ANTIGEN FOR 2019 NOVEL CORONAVIRUS AND DETECTION USE THEREOF

Abstract

Provided are a method for assaying a specific IgM antibody and a total antibody for the 2019 novel coronavirus (2019-nCOV), a method for determining whether subjects are infected with 2019-nCOV, and a virus antigen and a kit for carrying out the above-mentioned detection.

Claims

1. An isolated polypeptide, which comprises or consists of an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 1; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence shown in SEQ ID NO: 1; and (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence shown in SEQ ID NO: 1.

2. The isolated polypeptide according to claim 1, which bears a detectable label; preferably, the detectable label is selected from the group consisting of enzyme (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescence reagent (e.g., acridine ester compound), fluorescent dye, colloidal gold or biotin.

3. The isolated polypeptide according to claim 1, which is attached to a surface of a solid support, or has a modifying group that can be attached to the solid support; preferably, the modifying group is biotin or avidin; preferably, the solid support is selected from the group consisting of magnetic bead, microtiter plate (e.g., microplate or ELISA plate), and nitrocellulose membrane.

4. A fusion protein, which comprises the isolated polypeptide according to claim 1 and an additional polypeptide; preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the isolated polypeptide according to claim 1 optionally via a linker; preferably, the additional polypeptide is selected from a protein tag, such as His, Flag, GST, MBP, HA, Myc, GFP or Fc.

5. The fusion protein according to claim 4, which bears a detectable label; preferably, the detectable label is selected from the group consisting of enzyme (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescence reagent (e.g., acridine ester compound), fluorescent dye, colloidal gold or biotin.

6. The fusion protein according to claim 4, which is attached to a surface of a solid support, or has a modifying group that can be attached to the solid support; preferably, the modifying group is biotin or avidin; preferably, the solid support is selected from the group consisting of magnetic bead, microtiter plate (e.g., microplate or ELISA plate), and nitrocellulose membrane.

7. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the isolated polypeptide according to claim 1 or the fusion protein according to claim 4.

8. A vector, which comprises the isolated nucleic acid molecule according to claim 7.

9. A host cell, which comprises the isolated nucleic acid molecule according to claim 7 or the vector according to claim 8.

10. A kit, which comprises the isolated polypeptide according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 6.

11. The kit according to claim 10, which comprises a detection reagent, and the detection reagent being selected from the isolated polypeptide according to claim 2 or the fusion protein according to claim 5.

12. The kit according to claim 11, which further comprises a capture reagent, the capture reagent being selected from a reagent capable of specifically binding to IgM; preferably, the reagent capable of specifically binding to IgM is selected from an anti-IgM antibody or antigen-binding fragment thereof; preferably, the reagent capable of specifically binding to IgM is attached to a surface of a solid support, or has a modifying group (e.g., biotin or avidin) that can be attached to the solid support; preferably, the solid support is selected from the group consisting of magnetic bead, microtiter plate (e.g., microtiter plate or ELISA plate), and nitrocellulose membrane; preferably, the kit further comprises an instruction of using the capture reagent and the detection reagent to detect an IgM antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject, and optionally to determine whether the subject has a novel coronavirus (2019-nCoV) infection.

13. The kit according to claim 11, which further comprises a capture reagent, the capture reagent being selected from the isolated polypeptide according to claim 1 or the fusion protein according to claim 4; preferably, the capture reagent is selected from the isolated polypeptide according to claim 3 or the fusion protein according to claim 6; preferably, the kit further comprises an instruction of using the capture reagent and the detection reagent to detect an antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject, and optionally to determine whether the subject has a novel coronavirus (2019-nCoV) infection.

14. The kit according to any one of claims 10 to 13, which further comprises one or more reagents or devices selected from the group consisting of: (i) a device for collecting or storing a sample from a subject (e.g., blood collection device); (ii) an additional reagent required to perform the detection (e.g., buffer, diluent, blocking solution, positive control sample, and/or negative control sample).

15. Use of the isolated polypeptide according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 6 in the manufacture of a kit for detecting an IgM antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject, and/or for determining whether a subject has a novel coronavirus (2019-nCoV) infection; preferably, the sample is a blood sample, such as whole blood, plasma or serum; preferably, the subject is a human.

