A specific epitope polypeptide and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a specific epitope polypeptide and its application. The present invention provides a SARS-CoV-2 specific epitope polypeptide associated with neutralizing activity, and its amino acid sequence is shown in SEQ ID No.3. The present invention designs and constructs a SARS-CoV-2 polypeptide microarray, analyzes the IgG and IgM antibody responses induced by the SARS-CoV-2 inactivated vaccine, screens out a series of immune epitope polypeptides associated with the neutralizing activity of antibodies, and establishes a polypeptide indirect ELISA detection kit on this basis. The method has high sensitivity, good specificity, good repeatability and stability, and does not require a high-level biosafety laboratory. It is easy to operate and the result is objective. It can achieve the purpose of quickly evaluating the level of new crown vaccine antibodies, and provides new means and directions for new crown antibody detection methods.

Description

[0001] This invention patent application is a divisional application based on Chinese patent application No. 2024100313283, whose invention name is "A specific epitope polypeptide and a new coronavirus antibody detection method and kit" filed on January 9, 2024. Technical Field

[0002] The present invention relates to the field of biomedical technology, and in particular to a specific epitope polypeptide and application thereof. Background Art

[0003] The novel coronavirus infection (COVID-19) poses a serious threat to human health. The virus that causes this disease is called SARS-CoV-2, which is an enveloped positive-strand RNA novel coronavirus that encodes spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, 16 non-structural proteins and 5-8 accessory proteins. Symptoms of SARS-CoV-2 infection include cough, headache, dyspnea, myalgia, fever and severe pneumonia.

[0004] At present, in the evaluation of the effectiveness of the new crown vaccine, the protective efficacy of the vaccine, especially the level of neutralizing antibodies against different new crown variants, is the most critical evaluation indicator. Among the existing methods for detecting neutralizing antibodies against the new coronavirus, the "gold standard" is the plaque reduction neutralization test, which is to infect cells with real viruses, and then observe whether the serum of the vaccine recipients or recovered patients can block the binding of the virus to the cell receptors. However, the real virus neutralization test has very strict requirements on conditions and high biosafety risks. It can only be carried out in laboratories with biosafety level 3 (BSL-3) and above, and it is very time-consuming, usually taking 2 to 4 days to complete. Another method is a neutralization test based on pseudoviruses, that is, a protein of a virus such as varicella virus or herpes virus is replaced with the spike protein (S protein) of the new coronavirus for neutralization test. It can be operated in a biosafety level 2 laboratory (BSL-2), but it still requires the use of viruses and cells, usually takes 3 days to complete, which is very unfavorable for large-scale testing and evaluation.

[0005] Therefore, it is urgent to establish a method for detecting SARS-CoV-2 neutralizing antibodies that can respond quickly, is simple and easy to use, and is suitable for large-scale applications, so as to quickly and effectively assess the level of herd immunity and provide support for scientific data on the protective efficacy of the new crown vaccine. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a specific epitope polypeptide and its application.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a SARS-CoV-2 specific epitope polypeptide associated with neutralization activity, the amino acid sequence of which is shown in any one of SEQ ID No.1-5.

[0009] The present invention designs and constructs a SARS-CoV-2 polypeptide microarray, analyzes the IgG and IgM antibody responses induced by the SARS-CoV-2 inactivated vaccine, and screens out a series of immune epitope polypeptides related to the neutralizing activity of antibodies, which has a higher accuracy in judging the neutralization positivity after vaccine immunization.

[0010] In a second aspect, the present invention provides a SARS-CoV-2 specific antigen, comprising a polypeptide having a sequence as shown in at least one of SEQ ID No. 1-5.

[0011] As a preferred embodiment of the SARS-CoV-2 specific antigen described in the present invention, it includes polypeptides with sequences shown in SEQ ID No.1 and SEQ ID No.5.

[0012] As a preferred embodiment of the SARS-CoV-2 specific antigen described in the present invention, it includes polypeptides with sequences shown as SEQ ID No. 2 and SEQ ID No. 4.

[0013] As a preferred embodiment of the SARS-CoV-2 specific antigen described in the present invention, it includes a polypeptide with a sequence as shown in SEQ ID No.1-5.

[0014] In a third aspect, the present invention provides a novel coronavirus antibody detection kit coated with the specific antigen.

[0015] As a preferred embodiment of the kit described in the present invention, the kit is an ELISA kit.

