CN113945714A

CN113945714A - Method for detecting neutralizing capacity of novel coronavirus neutralizing antibody drugs

Info

Publication number: CN113945714A
Application number: CN202110804532.0A
Authority: CN
Inventors: 王立豪; 贾文双; 魏照阳; 郭剑鹏
Original assignee: Nanjing Vigorous Biotechnology Co ltd
Current assignee: Nanjing Vigorous Biotechnology Co ltd
Priority date: 2020-07-16
Filing date: 2021-07-16
Publication date: 2022-01-18
Anticipated expiration: 2041-07-16
Also published as: CN113945714B

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for detecting the neutralizing capacity of a novel coronavirus neutralizing antibody drug. The invention provides a method for detecting the ability of neutralizing a novel coronavirus in a sample, which is characterized in that a Cell-based ELISA experiment is carried out by using a novel coronavirus S protein ECD, a sample mixture and a HeLa Cell line which overexpresses human ACE2, the novel coronavirus is combined with a host Cell membrane surface receptor under the condition of reducing the physiological condition to the maximum extent, the biological activity of the novel coronavirus and the novel coronavirus combined receptor in the sample can be more accurately reflected, the use of the virus is avoided, the safety of the experiment is greatly improved, and the complexity and the period of the experiment are reduced; the method can also carry out quantitative analysis on the result by strictly controlling various variables in the implementation process of the detection method, thereby ensuring the stability of the result of the evaluation of the neutralizing capacity of the antibody drugs.

Description

Method for detecting neutralizing capacity of novel coronavirus neutralizing antibody drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly detecting neutralizing capacity of a novel coronavirus neutralizing antibody drug.

Background

According to technical data request (pharmacy) (request for comments) of novel coronavirus neutralizing antibody drugs issued by the drug evaluation Center (CDE) of the Chinese State drug administration^[1]The current detection method for biological activity of novel coronavirus neutralizing antibody drugs in vitro comprises the following steps: specific binding activity with novel coronavirus related proteins, ACE2 competitive binding activity, novel coronavirus/pseudovirus neutralizing or blocking biological activity, etc. In particular, the "evaluation of the neutralizing ability and specificity of the neutralizing antibody against the novel coronavirus" is important in the molecular screening stage, and it is obvious that the test for the specific binding activity of the neutralizing antibody against the novel coronavirus (e.g., receptor binding domain of S protein, hereinafter abbreviated as RBD) does not effectively reflect the neutralizing ability of the neutralizing antibody against the novel coronavirus under physiological conditions. The detection of the neutralizing activity of the novel coronavirus inevitably requires the use of the novel coronavirus in a living body, experimental operation must be carried out in a P3(Protection 3) laboratory which is filed by the national health and care Commission, and strict safety operation training must be carried out on laboratory personnel, so that only a few laboratories can obtain corresponding qualifications, and the novel coronavirus can not be popularized in a large range. Even a method for detecting a neutralizing antibody against a pseudovirus^[2]It is also required to perform experimental operations in the P2(Protection 2) laboratory, which usually lasts for more than one week, and the stability of the method is limited due to the difficulty in keeping the activity of the prepared virus consistent, and it is difficult to use the method as a quality release method in the drug production and quality control (CMC) stages.

According to the research of the literature, only angiotensin converting enzyme 2(ACE2) competition binding activity detection experiments can meet the requirements of a quality release method for neutralizing activity of novel coronavirus neutralizing antibody drugs, the experimental period is short, a special P2 laboratory is not needed, and the application range is wide. However, there is no corresponding method for accurately and stably measuring the neutralizing activity of the neutralizing antibody quantitatively, so that a stable test method for detecting the competitive binding activity of ACE2 is required to be developed for quality-release detection of the CMC stage of antibody drugs.

In summary, no report of a relevant detection method for neutralizing activity is found at present, and the reproducibility of physiological conditions and the stability of the method can be considered while the requirements of easy operation, high flux and rapid detection of an experiment are met.

Disclosure of Invention

In order to solve the problems, a rapid detection method for evaluating the neutralizing capacity of novel coronavirus neutralizing antibody drugs is established through an ELISA experiment based on HeLa cells and cells over-expressing ACE 2.

In one aspect, the present invention provides a method for detecting the ability to neutralize a novel coronavirus in a sample, said method comprising the steps of:

(1) contacting and incubating a sample with an ACE 2-overexpressing cell line with a marker-linked novel coronavirus spike protein (S) extracellular domain (ECD);

(2) obtaining the cells incubated in step (1), and incubating the cells in contact with a detection substance capable of reacting with the label to generate a detection signal;

(3) determining the ability of the sample to neutralize the novel coronavirus by detecting the intensity of the signal generated in step (2).

In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 80% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 90% identical to the amino acid sequence set forth in seq id No. 1 or 4. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 95% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 96% identical to the amino acid sequence set forth in seq id No. 1 or 4. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 97% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 98% identical to the amino acid sequence shown in seq id no. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 99% identity to the amino acid sequence set forth in seq id No. 1 or 4. In some embodiments, the novel coronavirus S protein ECD comprises SEQ ID NO:1 or 4. In a specific embodiment, the amino acid sequence of the ECD protein of the novel coronavirus S protein is shown in

SEQ ID NO

1 or 4.