16. The use according to claim 15, wherein the kit comprises a detection reagent and a capture reagent, the detection reagent is selected from the isolated polypeptide according to claim 2 or the fusion protein according to claim 5, and the capture reagent is selected from a reagent capable of specifically binding to IgM; preferably, the reagent capable of specifically binding to IgM is selected from an anti-IgM antibody or antigen-binding fragment thereof; preferably, the reagent capable of specifically binding to IgM is attached to a surface of a solid support; preferably, the solid support is selected from the group consisting of magnetic bead, microtiter plate (e.g., microplate or ELISA plate), and nitrocellulose membrane.

17. The use according to claim 16, wherein the kit is used for detecting an IgM antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject by the following method: (1) contacting the sample with the capture reagent to obtain an immune complex; (2) detecting the immune complex obtained in step (1) by using the detection reagent via an immunological detection; preferably, the immunological detection is selected from the group consisting of enzyme immunoassay (e.g., ELISA), chemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay, and immunocolloidal gold technique.

18. The use according to claim 17, wherein the kit is used to determining whether the subject has a novel coronavirus (2019-nCoV) infection by the following method: (i) detecting a level of IgM antibody specific to novel coronavirus (2019-nCoV) in the sample from the subject by the method of claim 17; and, (ii) comparing the level with a reference value to determine whether the subject has a novel coronavirus (2019-nCoV) infection.

19. Use of the isolated polypeptide according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 6 in the manufacture of a kit for detecting an antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject and/or for determining whether a subject has a novel coronavirus (2019-nCoV) infection; preferably, the sample is a blood sample, such as whole blood, plasma or serum; preferably, the subject is a human.

20. The use according to claim 19, wherein the kit comprises a capture reagent and a detection reagent, wherein the capture reagent is selected from the isolated polypeptide according to claim 3 or the fusion protein according to claim 6, and the detection reagent is selected from the isolated polypeptide according to claim 2 or the fusion protein according to claim 5.

21. The use according to claim 20, wherein the kit is used for detecting an antibody specific to novel coronavirus (2019-nCoV) in a sample from a subject by the following method: (1) contacting the sample with the capture reagent to obtain an immune complex; (2) detecting the immune complex obtained in step (1) by using the detection reagent via an immunological detection; preferably, the immunological detection is selected from the group consisting of enzyme immunoassay (e.g., ELISA), chemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay, and immunocolloidal gold technique.

22. The use according to claim 21, wherein the kit is used for determining whether the subject has a novel coronavirus (2019-nCoV) infection by the following method: (i) detecting a level of the antibody specific to novel coronavirus (2019-nCoV) in the sample from the subject by the method of claim 21; and, (ii) comparing the level with a reference value to determine whether the subject has a novel coronavirus (2019-nCoV) infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0128] FIG. 1 shows a schematic diagram of determination of the detection results obtained by the colloidal gold method.

SEQUENCE INFORMATION

[0129]

TABLE-US-00001 SEQ ID NO Description Sequence information 1 2019-nCoV S-RBD RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKR ISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR VVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

EXAMPLES

Example 1: Detection Kit and Method for Detecting 2019-nCoV IgM Antibody (Enzyme-Linked Immunosorbent Assay)

[0130] 1.1 Preparation of Detection Kit

[0131] The detection kit comprised: a 96-well ELISA plate coated with anti-human IgM, a 2019-nCoV-Ag enzyme labeled reagent labeled with horseradish peroxidase, a PBS buffer, a positive control containing 2019-nCoV-IgM positive sample, a negative control, a concentrated washing solution containing not less than 2.5% of surfactant, Developer A containing not less than 0.3 g/L of peroxide, Developer B containing not less than 0.2 g/L of TMB, and a termination solution containing not more than 2 mol/L of sulfuric acid.

[0132] Negative control: BSA 0.5 g/L, NaCl 137 mmol/L, KCl 2.7 mmol/L, Na.sub.2HPO.sub.4 4.3 mmol/L, KH.sub.2PO.sub.4 1.4 mmol/L.

[0133] Concentrated washing solution: TWeen-20 30 ml/L, NaCl 2 mol/L, KCl 54 mmol/L, Na.sub.2HPO.sub.4 80 mmol/L, KH.sub.2PO.sub.4 28 mmol/L.

[0134] Color developing solution A: sodium acetate 27.2 g/L, citric acid 3.2 g/L, 30% hydrogen peroxide 0.6 ml/L.

[0135] Color developing solution B: disodium EDTA 0.4 g/L, citric acid 2 g/L, glycerol 100 ml/L, 0.3 g/L TMB.

[0136] Termination solution: sulfuric acid 2 mol/L.

