodiment, when the amino acid sequence is a fusion protein of the spike protein S protein and the nucleocapsid protein N protein as shown in SEQ ID NO.2, the solid phase carrier is carboxyl magnetic beads, the first buffer is Tris buffer, the M reagent also contains EDC and Sulfo-NHS, the second buffer is MES buffer, the third buffer is PBS buffer, and the stabilizer is casein added in an amount of 1% of the total mass of the R1 reagent and polyethylene glycol added in an amount of 0.5% of the total mass of the R1 reagent.

[0029] The second aspect of the present invention provides a use of the SARS-CoV-2 neutralization detection kit in the detection of SARS-CoV-2 neutralizing antibodies for purposes other than disease diagnosis and treatment.

[0030] The detection method of the first design scheme is: add M reagent, R2 reagent, R1 reagent, and the sample to be tested to the reaction cup at the same time and incubate the reaction for 5 to 10 minutes. The NAbs in the sample to be tested and the competitive binding protein in the R2 reagent compete for the binding sites on the surface of the screening protein in the M reagent. After washing, the competitive binding protein-screening protein-AP complex and the neutralizing antibody-screening protein complex exist in the reaction cup at the same time. The added luminescent substrate releases photons under AP catalysis. The concentration of NAbs in the sample to be tested is inversely proportional to the number of photons generated. The kit is equipped with a fully automatic chemiluminescence instrument, and it only takes 15 minutes to complete a test.

[0031] The detection method of the second design scheme is: add M reagent, R2 reagent, R1 reagent, and the sample to be tested to the reaction cup at the same time and incubate the reaction for 5 to 10 minutes. The NAbs in the sample to be tested and the competitive binding protein in the M reagent compete for the binding site on the surface of the screening protein in the R2 reagent. After washing, the competitive binding protein-screening protein-AP complex remains in the reaction system. The added luminescent substrate releases photons under AP catalysis. The concentration of neutralizing antibodies in the sample to be tested is inversely proportional to the number of photons generated. The kit is equipped with a fully automatic chemiluminescence instrument, and it only takes 15 minutes to complete a test.

[0032] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0033] The kit developed by the present invention can efficiently, safely, quantitatively and accurately detect SARS-CoV-2 neutralizing antibodies, and can realize large-scale rapid detection. In addition, the RBD protein or S+N fusion protein recombined by the eukaryotic expression system is selected as the screening protein of the competitive reaction system, which can further improve the sensitivity and specificity of the detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Attached Figure 1 : Schematic diagram of the first design scheme of the SARS-CoV-2 neutralization detection kit of the present invention;

[0035] Attached Figure 2 : Schematic diagram of the second design scheme of the SARS-CoV-2 neutralization detection kit of the present invention. DETAILED DESCRIPTION

[0036] The following examples are used to describe the implementation methods of the present application. Those skilled in the art should recognize that these specific examples only illustrate the implementation technical solutions selected to achieve the purpose of the present application, and are not limitations on the technical solutions. According to the teachings of the present application, it is obvious to improve the technical solutions of the present application in combination with the prior art, and all of them belong to the scope of protection of the present application.

[0037] The known reagents and raw materials used in the following examples are all commercially available reagents and raw materials. The implementation conditions used in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are usually the conditions in routine experiments.

[0038] Embodiment 1

[0039] 1. Preparation of RBD recombinant protein

[0040] The RBD domain gene of SARS-CoV-2 (306-527 amino acid sequence of S protein (GeneID: 43740568)) was transformed into competent Escherichia coli DH5α, ice-bathed for 30 minutes, cultured in LB medium at 37°C for 60 minutes for recovery, and finally the bacterial solution was cultured on LB agar plates containing Amp antibiotics to select positive colonies for identification;

[0041] Select the correctly recombinant plasmid to transform E. coli DH10 Bac competent cells, recover for 2-4 hours, and culture in LB medium containing Kan+Gent+Tet to select positive colonies for identification;

[0042] The correctly recombinant bacmid was transformed into insect cells sf9, and the cell supernatant was collected by centrifugation after 3-5 days as the first-generation virus; the insect cells sf9 were infected again with the first-generation virus, and the supernatant was collected after 2-4 days as the second-generation virus for expression and purification of RBD protein.