[0016] In a fourth aspect, the present invention provides a method for detecting antibodies against the new coronavirus, wherein a polypeptide having an amino acid sequence as shown in any one of SEQ ID No. 1-5 is subjected to indirect ELISA detection.

[0017] As a preferred embodiment of the detection method of the present invention, the indirect ELISA detection is: combining the SARS-CoV-2 antibody in the serum to be tested with the polypeptide fixed on the ELISA plate, washing, adding an enzyme-labeled secondary antibody; adding a substrate that reacts with the enzyme to develop color, and then detecting the color according to the OD value of the microplate reader. 450 The value determines the antibody content in the sample.

[0018] As a preferred embodiment of the detection method described in the present invention, the indirect ELISA detection is as follows: take the polypeptide antigen to coat the enzyme-labeled plate and incubate; discard the coating solution, wash the plate, add the blocking solution to block; wash the plate, add the serum to be tested, and incubate; wash the plate, add the enzyme-labeled secondary antibody, and incubate; wash the plate, add the color developing solution, and terminate the reaction after color development.

[0019] Further preferably, the blocking solution is PBS buffer containing BSA.

[0020] More preferably, the enzyme-labeled secondary antibody is an HRP-IgG antibody.

[0021] Further preferably, the color developing solution is a sulfuric acid solution.

[0022] Further preferably, the conditions for the indirect ELISA detection are as follows: 2.0 μg / mL of the polypeptide antigen is coated on the ELISA plate, 100 μL per well, and incubated at 37°C for 2 h; the coating solution is discarded, the plate is washed 3 times with 1× washing solution, 200 μL of 5% BSA blocking solution is added, and the plate is blocked at 37°C for 2 h; the plate is washed 3 times with 1× washing solution, the serum to be tested is added, and the plate is incubated at 37°C for 30 min; the plate is washed 5 times with 1× washing solution, 100 μL of 1:1500 pre-diluted HRP-IgG antibody is added, and the plate is incubated at 37°C for 1 h; the plate is washed 5 times with 1× washing solution, 100 μL of TMB color developing solution is added to each well, and the reaction is protected from light at room temperature for 15 min. After color development, 50 μL of 2M H 2 SO 4 Terminate the reaction.

[0023] In the fifth aspect, the present invention applies the specific epitope polypeptide, the specific antigen, and the kit to the evaluation of the effectiveness of the new crown vaccine.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention designs and constructs a SARS-CoV-2 polypeptide microarray, analyzes the IgG and IgM antibody responses induced by the inactivated SARS-CoV-2 vaccine, and screens out a series of immune epitope polypeptides related to antibody neutralization activity, whose amino acid sequences are shown in any sequence of SEQ ID No. 1-5. On this basis, a polypeptide indirect ELISA detection kit is established. The method has high sensitivity, good specificity, good repeatability and stability, does not require a high-level biosafety laboratory, is easy to operate, and the results are objective. It can achieve the purpose of quickly evaluating the level of new crown vaccine antibodies, and provides new means and directions for new crown antibody detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the technical roadmap of the present invention.

[0027] Figure 2 The figure shows the coverage of linear peptides on the S, N and M proteins of SARS-CoV-2; the short lines in the figure indicate the positions of the peptide fragments in the protein.

[0028] Figure 3 IgG and IgM antibody responses for peptide microarrays; Figure 3 In the figure, A: IgG antibody response; B: IgM antibody response.

[0029] Figure 4 is the fluorescence intensity of the differential linear peptide antibody reaction; Figure 4 A: IgG antibody response; B: IgM antibody response. *Compared with the control group, P<0.05; #Compared with the neutralization negative group after immunization, P<0.05. The data in the figure are expressed as median (95% CI).

[0030] Figure 5 AUC value of the screened linear peptides related to neutralization activity.

[0031] Figure 6 shows the IgG response of five linear peptides (left) and their correlation with inhibition rate (right); including Figure 6-1 and Figure 6-2 ; In FIG6 , A: S544-573; B: S564-583; C: S574-603; D: S1106-1125; E: S1136-1165; the IgG log2 fluorescence values ​​in the figure are expressed as medians (95% CI).

[0032] Figure 7 The AUC of positive neutralization after vaccine immunization was determined by a combination of five linear peptides.

[0033] Figure 8 It is the critical value determination standard for the peptide indirect ELISA method.

[0034] Fig. 9 This is the sensitivity identification of the polypeptide indirect ELISA method in Example 4.