Preparation of 96-Well ELISA Plate with Anti-Human IgM

[0137] 1. Murine anti-human IgM (II chain) (Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was formulated into a coating solution with 1×PBS buffer, and placed at 100 ul/well in the support plate overnight at 2-8° C. [0138] 2. PBS buffer containing 0.1% BSA was used at 200/well to perform blocking at 2-8° C. overnight. [0139] 3. The blocking solution was discarded, and drying was performed in a dry environment. Then, it was packed in an aluminum foil bag with desiccant.

Preparation of 2019-nCoV-Ag Enzyme Labeled Reagent with Horseradish Peroxidase

[0140] 1. 2019-nCoV-Ag fusion protein containing SEQ ID NO: 1 and a protein tag was dialyzed into CB (pH9.6, 50 mM), in which the medium was changed more than 2 times. [0141] 2. The ddH.sub.2O solution of HRP and the ddH.sub.2O solution of NaIO.sub.4 were prepared. [0142] 3. The NaIO.sub.4 solution was slowly mixed with the HRP solution, and allowed to stand at 4° C. for 30 min. [0143] 4. Ethylene glycol was slowly added to the oxidized HRP solution, and allowed to stand for 30 minutes at room temperature in the dark. [0144] 5. The oxidized HRP was directly added to the fully dialyzed viral antigen protein. [0145] 6. Dialysis into CB (pH9.6, 50 mM) was continued, in which the medium was changed more than 2 times. [0146] 7. NaBH.sub.4 was added to it and allowed to stand at 4° C. for 2 hours. [0147] 8. 1×PBS buffer was allowed to stand overnight, the labeling solution was added with an equal volume of 50% glycerol, mixed well and stored at −20° C.

[0148] 1.2 Detection Method [0149] 1. Preparation of solution: Concentrated washing solution was diluted 20 times with distilled water or deionized water. [0150] 2. Numbering: Samples were numbered corresponding to microplates in sequence. For each plate, 3 wells of negative control, 2 wells of positive control and 1 well of blank control were set. (When dual-wavelength detection was used, blank control wells could be omitted). [0151] 3. Addition of diluent: 100 μl of sample diluent was added to each well, except for negative- and positive control wells and blank wells. [0152] 4. Addition of samples: 10 μl of serum or plasma sample, and 100 μl of each of negative and positive controls were added to the corresponding wells, respectively, and mixed with gentle shaking. [0153] 5. Incubation: After sealing the plate with sealing film, incubation was performed at 37° C. for 30 minutes. [0154] 6. Washing plate: The sealing film was carefully removed, and the plate was washed with a plate washer 5 times, and at the end of last washing, the plate was inverted and tapped to completely drain as much as possible. [0155] 7. Addition of enzyme: 100 μl of the enzyme labeled reagent was added to each well, except for the blank well. [0156] 8. Incubation: the operation was the same as step 5. [0157] 9. Washing plate: the operation was the same as step 6. [0158] 10. Color development: 50 μl of color Developer A solution and 50 μl of color Developer B solution were added to each well, gently shaken and mixed, and color development was performed at 37° C. for 15 minutes in the dark. [0159] 11. Detection: 50 μl of the termination solution was added to each well, gently shaken and mixed, and the detection results were obtained within 10 minutes. The wavelength of microplate reader was set at 450 nm (or detected with dual wavelengths of 450 nm/600-650 nm), and the A value of each well was measured after zeroing using the blank well.

[0160] Judgement of Detection Results: [0161] 1. Normal range of negative control: the A value of the negative control well was less than or equal to 0.1 (if the A value of the negative control in one well was greater than 0.1, it should be discarded; if the A values of the negative control in two or more wells were greater than 0.1, the experiment should be repeated). [0162] 2. Normal range of positive control: the A value of the positive control well was ≥0.8. [0163] 3. Calculation of cut-off value (CUTOFF): cut-off value=mean of A values of negative control wells (Nc)×2.1 (if the value of negative control well was lower than 0.05, it should be calculated as 0.05). Positive judgment: sample with A value≥cut-off value (CUTOFF) was positive for 2019-nCoV-IgM antibody; negative judgment: sample with A value<cut-off value (CUTOFF) was negative for 2019-nCoV-IgM antibody.