In some embodiments, the concentration of the marker-linked novel coronavirus S protein ECD added in step (1) is 0.8-3.2. mu.g/mL, preferably 1.2-2.0. mu.g/mL. In other embodiments, the novel coronavirus S protein ECD concentration is 0.8. mu.g/mL, 0.9. mu.g/mL, 1.0. mu.g/mL, 1.1. mu.g/mL, 1.2. mu.g/mL, 1.3. mu.g/mL, 1.4. mu.g/mL, 1.5. mu.g/mL, 1.6. mu.g/mL, 1.7. mu.g/mL, 1.8. mu.g/mL, 1.9. mu.g/mL, 2.0. mu.g/mL, 2.1. mu.g/mL, 2.2. mu.g/mL, 2.3. mu.g/mL, 2.4. mu.g/mL, 2.5. mu.g/mL, 2.6. mu.g/mL, 2.7. mu.g/mL, 2.8. mu.g/mL, 2.9. mu.g/mL, 3.0. mu.g/mL, 3.1. mu.g/mL, or 3.2. mu.g/mL. In some preferred embodiments, the ECD concentration of the novel coronavirus S protein is 1.2. mu.g/mL, 1.3. mu.g/mL, 1.4. mu.g/mL, 1.5. mu.g/mL, 1.6. mu.g/mL, 1.7. mu.g/mL, 1.8. mu.g/mL, 1.9. mu.g/mL, or 2.0. mu.g/mL. In a specific embodiment, the ECD concentration of the novel coronavirus S protein added in step (1) is 1.6. mu.g/mL.

In some embodiments, the tag in step (1) is selected from one or more of a His-tag, a Flag-tag, a c-Myc-tag, an HA-tag, a V5-tag, an HSV-tag, and biotin, preferably a His-tag. In some specific embodiments, the tag in step (1) is a His tag and a Flag tag.

In some embodiments, the ACE2 overexpressing cell line in step (1) is selected from HEK293, HeLa, Vero E6 or CHO, preferably an ACE2 overexpressing HeLa cell line. In some specific embodiments, the ACE 2-overexpressed cell line in step (1) is an ACE 2-overexpressed HeLa cell line.

In some embodiments, the ACE2 overexpressing cell line added has a cell concentration of 4 x 10⁵-1ⅹ10⁶Single cell/mL, preferably 5 x 10⁵Individual cells/mL. In other embodiments, the ACE2 overexpressing cell line added has a cell concentration of 4 x 10⁵、5ⅹ10⁵、6ⅹ10⁵、7ⅹ10⁵、8ⅹ10⁵、9ⅹ10⁵Or 1 x 10⁶Individual cells/mL. In a specific embodiment, the ACE2 overexpressing cell line was added at a cell concentration of 5 x 10⁵Individual cells/mL.

In some embodiments, the contact incubation in step (1) comprises incubation at 18 ℃ -37 ℃ for 0.5-3 hours, preferably at room temperature for 1-2 hours. In some specific embodiments, the contact incubation in step (1) comprises incubation at room temperature for 1-2 hours.

In some embodiments, the obtaining of the post-incubation cells of step (1) in step (2) comprises removing the post-incubation cell supernatant and washing the cells. The post-incubation cell supernatant and washing cell steps include removing the cell supernatant from the contact incubation of step (1), adding a laboratory buffer for washing, and removing the laboratory buffer. Wherein the laboratory buffer is a PBS solution containing 1% FBS.

In some embodiments, the detection substance in step (2) is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, or an acridinium compound-linked anti-marker antibody. In other embodiments, the substance detected in step (2) is selected from horseradish peroxidase-linked streptavidin (recognition biotin). The anti-marker antibody of the present invention is selected from anti-tag protein antibodies, such as anti-His tag antibody, anti-Flag tag antibody, anti-c-Myc tag antibody, anti-HA tag antibody, anti-V5 tag antibody or anti-HSV tag antibody.

In some embodiments, the tag comprises a His-tag and the detection substance comprises a horseradish peroxidase-linked anti-His-tag antibody.

In some embodiments, the contact incubation in step (2) comprises incubation at 18 ℃ -37 ℃ for 0.5-1.5 hours, preferably at room temperature for 1 hour. In some embodiments, the contacting incubation in step (2) comprises incubation at room temperature for 1 hour.

In some embodiments, the step (3) of detecting the signal comprises removing the supernatant of the cells after the incubation in the step (2), washing the cells, adding a chromogenic working solution for incubation, adding a chromogenic stop solution, and reading the signal value.

In some embodiments, the chromogenic working solution comprises a TMB peroxidase substrate and a peroxidase substrate solution B, and the chromogenic stop solution comprises a phosphoric acid solution.

In some embodiments, the sample is an anti-novel coronavirus antibody based drug. In some embodiments, the sample is a fusion protein or an antibody against a novel coronavirus. In a specific embodiment, the sample is an ACE2-Fc fusion protein. In another specific embodiment, the sample is an anti-novel coronavirus monoclonal antibody, an antibody fragment, or a bispecific antibody. Referring to technical data requirements (pharmacy) of novel coronavirus neutralizing antibody drugs (request for comments) published by the drug evaluation center of the State drug administration, the novel coronavirus neutralizing antibody drugs provided by the invention include, but are not limited to, monoclonal antibodies, antibody fragments, Fc fusion proteins, bispecific antibodies and the like, such as ACE2-Fc fusion proteins. The sample of the present invention can be derived from biologically prepared antibody or fusion protein against the novel coronavirus, or serum, plasma or whole blood obtained from a subject or animal (such as mouse, rabbit, etc.) immunized with the novel coronavirus.