[0035] Fig.10 It is a peptide indirect ELISA method for specific identification analysis.

[0036] Fig.11 This is the sensitivity identification of the polypeptide indirect ELISA method in Example 5. DETAILED DESCRIPTION

[0037] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0039] Example 1: Screening of SARS-CoV-2 specific epitopes with neutralizing activity using peptide microarray technology 1. Screening and synthesis of SARS-CoV-2 linear epitope peptides

[0040] The epitope information of SARS-CoV-2 was collected in the IEBD database (http: / / www.iedb.org / ) to obtain B cell antigen epitopes.

[0041] Parameter selection: Check Epitope as "Linear Epitope", Host as "Human", and Disease as "Infectious Disease". Retrieve and record each record one by one. Use the epitope clustering analysis tool (http: / / tools.iedb.org / ) to cluster the sequences, and form linear peptides with a minimum sequence identity of 70%. Then perform peptide synthesis.

[0042] A total of 634 SARS-CoV-2 epitope information was obtained from the IEDB database. The sequences were clustered using the epitope clustering analysis tool (http: / / tools.iedb), and finally 243 linear peptides were formed. The median length of the peptide fragments is 18 amino acids (9-30 amino acids). The linear peptides cover almost the entire S protein (coverage rate 97.6%), N protein (coverage rate 95.0%), and M protein (coverage rate 94.2%). The number of peptides for each protein and the peptide coverage of S, M, and N proteins are shown in the figure below. Figure 2 shown.

[0043] 2. Preparation of peptide microarray

[0044] Each peptide in the peptide library is randomly compiled with a code (A001, A002...B001, B002...), so that peptides with adjacent sequences are randomly spotted at different positions on the slide to improve the detection accuracy. The peptides are spotted on the slide carrier using a spotter (Scienion AG, USA) and duplicate wells are set up. Multiple BSA solutions or PBS without peptides are set up as negative controls and blank controls; Cy3_Cy5 mixed solution, gradient dilution of biotin-labeled anti-human IgG and IgM (concentrations of 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.625ng / mL, 7.813ng / mL, 3.906ng / mL and 1.953ng / mL) are set up as positive controls. Each peptide microarray has a total of 2×8 identical subarrays. After the peptide microarray is spotted, it is stored at -80°C and sealed to keep the slide completely dry.

[0045] 3. Detection of peptide microarray

[0046] Peptide microarray detection was performed using 54 negative control samples and 74 serum samples after immunization with the inactivated COVID-19 vaccine (12 of which were negative for pseudovirus neutralizing antibodies and 62 were positive for pseudovirus neutralizing antibodies). The detection steps are as follows:

[0047] 1) Balance and dry: After the slide is balanced at room temperature for 20 to 30 minutes, remove the sealing strip and then place it in a vacuum dryer or dry it at room temperature for 1 to 2 hours;

[0048] 2) Blocking: Add 100 μL of 1× blocking solution to each subarray well and incubate on a shaker at room temperature for 1 h to avoid bubbles;

[0049] 3) Primary antibody (serum sample) incubation: remove the blocking solution, add 100 μL sample to each well, one sample per array, and incubate overnight at 4°C;

[0050] 4) Washing: Wash the slides using a plate washer in two steps. First, wash with 1× Wash Solution I, 250 μL per well, wash 10 times, and shake for 10 seconds each time. Then wash with 1× Wash Solution II, 250 μL per well, wash 6 times, and shake for 10 seconds each time.

[0051] 5) Secondary antibody incubation: Prepare Cy3 goat anti-human IgG and Cy5 AffiniPure donkey anti-human IgM antibodies and incubate at room temperature with shaking for 2 h;

[0052] 6) Cleaning: same as step 4);

[0053] 7) Fluorescence detection: The fluorescence signal was scanned using a laser scanner InnoScan 300. Scanning parameters: Cy3 wavelength 532nm; Cy5 wavelength 640nm, resolution 10μm;

[0054] 8) Fluorescence signal value analysis: GenePix Pro7 software was used to extract the fluorescence signal intensity and background difference.

[0055] 4. IgG and IgM antibody reaction detection of linear peptides

[0056] Extract the fluorescence signal intensity values ​​of IgG and IgM from the peptide microarray, subtract the background fluorescence, and perform homogenization between the peptide microarrays using negative and positive controls. The signal intensity of each peptide is the average of the duplicate wells. Figure 3 ) show the fluorescence values ​​of IgG and IgM antibody reactions of each sample.