[0164] 1.3 Evaluation of Detection Performance [0165] (1) The above detection kit was used as candidate reagent to detect 2019-nCoV IgM antibody in plasma samples from 49 confirmed cases and 2 negative cases of Hubei Province. Among the 49 confirmed cases, 1 plasma sample was collected from each of 44 cases, and 2 plasma samples were collected at different periods from each of the remaining 5 cases, so that a total of 54 plasma samples were collected from the confirmed cases in this experiment. The candidate reagent detected 47 positive cases, and the detection rate of the candidate reagent compared with the confirmed cases was 95.92% (47/49). For the 2 negative cases, there were 1 sample from one subject, and 2 samples from another subject at two different periods, that was, there were a total of 3 negative samples. The results of the candidate reagent for the three negative samples were all negative. [0166] (2) The above detection kit was used as candidate reagent to detect 2019-nCoV IgM antibody in 312 serum samples from 81 confirmed cases of novel coronavirus pneumonia at different times after onset in Zhejiang Province, and 300 serum samples from 300 other cases.

[0167] The detection results of the candidate reagent and the clinically confirmed/excluded results were analyzed by contingency table (four-fold table) for Kappa consistency test. The results showed that the sensitivity of the candidate reagent was 91.36% (95% confidence interval was 83.00% to 96.45%), the specificity was 100.00% (95% confidence interval was 96.38% to 100.00%), the accuracy was 98.16% (95% confidence interval was 96.25% to 99.26%), the Kappa value was 0.94, and the consistency strength was the strongest. [0168] (3) Analysis of Detection Results of Samples of Different Times after Onset:

[0169] The above detection kit was used as candidate reagent to detect 2019-nCoV IgM antibody in plasma samples from confirmed cases of novel coronavirus pneumonia at different times after onset in Guangdong Province, and the detection results were taken together with those of samples of different times after onset in (2). The results were shown in the table below.

TABLE-US-00002 TABLE 2 Detection results of samples of different times after onset Days Candidate reagent after Total number of detected Number of positive of Positive rate onset samples (samples) detection (samples) of detection 1 3 2 66.67% 2 12 2 16.67% 3 21 5 23.81% 4 26 4 15.38% 5 28 7 25.00% 6 49 12 24.49% 7 47 18 38.30% 8 54 22 40.74% 9 62 23 37.10% 10 52 32 61.54% 11 54 41 75.93% 12 61 48 78.69% 13 42 35 83.33% 14 41 34 82.93% 15 40 33 82.50% 16 33 30 90.91% 17 42 34 80.95% 18 31 26 83.87% 19 33 31 93.94% ≥20 95 90 94.74%

[0170] It could be seen from the above table that the detection rate of the candidate reagent in the early stage of onset (before day 7) was 15% to 40%, reached up to 40% to 80% 7 days after onset and reached up to 80% to 95% 15 days after onset. According to different period after onset, there were 5 cases in the very early stage (≤3 days) and 13 cases in the early stage (4-7 days) that were detected to be negative in the nucleic acid detection, while the candidate reagent could detect 2 cases (40%) and 6 cases (46%) from them, respectively; there were 29 cases in the middle stage (8-14 days) and 23 cases in the late stage (≥15 days) that were detected to be negative in the nucleic acid detection, while the candidate reagent could detect 24 cases and 21 cases, respectively.

[0171] The above results show that the ELISA-based IgM antibody detection can be used for the screening of 2019-nCoV infection, and has a good complementary effect on the nucleic acid detection.

Example 2: Detection Kit and Method for Determining Total Antibody Against 2019-nCoV (Enzyme-Linked Immunosorbent Assay)

[0172] 2.1 Preparation of Detection Kit

[0173] The detection kit comprised: a 96-well ELISA plate coated with 2019-nCoV-Ag fusion protein containing SEQ ID NO: 1 and protein tag, a 2019-nCoV-Ag enzyme labeled reagent with horseradish peroxidase, a PBS buffer, a positive control containing 2019-nCoV antibody positive sample, a negative control, a concentrated washing solution containing not less than 2.5% of surfactant, Developer A containing not less than 0.3 g/L of peroxide, Developer B containing not less than 0.2 g/L of TMB, and a termination solution containing not more than 2 mol/L of sulfuric acid.

[0174] Negative control: BSA 0.5 g/L, NaCl 137 mmol/L, KCl 2.7 mmol/L, Na.sub.2HPO.sub.4 4.3 mmol/L, KH.sub.2PO.sub.4 1.4 mmol/L.

[0175] Concentrated washing solution: TWeen-20 30 ml/L, NaCl 2 mol/L, KCl 54 mmol/L, Na.sub.2HPO.sub.4 80 mmol/L, KH.sub.2PO.sub.4 28 mmol/L.

[0176] Color developing solution A: sodium acetate 27.2 g/L, citric acid 3.2 g/L, 30% hydrogen peroxide 0.6 ml/L.