In some embodiments of the invention, step (1) comprises contacting a sample containing 0.8-3.2. mu.g/mL of the novel coronavirus S protein ECD containing a marker with a concentration of 4 x 10⁵-1ⅹ10⁶cells/mL ACE 2-overexpressed cells were incubated at 18 ℃ to 37 ℃ for 0.5 to 3 hours.

In some embodiments of the invention, said step (2) comprises removing the cell culture supernatant of step (1), washing the cells, and incubating said cells with an anti-marker antibody conjugated to horseradish peroxidase for 0.5-1.5 hours at 18 ℃ -37 ℃.

In some embodiments of the invention, step (3) comprises removing the supernatant of the cells after incubation in step (2), washing the cells, adding TMB peroxidase substrate and peroxidase substrate solution B, incubating, adding a phosphate solution, and reading the signal.

In the step (1) of the present invention, the marker-linked novel coronavirus S protein ECD and the sample mixture may be incubated with ACE 2-overexpressed cells, or the sample may be added to be incubated with ACE 2-overexpressed cells, and then the marker-linked novel coronavirus S protein ECD may be added to be incubated.

In some embodiments of the invention, the method comprises: step (1) of mixing 0.8-3.2. mu.g/mL of the novel coronavirus S protein ECD containing a marker, samples and concentration of 4 x 10⁵-1ⅹ10⁶Each cell/mL of ACE2 overexpressed cells, and the cells are in contact culture at 18-37 ℃ for 0.5-3 hours; removing the cell culture supernatant in the step (1), washing the cells, and incubating the cells with an anti-marker antibody conjugated with horseradish peroxidase at 18-37 ℃ for 0.5-1.5 hours; and (3) removing the supernatant of the cells after the incubation in the step (2), washing the cells, adding a chromogenic substance, and reading the signal value.

In other embodiments of the present invention, the method comprises: step (1) of mixing 1.2-2.0. mu.g/mL of the novel His-tagged coronavirus S protein ECD, sample and concentration of 4 x 10⁵-1ⅹ10⁶Each cell/mL of HeLa cell over-expressed with ACE2, and culturing at 18-37 deg.C for 0.5-3 hr; step (2) removing the cell culture supernatant of step (1), washing the cells, and contacting the cells with horseradish peroxidase-conjugated anti-His tag antibodyIncubating at 18-37 deg.C for 0.5-1.5 hr; and (3) removing the supernatant of the cells after the incubation in the step (2), washing the cells, adding a TMB peroxidase substrate and a peroxidase substrate solution B for incubation, incubating for 10-30 minutes at 18-37 ℃, adding a phosphoric acid solution, and reading a signal value.

In a specific embodiment of the invention, the method comprises: step (1) 1.6. mu.g/mL of the novel His-tag-containing coronavirus S protein ECD and sample mixture were mixed at a concentration of 5 x 10⁵Each cell/mL of HeLa cell over-expressed with ACE2, and culturing at room temperature for 1-2 hr; removing the cell culture supernatant of step (1), washing the cells, and incubating the cells with horseradish peroxidase-conjugated anti-His tag antibody at room temperature for 1 hour; and (3) removing the supernatant of the cells after the incubation in the step (2), washing the cells, adding a TMB peroxidase substrate and a peroxidase substrate solution B for incubation, incubating at room temperature for 10-30 minutes, adding a phosphoric acid solution, and reading a signal value.

In another aspect, the present invention provides a kit for detecting the ability of neutralizing a novel coronavirus in a sample, wherein the kit comprises a marker-linked cell line in which the novel coronavirus S protein ECD and ACE2 are overexpressed, and a detection substance capable of reacting with the marker to generate a signal, wherein the ACE2 overexpressed cell line is an adherent cell.

In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence of at least 80% of the amino acid sequence set forth in seq id no. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 90% identical to the amino acid sequence set forth in seq id No. 1 or 4. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 95% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 96% identical to the amino acid sequence set forth in seq id No. 1 or 4. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 97% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, which is at least 98% identical to the amino acid sequence shown in seq id no. In some embodiments, the novel coronavirus S protein ECD comprises a sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 99% identity to the amino acid sequence set forth in seq id No. 1 or 4. In other embodiments, the novel coronavirus S protein ECD comprises SEQ ID NO:1 or 4. In a specific embodiment, the amino acid sequence of the ECD protein of the novel coronavirus S protein is shown in

SEQ ID NO

1 or 4.

In some embodiments, the tag comprises a His-tag and the detection substance comprises an anti-His-tag antibody linked to horseradish peroxidase, alkaline phosphatase, or an acridinium compound.

In some embodiments, the ACE2 overexpressing cell line has a cell concentration of 4 x 10⁵-1ⅹ10⁶Single cell/mL, preferably 5 x 10⁵Individual cells/mL.

In some embodiments, the ACE2 overexpressing cell line is selected from HEK293, HeLa, Vero E6 or CHO, preferably an ACE2 overexpressing HeLa cell.

In some embodiments, the solid support is a microwell plate, a microwell tube, a magnetic particle, a microbead or a plastic bead. In one embodiment, the solid support is a microplate. The solid phase carrier material is polystyrene, nylon, nitrocellulose, polyvinyl alcohol, etc.