[0043] The amino acid sequence of the RBD recombinant protein in this example is shown in SEQ ID NO.1.

[0044] 2. Preparation of S+N fusion recombinant protein

[0045] The SARS-CoV-2 N protein gene (GeneID: 43740575) and the SARS-CoV-2 RBD domain gene (306-527 amino acid sequence of the S protein (GeneID: 43740568)) were synthesized to construct plasmids;

[0046] Transformed into competent E. coli DH5α, placed in an ice bath for 30 minutes, cultured in LB medium at 37°C for 60 minutes for recovery, and finally cultured on LB agar plates containing Amp antibiotics to select positive colonies for identification;

[0047] Select the correctly recombinant plasmid to transform E. coli DH10 Bac competent cells, recover for 2-4 hours, and culture in LB medium containing Kan+Gent+Tet to select positive colonies for identification;

[0048] The correctly recombinant bacmid was transformed into insect cells sf9, and the cell supernatant was collected by centrifugation after 3-5 days as the first-generation virus; the insect cells sf9 were infected again with the first-generation virus, and the supernatant was collected after 2-4 days as the second-generation virus for expression and purification of S+N fusion protein.

[0049] The amino acid sequence of the S+N fusion recombinant protein in this example is shown in SEQ ID NO.2.

[0050] Embodiment 2

[0051] This embodiment provides a SARS-CoV-2 neutralization detection kit 1, comprising M reagent, R2 reagent, R1 reagent and a calibrator.

[0052] M reagent: the recombinant RBD protein prepared in Example 1 is coated on the surface of the tosyl magnetic beads, and the tosyl magnetic beads coated with the recombinant RBD protein are diluted with PBS buffer to a working concentration of 0.5 ug / mL (based on the concentration of the recombinant RBD protein);

[0053] R2 reagent: Alkaline phosphatase (AP) is labeled on the surface of ACE2 antigen, Sulfo-SMCC and 2-IT are used as joint cross-linking agents, and the ACE2 antigen labeled with AP enzyme is diluted to a working concentration of 1ug / mL with MES buffer;

[0054] R1 reagent: a stabilizer was added to the PBS buffer solution, wherein the stabilizer was 1% of aminoacetic acid and 0.5% of Tween 20 based on the total mass of the R1 reagent.

[0055] Calibrator: The recombinant RBD protein prepared in Example 1 was used as an immunogen to screen out high-affinity IgG monoclonal antibodies, which were coupled with human IgG antibodies to serve as calibrator raw materials; the raw materials of the calibration quality control products were diluted with citric acid buffer in a certain proportion to 0.5, 10, 50, 200, 1000, and 5000 ng / mL as calibrators, which were used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0056] Embodiment 3

[0057] This embodiment provides a SARS-CoV-2 neutralization detection kit 2, comprising M reagent, R2 reagent, R1 reagent and a calibrator.

[0058] M reagent: ACE2 antigen is coated on the surface of tosyl magnetic beads, and the tosyl magnetic beads coated with ACE2 antigen are diluted with PBS buffer to a working concentration of 0.5ug / mL (based on ACE2 antigen concentration);

[0059] R2 reagent: alkaline phosphatase (AP) was labeled on the surface of the recombinant RBD protein prepared in Example 1, Sulfo-SMCC and 2-IT were used as joint cross-linking agents, and the recombinant RBD protein labeled with AP enzyme was diluted to a working concentration of 1 ug / mL with MES buffer;

[0060] R1 reagent: add stabilizer to PBS buffer, the stabilizer is 1% of aminoacetic acid and 0.5% of Tween 20 based on the total mass of R1 reagent;

[0061] Calibrator: The recombinant RBD protein prepared in Example 1 was used as an immunogen to screen out high-affinity IgG monoclonal antibodies, which were coupled with human IgG antibodies to serve as calibrator raw materials; the raw materials of the calibration quality control products were diluted with citric acid buffer in a certain proportion to 0.5, 10, 50, 200, 1000, and 5000 ng / mL as calibrators, which were used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0062] Embodiment 4

[0063] This embodiment provides a SARS-CoV-2 neutralization detection kit 3, comprising M reagent, R2 reagent, R1 reagent and a calibrator.