[0177] Color developing solution B: disodium EDTA 0.4 g/L, citric acid 2 g/L, glycerol 100 ml/L, 0.3 g/L TMB.

[0178] Termination solution: sulfuric acid 2 mol/L.

Preparation of 96-Well ELISA Plate Coated with 2019-nCoV-Ag

[0179] 1. The 2019-nCoV antigen fusion protein containing SEQ ID NO: 1 and protein tag (National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University) was formulated into a coating solution with 1×PBS buffer, and placed at 100 ul/well in the support plate overnight at 2-8° C. [0180] 2. PBS buffer containing 0.1% BSA was used at 200/well to perform blocking at 2-8° C. overnight. [0181] 3. The blocking solution was discarded, and drying was performed in a dry environment. Then, it was packed in an aluminum foil bag with desiccant.

Preparation of 2019-nCoV-Ag Enzyme Labeled Reagent with Horseradish Peroxidase

[0182] 1. 2019-nCoV-Ag fusion protein containing SEQ ID NO: 1 and protein tag was dialyzed into CB (pH9.6, 50 mM), in which the medium was changed more than 2 times. [0183] 2. The ddH.sub.2O solution of HRP and the ddH.sub.2O solution of NaIO.sub.4 were prepared. [0184] 3. The NaIO.sub.4 solution was slowly mixed with the HRP solution, and allowed to stand at 4° C. for 30 min. [0185] 4. Ethylene glycol was slowly added to the oxidized HRP solution, and allowed to stand for 30 minutes at room temperature in the dark. [0186] 5. The oxidized HRP was directly added to the fully dialyzed viral antigen protein. [0187] 6. Dialysis into CB (pH9.6, 50 mM) was continued, in which the medium was changed more than 2 times. [0188] 7. NaBH.sub.4 was added to it and allowed to stand at 4° C. for 2 hours. [0189] 8. 1×PBS buffer was allowed to stand overnight, the labeling solution was added with an equal volume of 50% glycerol, mixed well and stored at −20° C.

[0190] 2.2 Detection Method [0191] 1. Preparation of solution: Concentrated washing solution was diluted 20 times with distilled water or deionized water. [0192] 2. Numbering: Samples were numbered corresponding to microplates in sequence. For each plate, 3 wells of negative control, 2 wells of positive control and 1 well of blank control were set. (When dual-wavelength detection was used, blank control wells could be omitted). [0193] 3. Addition of sample: 100 μl of sample to be detected, and 50 μl of each of negative- and positive controls were added to corresponding wells, respectively. [0194] 4. Incubation: After sealing the plate with sealing film, incubation was performed at 37° C. for 30 minutes. [0195] 5. Washing plate: The sealing film was carefully removed, and the plate was washed with a plate washer 5 times, and at the end of last washing, the plate was inverted and tapped to completely drain as much as possible. [0196] 6. Addition of enzyme: 100 μl of the enzyme labeled reagent was added to each well, except for the blank well. [0197] 7. Incubation: the operation was the same as step 4. [0198] 8. Washing plate: the operation was the same as step 5. [0199] 9. Color development: 50 μl of each of color Developer A and B solutions was added to each well, gently shaken and mixed, and color development was performed at 37° C. for 15 minutes in the dark. [0200] 10. Detection: 50 μl of the termination solution was added to each well, gently shaken and mixed, and the detection results were obtained within 10 minutes. The wavelength of microplate reader was set at 450 nm (or detected with dual wavelengths of 450 nm/600-650 nm), and the A value of each well was measured after zeroing using the blank well.

[0201] Judgement of Detection Results: [0202] 1. Normal range of negative control: the A value of the negative control well was less than or equal to 0.1 (if the A value of the negative control in one well was greater than 0.1, it should be discarded; if the A values of the negative control in two or more wells were greater than 0.1, the experiment should be repeated). [0203] 2. Normal range of positive control: the A value of the positive control well was ≥0.19. [0204] 3. Calculation of cut-off value (CUTOFF): cut-off value=0.16+mean of A values of negative control wells (if the value of negative control well was lower than 0.03, it should be calculated as 0.03). Positive judgment: sample with A value≥cut-off value (CUTOFF) was positive for 2019-nCoV antibody; negative judgment: sample with A value<cut-off value (CUTOFF) was negative for 2019-nCoV antibody.

[0205] 2.3 Evaluation of Detection Performance [0206] (1) The above detection kit was used as candidate reagent to detect the total antibody against 2019-nCoV in plasma samples of 49 confirmed cases from Hubei Province.