[0057] In the IgG antibody reaction, in the neutralization-positive group after immunization, the fluorescence intensity of 25 linear polypeptides was significantly higher than that of the control group, and 3 was significantly lower than that of the control group; the fluorescence intensity of 29 linear polypeptides was significantly higher than that of the neutralization-negative group after immunization, and 5 was significantly lower than that of the neutralization-negative group after immunization. Among them, the fluorescence intensity of 20 polypeptides was higher than that of both the control group and the neutralization negative group after immunization, namely ORF1ab1699-1713, ORF1ab2086-2099, ORF1ab4214-4226, ORF1ab5587-5601, ORF1ab6000-6013, ORF1ab6030-6042, S55-68, S144-163, S174-193, S544-573, S564-583, S574-603, S791-815, S801-825, S987-1000, S1106-1125, S1136-1165, S1186-1205, M1-19, and ORF7a40-63. Figure 4 As shown in A.

[0058] In the IgM antibody reaction, in the neutralization positive group after immunization, the fluorescence intensity of 14 linear peptides was significantly higher than that of the control group, and 9 were significantly lower than that of the control group; the fluorescence intensity of 8 linear peptides was significantly higher than that of the neutralization negative group after immunization, and 7 were significantly lower than that of the neutralization negative group after immunization. Among them, the fluorescence intensity of 4 peptides was higher than that of both the control group and the neutralization negative group after immunization, namely ORF1ab5587-5601, S574-603, S1136-1165 and S1186-1205. Figure 4 As shown in B.

[0059] 5. Screening of linear peptides with neutralizing activity

[0060] ROC was used to distinguish linear peptides related to neutralization activity, and linear peptides with AUC>0.7 were screened for further analysis. This example uses the IgG antibody reaction intensity for analysis, mainly because the IgM antibody reaction decays faster after immunization, while the IgG antibody reaction is more persistent; and in patients recovering from SARS-CoV-2 infection, there is a strong correlation between the true virus micro-neutralization titer and the anti-RBD IgG level of recovered patients (R 2 =0.8009), while the correlation with anti-RBD IgM / IgA titer was weaker than that with IgG.

[0061] The ROC curve results showed that there were 5 linear peptides with AUC>0.7 on the S protein, namely S544-573 (AUC0.761, P<0.001), S564-583 (AUC 0.783, P<0.001), S574-603 (AUC 0.810, P<0.001), S1106-1125 (AUC 0.789, P<0.001) and S1136-1165 (AUC 0.749, P<0.001). The specific results are shown in Tables 1 and Figure 5 As shown in the figure, no target linear peptide was screened out on ORF, M, N, and E proteins.

[0062] Table 1 ROC curve for judging linear peptides related to neutralization activity

[0063]

[0064]

[0065]

[0066]

[0067] The fluorescence intensity values ​​of the five linear polypeptides of the S protein were log2 transformed, and the inter-group differences in fluorescence intensity of the three groups were compared. The results showed that there was no statistical difference in the polypeptides between the negative control group and the neutralization negative group after vaccine immunization (P>0.05); but the fluorescence intensity values ​​of the S544-573, S564-583, S574-603, S1106-1125 and S1136-1165 polypeptides in the neutralization positive group after vaccine immunization were significantly higher than those in the neutralization negative group after vaccine immunization (P values ​​were 0.016, 0.003, 0.001, 0.007 and 0.020, respectively) and the negative control group (P<0.001), as shown in Table 2.

[0068] Table 2 Fluorescence intensity of three groups of IgG antibodies binding to linear peptides

[0069]

[0070] Note: Data are medians (upper and lower quartiles). * Comparison between the neutralization positive group and the negative control group after vaccination; Comparison between the neutralization positive group after vaccine immunization and the neutralization negative group after vaccine immunization.

[0071] In addition, linear correlation analysis was performed between the fluorescence intensity (log2 conversion value) of each linear peptide and the inhibition rate. The results showed that the correlation coefficients (r) between S544-573, S564-583, S574-603, S1106-1125 and S1136-1165 and the inhibition rate were 0.582, 0.522, 0.643, 0.568 and 0.546, respectively, with moderate linear correlation (P < 0.001, as shown in Figure 6).