The principle of the invention for detecting the novel coronavirus antibody is that a cell line over expressing ACE2 is plated, a novel coronavirus S protein ECD connected with a marker and a sample mixture are added for incubation, or the sample and the cell are added for incubation, and then the novel coronavirus S protein ECD connected with the marker is added for incubation. In the process, an ACE2 protein overexpressed in cells overexpressing ACE2 is combined with a novel coronavirus S protein ECD connected with a marker, then a detection antibody reacting with the marker is added for incubation, free reactants are removed by washing after each incubation, and finally a working solution capable of developing a detection substance is added, so that a color development reaction can occur when the cells overexpressing ACE2 are combined with the novel coronavirus S protein ECD connected with the marker; when a sample (neutralizing antibody drug, such as ACE2-Fc fusion protein) is bound to the novel coronavirus S protein ECD linked to a label, the conjugate is washed away and the color reaction is inhibited or does not occur. The combination condition of the novel coronavirus S protein ECD connected with the sample inhibition marker and cells over expressing ACE2 can be reflected by measuring the strength of the signal of the chromogenic substance. The signal intensity in the invention specifically refers to the optical density, and the capability of the sample for neutralizing the novel coronavirus can be qualitatively and quantitatively determined by measuring the optical density. The specific method for qualitative judgment comprises the following steps: when the measured optical density is high, it indicates that the binding capacity of the sample to the ECD, which is the ability of the sample to neutralize the novel coronavirus, of the novel coronavirus S protein linked to the marker is poor; when the measured optical density is low, the binding capacity of the sample and the novel coronavirus S protein ECD is strong, namely the sample has strong capability of neutralizing the novel coronavirus; the specific method for quantitative judgment comprises the following steps: fitting dose-effect curves based on logarithm values of signal values and concentrations by using analysis software, wherein fitting parameters are four-parameter fitting (4P-Fit), and parameters such as half effective concentration (EC50), lower asymptote (Bottom), upper asymptote (Top), slope (HillSlope), window (Span), LogEC50 and the like of each curve are obtained;

Y＝Bottom+(Top-Bottom)/(1+10^((LogEC50-X)×HillSlope))

recording the values of the parameters according to the fitted curve of the reference and the sample, calculating the relative activity of the sample:

relative activity (%) (control EC)₅₀Value/sample EC₅₀Value) × 100%

Other calculation formulas are respectively as follows:

relative activity mean value calculation formula:

standard Deviation (SD) calculation method:

coefficient of Variation (CV) or Relative Standard Deviation (RSD):

CV (%) ═ (standard deviation/average) × 100%

The method for detecting the ability of neutralizing the novel coronavirus in the sample, provided by the invention, particularly, the method uses an extra-membrane region (ECD) of the S protein of the novel coronavirus to replace the novel coronavirus/pseudovirus/RBD, so that the combination of the novel coronavirus and a host cell membrane surface receptor under physiological conditions is reduced to the maximum extent, the use of the virus is avoided, the safety of the experiment is greatly improved, and the complexity and the period of the experiment are reduced; secondly, the method uses a HeLa Cell line which over-expresses human ACE2 to replace a human ACE2 protein coated plate to carry out Cell-based ELISA experiments, and the conformational reduction degree of an ACE2 receptor under physiological conditions is higher, so that the relative activity of the detected neutralizing antibody can accurately reflect the biological activity of the antibody for neutralizing a new coronavirus binding receptor; in addition, the detection method can strictly control various variables in the implementation process of the detection method through a Cell-based ELISA experiment, including the concentration and activity of related proteins, the plating density of a HeLa/ACE2 Cell line and the like, and quantitatively analyze the result, so that the stability of the result of the evaluation of the neutralizing capacity of the antibody is ensured.

The method for detecting the capability of neutralizing the novel coronavirus in the sample has the following beneficial technical effects that:

1. the full-length outside the membrane (novel coronavirus S protein ECD) of the novel coronavirus S protein is used for replacing a receptor binding domain RBD, compared with the technology using the RBD, the method is closer to the real physiological state of virus infected cells, and can more accurately reflect the physiological process that the drug blocks the binding of the virus and the host cell receptor.

2. The novel coronavirus/pseudovirus is replaced by the full-length extramembranous (novel coronavirus S protein ECD) of the novel coronavirus S protein, the experiment period is short compared with the conventional virus/pseudovirus neutralization experiment, the operation is simple, and the experiment can be carried out in a common laboratory without a P2/P3 laboratory.

3. By using the HeLa cell line which stably over-expresses the ACE2 receptor, the receptor expression amount is high, and the natural conformation of the receptor can be reflected better than that of the prior art which uses the ACE2 protein plating, so that the physiological process of blocking the combination of virus and receptor by drugs can be simulated better

4. The stable Cell-based ELISA method has strong experimental repeatability, can accurately reflect the biological activity of neutralizing antibody drugs, and can be used for release detection in the drug production process.

The noun explains:

the term "novel coronavirus" (SARS-CoV-2), also known as 2019-nCoV, refers to a pathogenic microorganism causing pneumonia of unknown cause reported first in 12 months in 2019, which belongs to the genus beta coronavirus, has an envelope, and has a virus particle in a circular or elliptical shape with a diameter of 60-140 nm. The genome similarity of SARS (SARS-CoV-1) and its genome is only 80%, and the similarity of gene sequence of coronavirus (Bat coronavirus RaTG13) separated from Hepialus asteroides (Rhinolophus affinis) is up to 96%.