[0207] Among the 49 confirmed cases, 1 plasma sample was collected from each of 44 cases respectively, and 2 plasma samples of two different periods were collected from each of the remaining 5 cases respectively, so that a total of 54 plasma samples were collected from the confirmed cases in this experiment. The candidate reagent detected 49 positive cases, and the detection rate of the candidate reagent compared with the confirmed cases was 100.00% (49/49). For the 2 samples of two different periods for 1 case subject, the candidate reagent detected 1 negative and 1 positive, and this result was determined as a positive result of the candidate reagent. [0208] (2) The above detection kit was as candidate reagent to detect the total antibody against 2019-nCoV in 312 serum samples of different times after onset from 81 confirmed cases of novel coronavirus infection pneumonia in Zhejiang Province and 300 serum samples from 300 other cases.

[0209] The detection results of the candidate reagent and the clinically confirmed/excluded results were analyzed by contingency table (four-fold table) for Kappa consistency test. The results showed that the sensitivity of the candidate reagent was 96.30% (95% confidence interval was 89.56% to 99.23%), the specificity was 100% (95% confidence interval was 96.38% to 100.00%), the accuracy was 99.21% (95% confidence interval was 99.72% to 99.84%), the Kappa value was 0.98, and the consistency strength was the strongest. [0210] (3) Detection Results of Samples of Different Times after Onset:

[0211] The above detection kit was used as candidate reagent to detect 2019-nCoV total antibody in plasma samples from confirmed cases of novel coronavirus pneumonia with different times after onset in Guangdong Province, and the detection results were taken together with those of samples of different times after onset in (2). The results were shown in the table below.

TABLE-US-00003 TABLE 3 Detection results of samples of different times after onset Days Candidate reagent after Total number of detected Number of positive of Positive rate onset samples (samples) detection (samples) of detection 1 3 1 33.33% 2 12 3 25.00% 3 21 4 19.05% 4 26 9 34.62% 5 28 9 32.14% 6 50 23 46.00% 7 46 24 52.17% 8 54 32 59.26% 9 62 44 70.97% 10 54 43 79.63% 11 54 48 88.89% 12 62 59 95.16% 13 43 41 95.35% 14 44 42 95.45% 15 43 43 100.00% 16 35 35 100.00% 17 42 38 90.48% 18 33 32 96.97% 19 34 33 97.06% ≥20 96 94 97.92%

[0212] The detection rate of the candidate reagent was 20% to 50% in the early stage of onset (before day 7), more than 90% after 12 days of onset, and almost 100% after 15 days of onset. According to different periods after onset, in the very early stage (≤3 days) and early stage (4-7 days), there were 5 and 13 patients who were negative in the nucleic acid detection, respectively, and the candidate reagent could detect 2 cases (40%) and 6 cases (46%) from them; in the middle stage (8-14 days) and late stage (≥15 days), there were 29 and 23 patients who were negative in the nucleic acid detection, and the candidate reagent could detect all of them, respectively, that was, the positive rate of detection was up to 100%.

[0213] The above results show that the ELISA-based detection for total antibody can be used for the screening of 2019-nCoV infection, and shows a good complementary effect on the nucleic acid detection.

Example 3: Detection Kit and Method for Detecting 2019-nCoV IgM Antibody (Colloidal Gold Method)

[0214] 3.1 Preparation of Detection Kit

[0215] The detection kit comprised: a glass fiber with gold-labeled novel coronavirus (2019-nCoV) antigen, a nitrocellulose membrane coated with anti-human IgM (anti-μ chain), a glass fiber, a plastic backing, a phosphate buffer-containing sample diluent, etc.

[0216] Sample diluent: NaCl 137 mmol/L, KCl 2.7 mmol/L, Na.sub.2HPO.sub.4 4.3 mmol/L, KH.sub.2PO.sub.4 1.4 mmol/L.

Preparation of Nitrocellulose Membrane Coated with Anti-Human IgM (Anti-μChain)

[0217] 1. Murine anti-human IgM (μ chain) (Beijing Wantai Bio-Pharmaceutical Co., Ltd.) as the material for coating test line was formulated with PBS buffer to prepare the test line coating solution; the material for coating control line was formulated with PBS buffer to prepare the control line coating solution. [0218] 2. The test line coating solution was taken and coated on the test line of the nitrocellulose membrane; the control line coating solution was taken and coated on the control line of the nitrocellulose membrane. [0219] 3. The coated nitrocellulose membrane was fully dried, packaged in an aluminum foil bag, and sealed by machine.