[0072] The ROC curve was used to analyze the value of peptide combinations in judging positive neutralization after vaccine immunization. When S544-573, S564-583, S574-603, S1106-1125 and S1136-1165 were used in combination, the AUC was 0.861 (0.794-0.929), and the sensitivity and specificity were both 84.4%, indicating that the combination of linear peptides has a higher accuracy in judging positive neutralization after vaccine immunization (such as Figure 7 shown).

[0073] Table 3 Peptide combination sequence

[0074] Peptide naming Peptides sequence ID Number A1 S544-573 NGLTGTGVLTESNKKFLPFQQFGRDIADTT SEQ ID No.1 A2 S564-583 QFGRDIADTTDAVRDPQTLE SEQ ID No.2 A3 S574-603 DAVRDPQTLEILDITPCSFGGVSVITPGTN SEQ ID No.3 A4 S1106-1125 RNFYEPQIITTDNTFVSGN SEQ ID No.4 A5 S1136-1165 TVYDPLQPELDSFKEELDKYFKNHT SPDVD SEQ ID No.5

[0075] Example 2: Establishment of IgG antibody detection method using different peptide combinations

[0076] Different combinations of peptides were used to perform indirect ELISA to detect antibodies against the novel coronavirus. Considering the diagnostic efficacy, conservatism, and P / N values ​​of peptides A1, A2, A3, A4, and A5, the P / N values ​​of different peptides and their combinations are shown in Table 4. The combination of peptides A2 and A4 had the highest P / N value, reaching 9.879.

[0077] Table 4 Best selection of different peptides and combinations

[0078] Peptide combination P N P / N A1 1.318 0.793 1.660 A2 1.369 0.513 2.668 A3 1.577 0.900 1.753 A4 1.464 0.435 3.367 A5 1.658 0.426 3.894 A1-A3 1.213 0.497 2.441 A1-A4 1.158 0.528 2.195 A1-A5 1.621 0.187 8.666 A2-A4 1.814 0.184 9.879 A2-A5 1.663 0.925 1.799 A3-A4 1.383 0.378 3.661 A3-A5 1.092 0.711 1.536 A4-A5 1.647 0.415 3.972 A1-A3-A4 1.444 0.509 2.838 A2-A4-A5 1.828 0.903 2.024 A3-A4-A5 1.790 0.360 4.972 A1-A3-A4-A5 1.435 0.546 2.628

[0079] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value.

[0080] Example 3: Establishment of a peptide indirect ELISA method for detecting antibodies against the new coronavirus

[0081] The combined polypeptides A2-A4 in Example 2 were diluted to 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL and 4.0 μg / mL as coating antigens, respectively. The serum to be tested was diluted at 1:50, 1:100, 1:200 and 1:400. The polypeptide coating concentration and serum dilution concentration in the polypeptide indirect ELISA method were determined by the square matrix method. The P / N values ​​were compared, and the group with the highest P / N value was selected as the optimal working concentration. The results are shown in Table 5. The optimal working concentration combination of the specific polypeptide indirect ELISA method was 2.0 μg / mL when the polypeptide was 2.0 μg / mL and the serum was diluted 1:50. The P / N measurement value was the highest, which was 20.117.

[0082] Table 5 Optimal selection of different coating concentrations and serum dilution concentrations

[0083]

[0084] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value

[0085] 2. Optimization of blocking solution composition and blocking time

[0086] In order to determine the most suitable blocking solution composition and blocking time, based on the above optimized coating antigen and serum dilution concentration, different blocking conditions were set for testing, the P / N values ​​were compared, and the group with the highest P / N value was selected as the most suitable blocking parameter. The results are shown in Table 6. The most suitable blocking parameter was set at 37°C and 5% BSA blocking for 2h.

[0087] Table 6 Best choice for different blocking solution components and blocking time optimization

[0088]

[0089]

[0090] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value

[0091] 3. Optimization of serum incubation time

[0092] In order to determine the optimal incubation time of the sample to be tested, based on the above optimized conditions, the serum to be tested was incubated for 30min, 45min, 60min and 90min respectively, and then the P / N values ​​were compared, and the group with the highest P / N value was selected as the best optimized condition. The results are shown in Table 7, and the incubation time of the serum to be tested should be set to 30min. The test results show that the serum incubation time is 30min, and the maximum P / N value is 15.828 at this time. Therefore, the optimal serum incubation time is determined to be 30min.