The term "novel coronavirus S protein ECD" refers to the extra-membrane domain (ECD) of Spike protein (Spike protein) on the surface of the novel coronavirus particle. The novel coronavirus S protein ECD comprises a Receptor Binding Domain (RBD) of the novel coronavirus, and the RBD structural domain is an interaction site with a human ACE2 (angiotensin converting enzyme 2) receptor and plays an important role in the infection process of the virus. In some embodiments, the novel coronavirus S protein ECD comprises the amino acid sequence set forth in SEQ ID NO. 4.

The "ACE 2" or "ACE 2 protein" in the invention may be embodied as recombinant ACE2 protein, may be embodied as soluble ACE2 protein, and may be embodied as Fc-tag ACE2 protein. The ACE2 overexpressed cell line in the present invention refers to the expression of ACE2 protein produced on the cell surface by recombinant expression methods conventional in the art. In some embodiments, the ACE2 comprises the amino acid sequence set forth in SEQ ID No. 2 or 3.

The term "neutralization" refers to the process by which binding of a sample to the S protein of SARS-CoV-2 virus prevents its adhesion to, fusion with, and entry into cells. The term "neutralizing antibody" as used herein refers to antibodies that prevent a cell from being invaded by an antigen or infectious agent by binding to viral molecules, by inhibiting or even neutralizing some of their biochemical effects.

"percent (%) identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

The term "EC 50," also known as half maximal effect concentration, refers to a concentration that causes 50% of the maximal effect.

The term "IC 50," also known as half maximal inhibitory concentration, refers to a concentration that causes 50% of the maximal inhibitory effect.

Drawings

FIG. 1 is a schematic diagram of the principle of ELISA detection of neutralizing activity of novel coronavirus neutralizing antibody drugs. (A) His-ECD protein and HeLa/ACE2 cell binding activity diagram. (B) After the ACE2-Fc fusion protein (referring to neutralizing antibody drugs) is added, a schematic diagram of a neutralization effect ELISA detection principle is shown;

FIG. 2 is a dose-response curve of RBD/ECD binding to HeLa/ACE2 cells;

FIG. 3 is a dose-response curve of the neutralization of ECD and HeLa/ACE2 cell binding by ACE2-Fc fusion protein, wherein FIG. 3a is a first experiment and FIG. 3b is a second experiment; figure 3c is a third experiment.

Detailed Description

ELISA is a highly sensitive test technique which is based on immunological reactions and combines the specific reactions of antigens and antibodies with the high-efficiency catalytic action of enzyme on substrates. Because the reaction of antigen and antibody is carried out in the well of a solid phase carrier-polystyrene microtiter plate, after each incubation with a reagent, the excess free reactant can be removed by washing, thereby ensuring the specificity and stability of the test result. In the project, HeLa cells over expressing ACE2 are inoculated, gradient diluted neutralizing antibody drugs (such as ACE2-Fc fusion protein) and fixed concentration mixed solution of novel coronavirus ECD protein are added, and finally the binding capacity of the drugs (such as ACE2-Fc fusion protein) and the novel coronavirus ECD protein to target cells is evaluated through detection reagents.

Adjusting the concentration of the ECD protein of the novel coronavirus according to the preliminarily determined experimental parameters according to the preliminary experimental results, and optimizing the experimental parameters. According to the preliminarily determined experimental parameters, different experimenters carry out repeated experiments, and the experimental parameters are preliminarily determined so as to establish the evaluation method of the neutralizing activity of the novel coronavirus antibody drugs. The experimental principle is shown in figure 1, a HeLa cell line over expressing ACE2 is plated, His-ECD protein and sample mixture, anti-His detection antibody coupled with horseradish peroxidase (HRP) and reaction substrate are sequentially added, free reactants are removed through washing after each incubation step, and finally the bonding condition of the ECD protein and ACE2 receptor can be reflected through the optical density value measured through catalyzing to generate colored substrate. If the concentration of the ECD protein is controlled, the activity of neutralizing antibody drugs (ACE2-Fc fusion protein) for neutralizing the interaction between the ECD protein and an ACE2 receptor can be accurately determined, and the purpose of quantitatively measuring the neutralizing activity of neutralizing antibodies is achieved.

Example 1 construction of ACE2 Single overexpression HeLa cell line

After the DNA sequence of human ACE2 protein is subjected to gene synthesis, the same restriction enzyme is used for enzyme digestion of plasmid vector pLVX-Puro (Clontech, Cat. No.632164), the obtained ACE2-Flag protein ORF DNA fragment (the coded amino acid sequence is shown as SEQ ID NO: 2) after enzyme digestion and the plasmid vector with sticky endBulk fragment, CloneEZ using Genscript^TMAnd connecting the PCR cloning kit, and transforming the escherichia coli competent cells to obtain the plasmid pLVX-Puro-ACE 2.

Lentivirus production: HEK293T cells were trypsinized, resuspended in 10% FBS DMEM and plated at 6-10X 10⁶HEK 293T/Petri dish (10 cm). Lipofectamine 3000(Thermo Fisher, Cat. No. L3000001) was mixed with the three plasmids and added to each dish following transfection of 7-10. mu.g of psPAX2, 5-8. mu.g of PMD2.G-VSV-G, and 9-13. mu.g of pLVX-Puro-ACE 2. Fresh culture solution is replaced 6-8 hours after transfection. Viral supernatants were collected 48-56 hours after transfection, filtered through a 0.45 μm filter and ultracentrifuged. The viral pellet was resuspended in 500. mu.l fresh medium and stored at-80 ℃.