[0064] M reagent: the S+N fusion protein prepared in Example 1 is coated on the surface of carboxyl magnetic beads, EDC and Sulfo-NHS are used as dual activators, and the carboxyl magnetic beads coated with S+N fusion protein are diluted with Tris buffer to a working concentration of 0.5 ug / mL (based on the concentration of S+N fusion protein);

[0065] R2 reagent: Alkaline phosphatase (AP) is labeled on the surface of ACE antigen, DMP is used as a cross-linking agent, and the ACE antigen labeled with AP enzyme is diluted into a working solution of 1ug / mL with MES buffer;

[0066] R1 reagent: Add stabilizer to PBS buffer, the stabilizer is 1% casein and 0.5% PEG based on the total mass of R1 reagent;

[0067] Calibrator: The S+N recombinant fusion protein prepared in Example 1 was used as an immunogen to screen out high-affinity IgG monoclonal antibodies, which were coupled with human IgG antibodies to serve as calibrator raw materials; the raw materials of the calibration quality control products were diluted with citric acid buffer in a certain proportion to 0.5, 10, 50, 200, 1000, and 5000 ng / mL as calibrators, which were used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0068] Embodiment 5

[0069] This embodiment provides a SARS-CoV-2 neutralization detection kit 4, comprising M reagent, R2 reagent, R1 reagent and a calibrator.

[0070] M reagent: ACE2 antigen is coated on the surface of carboxyl magnetic beads, EDC and Sulfo-NHS are used as dual activators, and the carboxyl magnetic beads coated with ACE2 antigen are diluted with Tris buffer to a working concentration of 0.5ug / mL (based on ACE2 antigen concentration);

[0071] R2 reagent: alkaline phosphatase (AP) was labeled on the surface of the S+N fusion protein prepared in Example 1, DMP was used as a cross-linking agent, and the S+N fusion protein labeled with AP enzyme was diluted into a working solution of 1 ug / mL with MES buffer;

[0072] R1 reagent: Add stabilizer to PBS buffer, the stabilizer is 1% casein and 0.5% PEG based on the total mass of R1 reagent;

[0073] Calibrator: The S+N recombinant fusion protein prepared in Example 1 was used as an immunogen to screen out high-affinity IgG monoclonal antibodies, which were coupled with human IgG antibodies to serve as calibrator raw materials; the raw materials of the calibration quality control products were diluted with citric acid buffer in a certain proportion to 0.5, 10, 50, 200, 1000, and 5000 ng / mL as calibrators, which were used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0074] Comparative Example 1

[0075] This comparative example provides a SARS-CoV-2 neutralization detection kit 5, comprising M reagent, R2 reagent, R1 reagent and a calibrator.

[0076] M reagent: Biotin-labeled RBD protein is coated on the surface of streptavidin magnetic beads. The biotin-labeled RBD protein is purchased from outside. The magnetic bead antigen complex is diluted to a working concentration of 0.5ug / mL (based on RBD protein concentration) with PBS buffer;

[0077] R2 reagent: label alkaline phosphatase (AP) on the surface of RBD antibody, use DMP as cross-linking agent, and dilute the labeled AP enzyme conjugate into 1ug / mL working solution with MES buffer;

[0078] R1 reagent: Add stabilizer to PBS buffer, the stabilizer is 1% casein and 0.5% PEG based on the total mass of R1 reagent;

[0079] Calibrator: RBD antibody is used as the raw material of the calibrator; the raw material of the calibration quality control product is diluted with citrate buffer in a certain proportion to 0.5, 10, 50, 200, 1000, 5000 ng / mL as the calibrator, which is used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0080] Kit Performance Analysis