[0093] Table 7 Selection of incubation time for serum to be tested

[0094] Different serum incubation time P N P / N 30min 1.567 0.099 15.828 45min 1.856 0.123 15.089 60min 1.883 0.129 14.597 90min 1.883 0.216 8.718

[0095] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value

[0096] 4. Optimal dilution of HRP-IgG antibody

[0097] In order to determine the optimal dilution concentration and incubation time of enzyme-labeled IgG antibody, based on the above optimized conditions, the setting conditions of enzyme-labeled antibody are shown in Tables 8 and 9, and the P / N values ​​are compared, and the group with the highest P / N value is selected as the best optimization condition. The results show that when the dilution of enzyme-labeled antibody is 1:500 and the incubation time is set to 60min, the P / N value is the largest, which is 14.683 and 16.366 respectively.

[0098] Table 8 OD values ​​of different HRP-IgG antibody release times

[0099] Dilution multiple P N P / N 1:500 1.857 0.126 14.683 1:1000 1.451 0.158 9.205 1:2000 1.581 0.190 8.336 1:4000 1.405 0.153 9.157

[0100] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value

[0101] Table 9 OD values ​​of different HRP-IgG antibody incubation times

[0102] Antibody incubation time P N P / N 30min 1.430 0.104 13.750 60min 1.653 0.101 16.366 90min 1.647 0.113 14.575 120min 1.751 0.145 12.076

[0103] Note: P is positive serum OD 450 Measured value; N is negative serum OD 450 Measured value

[0104] 5. TMB color development time

[0105] In order to determine the optimal color development time of TMB substrate, based on the above optimized conditions, TMB was protected from light for 5min, 10min, 15min and 20min, respectively, and then tested, the P / N values ​​were compared, and the group with the highest P / N value was selected as the optimal optimization condition. The results are shown in Table 10, and the optimal color development time of TMB should be set to 15min.

[0106] Table 10 OD values ​​of different TMB color development time

[0107] Color development time (min) P N P / N 5min 0.678 0.084 8.071 10min 0.956 0.079 12.101 15min 1.897 0.124 15.298 20min 2.152 0.338 6.367

[0108] Note: P is positive serum OD 450 Measured value; N is negative serum OD450 Measured value

[0109] After exploring the above working conditions, the optimal working conditions of the kit of Example 2 were finally set as follows: 2.0 μg / mL polypeptide (A2-A4) antigen coated ELISA plate, 100 μL per well, incubated at 37°C for 2 h. Discard the coating solution (pH 9.6 0.05 M carbonate buffer), 1× washing solution (KH 2 PO 4 4g, Na 2 HPO 4 12H 2 O58g, NaCl 160g, KCl 4g, Tween-20 10mL add water to 20L) wash the plate 3 times, add 200μL 5% BSA blocking solution, block at 37℃ for 2h. Wash the plate 3 times with 1× washing solution, add the serum to be tested, incubate at 37℃ for 30min. Wash the plate 5 times with 1× washing solution, add 100μL 1:1500 pre-diluted HRP-IgG antibody, incubate at 37℃ for 1h. Wash the plate 5 times with 1× washing solution, add 100μL TMB colorimetric solution (P0209, Biyuntian) to each well, react at room temperature in the dark for 15min, add 50μL 2M H 2 SO 4 Terminate the reaction.

[0110] Example 4: Determination of the method for detecting novel coronavirus antibodies using peptide indirect ELISA

[0111] 1. Determination of positive and negative critical values ​​by peptide indirect ELISA method

[0112] Using the optimal working conditions finally optimized in Example 2, the combined polypeptides A2-A4 in Example 2 were used to establish a polypeptide indirect ELISA method to detect 50 serum samples that were determined to be negative in the new coronavirus pseudovirus neutralizing antibody detection experiment (30 of them were not immunized with the new coronavirus vaccine, and 20 were immunized with the new coronavirus vaccine but had negative neutralizing antibodies).

[0113] The results are as follows Figure 8 As shown in Figure 2, the Cut-off value is calculated according to the following formula: Cut-off = OD of negative sample 450 Mean value + 2 × standard deviation. OD of 50 COVID-19 negative serum samples 450 The average value is 0.148, and the 2× standard deviation is 0.194, that is, the cut-off value of the peptide indirect ELISA method established in this study is 0.352. The judgment criteria are as follows: when the OD 450 When the value is ≥0.352, it is judged as a positive sample for the new crown neutralizing antibody; the sample OD 450 When the value is <0.352, it is judged as a negative sample for the new coronavirus neutralizing antibody.