Infection of the target cells: HeLa cells were plated on 12-well plates, and the number of cells was 50% on the next day, followed by overnight culture. A mixture of complete medium and Polybrene (Sigma, Cat. No. H9268-10G) was prepared at a final Polybrene concentration of 3-6. mu.g/ml. The medium was removed and 0.5ml of Polybrene/medium mixture was added to each well. Before infection, the virus was removed from the freezer and thawed, the original cell culture medium was aspirated, 1/2 volumes of fresh medium were added, and the virus stock was added to Hela cells and mixed well. The next day after infection (about 24 hours), the virus-containing culture medium was aspirated, replaced with fresh complete culture medium, and culture was continued at 37 ℃.

Puromycin (Puromycin) resistance screening: adding 1-3 mu g/ml of Puromycin into a cell culture medium, and changing a complete culture solution containing the Puromycin once for 2-3 days until the cells of a non-infection screening control group are killed by the Puromycin. And continuously screening until a stable cell strain is obtained.

FACS detection of ACE2-Flag expression: a portion of the obtained stable cells was taken into FACS tubes and the supernatant was centrifuged off. 4% paraformaldehyde was added and fixed for 20 minutes at room temperature. After 20 minutes the supernatant was removed and BD fire/Permeabilization (BD Biosciences, Cat. No.554714) was added and incubated at 4 ℃ for 20 minutes. After 20 minutes the supernatant was washed off and incubated for 30 minutes with PE anti-DYKDDDDK Tag Antibody (Biolegend, Cat. No. 637310). After 30 minutes, the supernatant was washed, resuspended in FACS buffer, and the ACE2 expression level was measured on the machine to screen for cells with high ACE2 expression. Then, the cells highly expressing ACE2 were selected by single cloning (the specific steps are as follows), and a single clone cell line was selected. The single clone highly expressing ACE2 was expanded until a stable cell line was established.

And (3) selecting a single clone: the cell pool was diluted to the limit into 96-well plates, and after 7 days the 96-well plates were observed under a microscope and wells with single clones were marked. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates.

Example 2 ECD and HeLa/ACE2 binding experiments

2.1 reagent preparation:

sample working solution: both His-RBD sample (Cat. No. Z03479-1) and His-ECD sample (Cat. No. Z03481-1) were synthesized by GenScript. Before the start of the experiment, the His-RBD sample and the His-ECD sample were each diluted in gradient to the concentrations shown in Table 1 using an experiment buffer (PBS solution containing 1% FBS, the same applies hereinafter) to prepare a sample working solution, and were left at 4 ℃ for use.

TABLE 1 concentration of His-RBD sample working solution and His-ECD sample working solution for gradient dilution respectively

Detecting an antibody working solution: THE detection antibody is THE^TM His Tag Antibody[HRP]mAb, Mouse (GenScript, cat. No. aj00612), stock concentration 0.5mg/mL, was diluted 500-fold with assay buffer to prepare detection antibody working solution.

Developing working solution: TMB peroxidase substrate and peroxidase substrate solution B (KPL, cat. No.50-76-00) were removed and returned to room temperature as 1:1 to prepare the color developing working solution.

2.2 Experimental procedures

ACE2 high expression HeLa cell line prepared in example 1 was collected by digestion with 2mL StemPro Accutase (Gibco, Cat. No. A1105-01), resuspended and countedCell number and viability, cell suspension was diluted with complete medium to a cell density of 500,000 cells/mL. Transferring 100. mu.L of cell suspension to corresponding wells of a 96-well cell culture plate, transferring the ELISA plate to a cell culture box, incubating overnight for adherence (37 ℃, introducing 5% CO)₂And the culture time is 12-18 hours). The microplate was removed from the cell culture chamber and the remaining supernatant was removed. Transferring 300 mu L of experiment buffer solution into corresponding holes, uniformly mixing the liquid in the holes, removing the plate washing buffer solution, and repeating for 2-3 times. Transferring 50-100 mu L of sample working solution to corresponding holes, and incubating for 1-2 hours at room temperature.

The microplate is removed, the supernatant removed and the remaining liquid removed. The plate was washed 3 times with 300. mu.L of assay buffer. Transfer 100 μ L of the test antibody working solution to the corresponding well and incubate at room temperature for about 1 hour. The microplate is removed, the supernatant removed and the remaining liquid removed. The plate was washed 3 times with 300. mu.L of assay buffer. Transferring 100 mu L of the color development working solution to corresponding holes, and incubating for 10-30 minutes at room temperature. After completion of the incubation, 100. mu.L of a color development stop solution (2M phosphoric acid solution, the same applies hereinafter) was transferred to the corresponding well to stop the color development.

The optical density signal values were read using a microplate reader. The detection wavelength and the reference wavelength are respectively selected from 450nm and 630nm, and the original experimental data is the difference value between the signal value read by the detection wavelength and the signal value of the reference wavelength. And (5) analyzing experimental data and fitting a dose-effect curve.