Preparation of Glass Fiber with Gold-Labeled Novel Coronavirus (2019-nCoV) Antigen

[0220] 1. The 2019-nCoV antigen fusion protein containing SEQ ID NO: 1 and protein tag (National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University) as the material for gold-labeling was mixed with colloidal gold solution, added with the blocking solution to perform blocking to obtain gold-labeled solution. [0221] 2. The gold-labeled solution was added to the glass fiber, fully dried, packaged in an aluminum foil bag, and sealed by machine.

[0222] 3.2 Detection Method

[0223] At room temperature, 10 μL of serum or plasma sample to be tested was added to the sample hole of test strip, then added dropwise with 2 drops of sample diluent, the test strip was horizontally placed on table, and observed to obtain results 15 minutes after adding the sample.

[0224] The judgement of the detection results was shown in FIG. 1. A: When the control line developed color and the test line also developed color, the result was judged as positive. B: When the control line developed color, while the test line did not develop color, the result was judged as negative. C: When the control line did not develop color, regardless of whether the test line developed color, the experiment was judged as invalid, and a repeated experiment was required.

[0225] 3.3 Evaluation of Detection Performance [0226] (1) The above detection kit as candidate reagent was used to detect 2019-nCoV IgM antibody in plasma samples from 49 confirmed cases and 5 negative cases in Hubei Province.

[0227] Among the 49 confirmed cases, 1 plasma sample was collected from each of 44 cases respectively, 2 plasma samples of two different periods were collected from each of 2 cases respectively, and thus a total of 40 plasma samples were collected from the confirmed cases in this experiment. The candidate reagent detected 45 positive cases, and the detection rate of the candidate reagent compared with the confirmed cases was 91.84% (45/49). Among the 5 negative cases, there were 3 samples from 3 case subjects and 2 samples of two different periods from 1 case subject, with a total of 5 negative samples. The results of the five negative samples detected by the candidate reagent were all negative. [0228] (2) The above detection kit was used as candidate reagents to detect 2019-nCoV IgM antibody in 312 serum samples and 49 whole blood samples of different times after onset from 90 confirmed cases of novel coronavirus pneumonia, and 209 serum samples and 99 whole blood samples from 308 other cases in Zhejiang Province.

[0229] The detection results of the candidate reagent and the clinically confirmed/excluded results were analyzed by contingency table (four-fold table) for Kappa consistency test. The results showed that the sensitivity of the candidate reagent was 93.33% (95% confidence interval was 86.05% to 97.51%), the specificity was 98.70% (95% confidence interval was 96.71% to 99.65%), and the accuracy was 97.49% (95% confidence interval was 95.43% to 98.79%), the Kappa value was 0.93, and the consistency strength was the strongest.

[0230] The detection results in (2) were analyzed according to different times after onset, and the results were shown in the following table.

TABLE-US-00004 TABLE 4 Detection results of samples of different times after onset Days Candidate reagent after Total number of detected Number of positive of Positive rate onset samples (samples) detection (samples) of detection 1 1 0 0.00% 2 4 0 0.00% 3 4 0 0.00% 4 9 2 22.22% 5 7 0 0.00% 6 12 0 0.00% 7 18 4 22.22% 8 12 2 16.67% 9 18 6 33.33% 10 22 9 40.91% 11 25 19 76.00% 12 22 13 59.09% 13 20 16 80.00% 14 21 18 85.71% 15 20 18 90.00% 16 15 12 80.00% 17 19 15 78.95% 18 19 18 94.74% 19 16 14 87.50% ≥20 77 68 88.31%

[0231] It could be seen from the detection results of samples of different times after onset that with progression after onset, the detection rate of the candidate reagent increased rapidly 10 days after onset, and reached up to 80% to 90% after 13 days. The above results indicate that the IgM antibody detection based on colloidal gold method can also be used for the screening of 2019-nCoV infection.

Example 4: Detection Kit and Method for Determining 2019-nCoV Total Antibody (Colloidal Gold Method)

[0232] 4.1 Preparation of Detection Kit

[0233] The detection kit comprised: a glass fiber containing gold-labeled 2019-nCoV Ag, a nitrocellulose membrane coated with 2019-nCoV Ag, a glass fiber, a plastic backing, and a phosphate buffer-containing sample diluent.

[0234] Sample diluent: NaCl 137 mmol/L, KCl 2.7 mmol/L, Na.sub.2HPO.sub.4 4.3 mmol/L, KH.sub.2PO.sub.4 1.4 mmol/L.