[0114] 2. Sensitivity of peptide indirect ELISA

[0115] The optimized peptide indirect ELISA method was used to detect serum samples positive for the new coronavirus neutralizing antibody. The samples were diluted in a two-fold gradient of 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200. The results are as follows Fig. 9 As shown, when the positive serum of the new coronavirus neutralizing antibody was diluted to 1:800, OD 450 The measured value was still higher than the Cut-off value and was judged to be positive, indicating that the peptide indirect ELISA method had a high sensitivity.

[0116] 3. Analysis of specificity test results of peptide indirect ELISA method

[0117] The optimized peptide indirect ELISA method was used to detect influenza positive sera that may have potential cross-reactions. The positive serum samples of the new coronavirus neutralizing antibody and the healthy physical examination sera were used as positive and negative controls. The results are as follows Fig.10 As shown, influenza-positive sera were all judged as negative according to the Cut-off value, with no cross-reaction and high specificity.

[0118] 4. Analysis of the accuracy test results of peptide indirect ELISA method

[0119] Three positive samples were selected for verification. The same experimenter performed three repeated experiments in the same batch. The results are shown in Table 11. The CV range of the intra-batch repeatability test was 5% to 8%, and the CV was less than 10%, indicating that the established polypeptide indirect ELISA method has good repeatability and stability.

[0120] Table 11 Accuracy test of peptide indirect ELISA method

[0121]

[0122]

[0123] Example 5: Establishment of a peptide indirect ELISA method for detecting antibodies against the new coronavirus

[0124] The combined polypeptides A1-A5 in Example 2 were taken, and the serum samples positive for the new coronavirus neutralizing antibodies were detected according to the optimal working conditions determined in Example 3, and the cut-off (0.352) determined in Example 5. The samples were diluted in a two-fold gradient of 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200. The results are as follows: Fig.10 As shown, when the positive serum of the new coronavirus neutralizing antibody was diluted to 1:400, OD 450 If the measured value is higher than the cut-off value, it is judged as positive, and the sensitivity is good. Fig.11 shown.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A SARS-CoV-2 specific epitope polypeptide associated with neutralization activity, characterized in that: Its amino acid sequence is shown in SEQ ID No.

3.

2. A method for detecting SARS-CoV-2 antibodies for non-disease diagnosis or treatment purposes, characterized in that: The polypeptide with the amino acid sequence shown in SEQ ID No. 3 was used for indirect ELISA detection.

3. The detection method according to claim 2, characterized in that: The indirect ELISA test is as follows: the SARS-CoV-2 antibody in the serum to be tested is combined with the polypeptide fixed on the ELISA plate, washed, and an enzyme-labeled secondary antibody is added; a substrate that reacts with the enzyme is added and then color is developed, and the color is displayed according to the OD value of the microplate reader. 450 The value determines the antibody content in the sample.

4. The detection method according to claim 3, characterized in that: The indirect ELISA detection is as follows: take the polypeptide to coat the enzyme-labeled plate and incubate; discard the coating solution, wash the plate, add the blocking solution to block; wash the plate, add the serum to be tested, and incubate; wash the plate, add the enzyme-labeled secondary antibody, and incubate; wash the plate, add the color developing solution, and terminate the reaction after color development.

5. The detection method according to claim 4, characterized in that: The conditions for the indirect ELISA detection are as follows: 2.0 μg / mL of the polypeptide is coated on the ELISA plate, 100 μL per well, and incubated at 37°C for 2 h; the coating solution is discarded, the plate is washed 3 times with 1× washing solution, 200 μL of 5% BSA blocking solution is added, and the plate is blocked at 37°C for 2 h; the plate is washed 3 times with 1× washing solution, the serum to be tested is added, and the plate is incubated at 37°C for 30 min; the plate is washed 5 times with 1× washing solution, 100 μL of 1:1500 pre-diluted HRP-IgG antibody is added, and the plate is incubated at 37°C for 1 h; the plate is washed 5 times with 1× washing solution, 100 μL of TMB color developing solution is added to each well, the reaction is carried out in the dark at room temperature for 15 min, and 50 μL of 2M H2SO4 is added after color development to terminate the reaction.

6. Use of the specific epitope polypeptide according to claim 1 in preparing a kit for evaluating the effectiveness of a SARS-CoV-2 vaccine.

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