2.3 results of the experiment

The binding experiment of ECD and HeLa/ACE2 was carried out according to the above experimental procedures, and the measured dose-response curves of binding of viral RBD protein and ECD protein to HeLa/ACE2 cells, respectively, are shown in FIG. 2. The results show that the binding capacities of the RBD protein and the ECD protein of the virus and HeLa/ACE2 cells are greatly different, and EC50 of the RBD protein and ECD protein which are bound with the cells is obtained simultaneously and is 0.0813 mug/mL and 1.632 mug/mL respectively. Considering that ECD is better able to mimic the new coronavirus S protein structure in physiological state than RBD, we chose the EC50 concentration of ECD for neutralizing the protein concentration of experimental ECD.

Example 3 neutralization of ECD with novel Corona Virus neutralizing antibody drugs binding experiment with HeLa/ACE2

3.1 reagent preparation:

neutralizing a sample working solution: His-ECD stock solution was diluted to 3.2 μ g/mL (EC50 concentration obtained in example 2 after 1:1 dilution) using the experimental buffer to prepare His-ECD working solution, ACE2-Fc fusion protein (GenScript, cat. No. z03484-1) sample was gradient-diluted to the concentrations shown in table 2 to prepare sample working solution, and the His-ECD working solution and ACE2-Fc fusion protein sample working solution of corresponding concentrations were mixed in a volume ratio of 1:1 to prepare neutralized sample working solution.

TABLE 2 concentration of His-ECD in the neutralized sample working solution and concentration gradient of the corresponding ACE2-Fc fusion protein

Detecting an antibody working solution: the reagent preparation method of the embodiment 2.1;

developing working solution: the same procedure as in example 2.1 was used to prepare the reagents.

3.2 Experimental procedures

The ACE2 high expression HeLa cell line prepared in example 1 was collected by digestion with 2mL StemPro Accutase (Gibco, Cat. No. A1105-01), cell resuspended and cell number and viability calculated, and the cell suspension was diluted with complete medium to a cell density of 500,000 cells/mL. Transferring 100. mu.L of cell suspension to corresponding wells of a 96-well cell culture plate, transferring the ELISA plate to a cell culture box, incubating overnight for adherence (37 ℃, introducing 5% CO)₂And the culture time is 12-18 hours). The microplate was removed from the cell culture chamber and the supernatant removed. Transferring 300 mu L of experiment buffer solution into corresponding holes, uniformly mixing liquid in the holes, removing the plate washing buffer solution, removing residual liquid, and repeating for 2-3 times. Transferring 100 mu L of the neutralization sample working solution to corresponding holes, and incubating for 1-2 hours at room temperature.

The microplate is removed and the supernatant removed to remove any remaining liquid. The plate was washed 3 times with 300. mu.L of assay buffer. Transfer 100 μ L of assay neutralizing antibody sample working solution to the corresponding well and incubate for about 1 hour at room temperature. The microplate is removed, the supernatant removed and the remaining liquid removed. The plate was washed 3 times with 300. mu.L of assay buffer. Transferring 100 mu L of the color development working solution to corresponding holes, and incubating for 10-30 minutes at room temperature. After completion of incubation, 100. mu.L of the color development stop solution was transferred to the corresponding well to stop the color development.

3.3 results of the experiment

Different experimenters A and B respectively use ACE2-Fc fusion protein as a test sample, three independent repetitions are carried out on different experimental dates according to the operation steps of example 3, a Reference Substance (RS) and a quality control substance (QC) are set for each repetition, and the method is preliminarily confirmed, and the result is shown in FIG. 3. The RS and QC samples are ACE2-Fc fusion proteins independently prepared according to the operation steps of example 3, and the accuracy and stability of the experimental method can be measured by comparing the difference of the detection results of the QC sample and the RS sample. Wherein, fig. 3a is a first experiment, human IgG1 protein is set as a negative control of a neutralization experiment, dose-effect curves of a second experiment and a third experiment are shown in fig. 3b and fig. 3c, data of three independent repeated experiments are summarized in table 3, and experimental results show that the relative activities of the QC of the three independent experiments fluctuate within the range of 80-120%, the average value of the relative activities of QC samples of the three experiments is 96%, so that the detection method has good system applicability, and the detection method obtained by the detection method has high accuracy; the Coefficient of Variation (CV) of the relative activity is 13.6 percent and is less than 15 percent, which indicates that the method is more stable, the fluctuation of the detection result obtained by applying the method is smaller, and the precision of the experimental result is high. The non-related antibody human IgG1 showed no neutralizing ability, indicating that the method has good specificity and can specifically detect the ability of the antibody to neutralize the binding of ECD and ACE 2.

TABLE 3 comparison of data from experiments on the neutralization of ECD by ACE2-Fc fusion protein and the binding capacity of HeLa/ACE2 cells

Novel coronavirus S protein ECD (containing tag protein) of SEQ ID NO. 1

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAASVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPHHHHHHHHDYKDDDDK

2 ACE2-Flag sequence:

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSFDYKDDDDK

3 ACE2 sequence

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

Novel coronavirus S protein ECD of SEQ ID NO. 4

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAASVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP

Reference documents:

[1] the national drug administration drug evaluation center, novel technical data on drugs for neutralizing antibodies against coronavirus, request for pharmacy (manuscript of comments), included in the book, 5 months in 2020.

[2]Nie,Jianhui,et al."Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2."Emerging microbes&infections 9.1(2020):680-686.