[0081] 1. Minimum detection limit

[0082] Use zero concentration calibrator (sample diluent) and repeat the measurement 20 times according to the operating steps in the kit instructions to obtain the relative luminescence value of the 20 measurement results, calculate the average value and standard deviation, and obtain The adjacent concentration calibrator (main calibrator S1) was measured three times, and its relative luminescence value (Xi) was recorded. Its average value was calculated. A linear equation was obtained by performing a two-point regression fitting based on the concentration-relative luminescence value results between the zero concentration calibrator and the adjacent calibrator. The corresponding relative luminescence value is substituted into the above equation to calculate the corresponding concentration, which is the minimum detection limit.

[0083] Test results: The minimum detection limit of kit 1 was 0.0321 ng / mL; the minimum detection limit of reagent 2 was 0.0278 ng / mL; the minimum detection limit of kit 3 was 0.0483 ng / mL; the minimum detection limit of kit 4 was 0.0256 ng / mL; and the minimum detection limit of kit 5 was 2.85 ng / mL.

[0084] 2. Linear

[0085] The high-value sample (5000 ng / mL) close to the upper limit of the linear range was diluted at a certain ratio to prepare samples of 8 concentrations (5000, 1250, 313, 78, 20, 4.9, 1.22, and 0.3 ng / mL). Each concentration of the sample was tested 3 times, and the average value of each concentration of the sample was calculated. The average value of the result and the dilution ratio were fitted with a linear fit using the least squares method, and the slope and linear correlation coefficient r were calculated.

[0086] Test results: The slope of kit 1 was 0.928 and the r value was 0.9989; the slope of kit 2 was 0.951 and the r value was 0.9991; the slope of kit 3 was 0.914 and the r value was 0.9982; the slope of kit 4 was 0.975 and the r value was 0.9993; the slope of kit 5 was 1.245 and the r value was 0.9805.

[0087] 3. Accuracy

[0088] The enterprise accuracy reference materials with concentration values ​​of 10 ng / mL and 1000 ng / mL were prepared and measured. The test was repeated 3 times and the relative deviations were calculated respectively.

[0089] Test results: Kit 1 had a low value deviation of 3.1% and a high value deviation of 3.7%; Kit 2 had a low value deviation of 2.2% and a high value deviation of 3.9%; Kit 3 had a low value deviation of 3.6% and a high value deviation of 2.5%; Kit 4 had a low value deviation of 2.4% and a high value deviation of 1.7%; Kit 5 had a low value deviation of 4.5% and a high value deviation of 4.9%.

[0090] 4. Precision

[0091] Repeat the test of low-value enterprise reference product (10 ng / mL) and high-value enterprise reference product (1000 ng / mL) 10 times, calculate the mean and standard deviation of the 10 test results, and calculate the coefficient of variation according to the formula.

[0092] Test results: The low value coefficient of variation of kit 1 is 4.3%, and the high value coefficient of variation is 3.2%; the low value coefficient of variation of kit 2 is 0.8%, and the high value coefficient of variation is 1.5%; the low value coefficient of variation of kit 3 is 2.6%, and the high value coefficient of variation is 3.3%; the low value coefficient of variation of kit 4 is 1.7%, and the high value coefficient of variation is 0.9%; the low value coefficient of variation of kit 5 is 5.2%, and the high value coefficient of variation is 3.9%.

[0093] 5. Clinical evaluation

[0094] 200 samples from healthy people who were not infected with the COVID-19, 30 samples from people who had recovered from the COVID-19, and 30 samples from people who had been vaccinated were tested, and the test results were analyzed using SPSS statistical software.

[0095] Test results:

[0096] The detection levels of kit 1 in people who have recovered from COVID-19 and people who have been vaccinated were 420.68 ng / mL and 243.57 ng / mL, respectively, which were significantly higher than the detection level of 3.52 ng / mL in healthy people. The sensitivity and specificity analysis results of the kit were 98.33% and 98.67%, respectively.