Preparation of Nitrocellulose Membrane Coated with 2019-nCoV Ag

[0235] 1. 2019-nCoV antigen fusion protein containing SEQ ID NO: 1 and protein tag as the material for coating test line was formulated with PBS buffer to prepare the test line coating solution; the material for coating control line was formulated with PBS buffer to prepare the control line coating liquid. [0236] 2. The test line coating solution was taken and coated on the test line of the nitrocellulose membrane; the control line coating solution was taken and coated on the control line of the nitrocellulose membrane. [0237] 3. The coated nitrocellulose membrane was fully dried, packaged in an aluminum foil bag, and sealed by machine.

Preparation of Glass Fiber with Gold-Labeled Novel Coronavirus (2019-nCoV) Antigen

[0238] 1. 2019-nCoV antigen fusion protein containing SEQ ID NO: 1 and protein tag (National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University) as the material for T gold-labeling was mixed with colloidal gold solution, added with the blocking solution to perform blocking to obtain gold-labeled solution. [0239] 2. The gold-labeled solution was added to the glass fiber, fully dried, packaged in an aluminum foil bag, and sealed by machine.

[0240] 4.2 Detection Method

[0241] At room temperature, 10 μL of serum or plasma sample to be tested was added to the sample hole of test strip, then 2 drops of sample diluent were added dropwise, the test strip was horizontally placed on table, and observed to obtain results 15 minutes after adding the sample.

[0242] The judgement of the detection results was shown in FIG. 1. A: When the control line developed color, and the test line also developed color, the result was judged as positive. B: When the control line developed color, while the test line did not develop color, the result was judged as negative. C: When the control line did not develop color, regardless of whether the test line developed color, the experiment was judged as invalid, and a repeated experiment was required.

[0243] 4.3 Evaluation of Detection Performance [0244] (1) The above detection kit was used as candidate reagent to detect the total antibody against of 2019-nCoV in plasma samples from 49 confirmed cases and 1 negative case in Hubei Province.

[0245] Among the 49 confirmed cases, 1 plasma sample was collected from each of 44 cases respectively, and 2 plasma samples of two different periods were collected from each of remaining 5 cases respectively, so that a total of 54 plasma samples from confirmed cases were collected in this experiment. The candidate reagent detected 48 positive cases, and the detection rate of the candidate reagent compared with the confirmed cases was 97.96% (48/49). For the 2 samples of two different periods from 1 case subject, the candidate reagent detected 1 negative and 1 positive, and this result was determined as a positive result of the candidate reagent. The negative sample included 1 negative sample from 1 negative case, and its detection result of the candidate reagent was negative. [0246] (2) The above detection kit was used as candidate reagent to detect 2019-nCoV total antibody in 312 serum samples and 49 whole blood samples of different times after onset from 90 confirmed cases of novel coronavirus pneumonia, and 209 serum samples and 99 whole blood samples from 308 other cases in Zhejiang Province.

[0247] The detection results of the candidate reagent and the clinically confirmed/excluded results were analyzed by contingency table (four-fold table) for Kappa consistency test. The results showed that the sensitivity of the candidate reagent was 100.00% (95% confidence interval was 95.98% to 100.00%), the specificity was 96.75% (95% confidence interval was 94.11% to 98.43%), the accuracy was 97.49% (95% confidence interval was 95.43% to 98.79%), the Kappa value was 0.93, and the consistency strength was the strongest.

[0248] The detection results in (2) were analyzed according to different times after onset, and the results were shown in the following table.

TABLE-US-00005 TABLE 5 Detection results of samples of different times after onset Days Candidate reagent after Total number of detected Number of positive of Positive rate onset samples (samples) detection (samples) of detection 1 1 1 100.00% 2 4 2 50.00% 3 4 1 25.00% 4 9 4 44.44% 5 7 3 42.86% 6 12 8 66.67% 7 18 11 61.11% 8 12 7 58.33% 9 18 13 72.22% 10 22 20 90.91% 11 25 22 88.00% 12 22 20 90.91% 13 20 19 95.00% 14 21 20 95.24% 15 20 18 90.00% 16 15 15 100.00% 17 19 18 94.74% 18 19 19 100.00% 19 16 16 100.00% ≥20 77 76 98.70%

[0249] It could be seen from the detection results of samples of different times after onset that the detection rate of the candidate reagents in the early stage of onset (before day 7) was 40% to 60%, and with progression after onset, it exceeded 90% after 10 days of onset. The above results indicate that the detection of total antibody based on the colloidal gold method can also be used for the screening of 2019-nCoV infection.

[0250] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The whole of the present invention is given by the appended claims and any equivalents thereof.