SEQUENCE LISTING

<110> Nanjing Kinsrui Biotechnology Ltd

<120> method for detecting neutralizing ability of novel coronavirus neutralizing antibody drug

<130> BD-C21211CN

<150> CN202010685450.4

<151> 2020-07-16

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1229

<212> PRT

<213> Artificial Sequence

<220>

<223> novel coronavirus S protein ECD (containing tag protein)

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Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

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His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

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Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

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Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

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Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

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Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

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Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

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Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

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Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

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Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

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Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

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Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

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Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

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Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

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Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

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Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

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Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

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Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

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Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

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Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

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Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

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Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

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Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

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Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

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Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

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Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

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Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

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Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

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Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

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Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

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Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

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Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

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His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

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Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

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Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

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Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Ala Ser Val Ala

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Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

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Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

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Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

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Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

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Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

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Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

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Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

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Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

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Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

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Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

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Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

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Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

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Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

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Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

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Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

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Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

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Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

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Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

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Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

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Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

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Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

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Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

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His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

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Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

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Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

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His His His Asp Tyr Lys Asp Asp Asp Asp Lys

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Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

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Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

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Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

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Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

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Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

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Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

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Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

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Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

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Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

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Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

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His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

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Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

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Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

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Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

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Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

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Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

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Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

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Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

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Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

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Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

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Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe Asp Tyr Lys Asp Asp Asp Asp Lys

805 810

<210> 3

<211> 805

<212> PRT

<213> Artificial Sequence

<220>

<223> ACE2 sequences

<400> 3

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

<210> 4

<211> 1213

<212> PRT

<213> Artificial Sequence

<220>

<223> novel coronavirus S protein ECD

<400> 4

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Ala Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro

1205 1210

Claims

1.A method for detecting the ability to neutralize a novel coronavirus in a sample, said method comprising the steps of:

(1) contacting and incubating a sample with an ACE 2-overexpressing cell line with a marker-linked extracellular domain of a novel coronavirus spike protein;

2. The method of claim 1, wherein the novel coronavirus spike protein extracellular domain comprises an amino acid sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 80% identity to the amino acid sequence set forth in seq id No. 1 or 4.

3. The method according to claim 1 or 2, wherein the concentration of the extracellular domain of the spike protein of the novel coronavirus added in step (1) is 0.8-3.2 μ g/mL, preferably 1.2-2.0 μ g/mL.

4. The method according to any one of claims 1 to 3, wherein the marker in step (1) is selected from one or more of a His-tag, a Flag-tag, a c-Myc-tag, a HA-tag, a V5-tag, an HSV-tag and biotin, preferably a His-tag.

5. The method according to any one of claims 1 to 4, wherein the ACE2 overexpressing cell line in step (1) is selected from HEK293, HeLa, Vero E6 or CHO, preferably an ACE2 overexpressing HeLa cell.

6. The method according to claim 5, wherein the ACE2 was added at an over-expressed cell line cell concentration of 4 x 10⁵-1ⅹ10⁶Single cell/mL, preferably 5 x 10⁵Individual cells/mL.

7. The method according to any one of claims 1 to 6, wherein the contact culture in step (1) comprises incubation at 18 ℃ -37 ℃ for 0.5-3 hours, preferably at room temperature for 1-2 hours.

8. The method of any one of claims 1-7, wherein obtaining the cells after incubation in step (1) in step (2) comprises the steps of removing culture supernatant and washing the cells.

9. The method according to any one of claims 1 to 8, wherein the detection substance in step (2) is selected from horseradish peroxidase, alkaline phosphatase, or an acridinium compound-linked anti-marker antibody.

10. The method of claim 9, wherein the tag comprises a His-tag and the detection substance comprises horseradish peroxidase-linked anti-His-tag antibody.

11. The method according to any one of claims 1 to 10, wherein the contact incubation in step (2) comprises incubation at 18 ℃ -37 ℃ for 0.5-1.5 hours, preferably for 1 hour at room temperature.

12. The method according to any one of claims 1 to 11, wherein the step (3) of detecting the signal comprises removing the supernatant of the cells after the incubation in the step (2), washing the cells, adding a chromogenic working solution for incubation, adding a chromogenic stop solution, and reading the signal value.

13. The method of claim 12, wherein the chromogenic working solution comprises a TMB peroxidase substrate and a peroxidase substrate solution B, and the chromogenic stop solution comprises a phosphoric acid solution.

14. The method of any one of claims 1-13, wherein the sample is an anti-novel coronavirus antibody-based drug.

15. A kit for detecting the ability to neutralize a novel coronavirus in a sample, said kit comprising a marker linked to the extracellular domain of the novel coronavirus spike protein, an ACE 2-overexpressed cell line, and a detection substance capable of reacting with said marker to generate a signal, wherein said ACE 2-overexpressed cell line is an adherent cell line.

16. The method of claim 15, wherein the novel coronavirus spike protein extracellular domain comprises an amino acid sequence identical to SEQ ID NO:1 or 4, or a sequence having at least 80% identity to the amino acid sequence set forth in seq id No. 1 or 4.

17. The test kit of any one of claims 15-16, wherein the tag comprises a His-tag and the detection substance comprises an anti-His-tag antibody linked to horseradish peroxidase, alkaline phosphatase, or an acridinium compound.

18. The test kit according to any one of claims 15-17, wherein the ACE2 overexpressing cell line is selected from HEK293, HeLa, Vero E6 or CHO, preferably an ACE2 overexpressing HeLa cell.

19. The test kit according to claim 18, wherein the cell line overexpressing ACE2 has a cell concentration of 4 x 10⁵-1ⅹ10⁶Single cell/mL, preferably 5 x 10⁵Individual cells/mL.

20. The test kit according to any one of claims 15 to 19, wherein the solid support is a microplate, a microtube, a magnetic particle, a microbead or a plastic bead.