[0097] The detection levels of Test Kit 2 in people who have recovered from COVID-19 and people who have been vaccinated were 557.92 ng / mL and 313.06 ng / mL, respectively, which were significantly higher than the detection level of 3.23 ng / mL in healthy people. The sensitivity and specificity analysis results of the kit were 99.07% and 98.88%, respectively.

[0098] The detection levels of Kit 3 in people who have recovered from COVID-19 and people who have been vaccinated were 582.41 ng / mL and 337.05 ng / mL, respectively, which were significantly higher than the detection level of 2.56 ng / mL in healthy people. The sensitivity and specificity analysis results of the kit were 99.27% ​​and 99.44%, respectively.

[0099] The detection levels of Test Kit 4 in people who have recovered from COVID-19 and people who have been vaccinated were 679.04 ng / mL and 492.18 ng / mL, respectively, which were significantly higher than the detection level of 1.03 ng / mL in healthy people. The sensitivity and specificity analysis results of the kit were 100% and 100%, respectively.

[0100] The detection levels of Kit 5 in people who have recovered from COVID-19 and people who have been vaccinated were 295.14 ng / mL and 155.06 ng / mL respectively, and the detection level in healthy people was 14.88 ng / mL. The sensitivity and specificity analysis results of the kit were 83.53% and 81.07% respectively.

[0101] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Suzhou Xiechuang Biotechnology Co., Ltd. <120> A SARS-CoV-2 neutralizing antibody detection kit <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 225 <212> PRT <213> Artificial sequence <400> 1 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 1 5 10 15 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 20 25 30 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 35 40 45 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 50 55 60 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 65 70 75 80 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 85 90 95 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 100 105 110 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 115 120 125 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 130 135 140 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 145 150 155 160 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 165 170 175 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 180 185 190 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 195 200 205 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 210 215 220 Ser 225 <210> 2 <211> 649 <212> PRT <213> Artificial sequence <400> 2 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala Gly Gly Gly Gly Ser Phe Thr Val Glu Lys Gly Ile Tyr 420 425 430 Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 435 440 445 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 450 455 460 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 465 470 475 480 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 485 490 495 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 500 505 510 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 515 520 525 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 530 535 540 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 545 550 555 560 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 565 570 575 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 580 585 590 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 595 600 605 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 610 615 620 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 625 630 635 640 Ala Thr Val Cys Gly Pro Lys Lys Ser 645

Claims

1. A SARS-CoV-2 neutralization detection kit, characterized in that: The kit includes reagent M, reagent R2, and reagent R1. ACE2 antigen is coated on the surface of carboxyl magnetic beads, EDC and Sulfo-NHS are used as dual activators, and the carboxyl magnetic beads coated with ACE2 antigen are diluted with Tris buffer to obtain the M reagent. The R2 reagent is obtained by diluting the screening protein labeled with AP enzyme using DMP as a cross-linking agent with MES buffer, wherein the screening protein is a fusion protein of S protein and N protein with an amino acid sequence as shown in SEQ ID NO.2, The stabilizer is added to the PBS buffer to obtain the R1 reagent, wherein the stabilizer is casein and PEG. In the M reagent, the working concentration of the carboxyl magnetic beads coated with the ACE2 antigen is 0.5 μg / mL; in the R2 reagent, the working concentration of the screening protein labeled with the AP enzyme is 1 μg / mL; in the R1 reagent, the added amount of casein is 1% of the total mass of the R1 reagent, and the added amount of PEG is 0.5% of the total mass of the R1 reagent.

2. The SARS-CoV-2 neutralization detection kit according to claim 1, characterized in that: The kit also includes a calibrator, and the raw materials of the calibrator include anti-screening protein human IgG monoclonal antibody.

3. Use of a SARS-CoV-2 neutralization detection kit as claimed in claim 1 or 2 in the detection of SARS-CoV-2 neutralizing antibodies for the purpose of non-disease diagnosis and treatment.

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