Kit and method for detecting SARS-CoV-2 neutralizing antibody
阅读说明：本技术 一种检测SARS-CoV-2中和抗体的试剂盒及方法 (Kit and method for detecting SARS-CoV-2 neutralizing antibody ) 是由 时亚斌 陈志才 马可 徐晓昱 于 2020-11-11 设计创作，主要内容包括：本申请涉及一种检测SARS-CoV-2中和抗体的试剂盒及方法。本申请涉及生物医药领域,提供一种用于检测SARS-CoV-2中和抗体的检测试剂盒及方法,采用蛋白标签对RBD及ACE2修饰使其二者具有更好的稳定性和反应性,提高了检测灵敏度。(The application relates to a kit and a method for detecting SARS-CoV-2 neutralizing antibody. The application relates to the field of biological medicine, and provides a detection kit and a method for detecting a SARS-CoV-2 neutralizing antibody, wherein a protein label is adopted to modify RBD and ACE2 so that the RBD and the ACE2 have better stability and reactivity, and the detection sensitivity is improved.)
1. A detection kit for SARS-CoV-2 neutralizing antibodies comprising:
1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and
2) a competitive binding agent comprising a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
2. The kit of claim 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region, RBD.
3. The kit of claim 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
4. The kit of claim 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-CoV-2 spike protein.
5. The kit of claim 2, wherein said RBD is linked directly or indirectly to an Fc region.
6. The kit of claim 5, wherein the RBD is fused in frame with the Fc.
7. The kit of claim 5, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
8. The kit of claim 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 7.
9. The kit of claim 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 8.
10. The kit of claim 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 9.
11. The kit of claim 3, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 10.
12. The kit of claim 4, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 11.
13. The kit of any one of claims 1-12, wherein the competing binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
14. The kit of claim 13, wherein the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
15. The kit of claim 13, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the extracellular domain ECD.
16. The kit of claim 13, wherein the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
17. The kit of claim 16, wherein the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
18. The kit of claim 16, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
19. The kit of claim 16, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 2.
20. The kit of claim 16, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 3.
21. The kit of claim 16, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 4.
22. The kit of claim 13, wherein the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
23. The kit of claim 22, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
24. The kit of claim 1, comprising a sample diluent having a pH of 6.0-9.6.
25. The kit of any one of claims 1, further comprising a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof.
26. The kit of claim 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
27. The kit of claim 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
28. The kit of claim 25, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35;
the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or
The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO 45-47.
29. The kit of claim 25, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22;
the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or
The VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
30. The kit of claim 25, wherein the reference antibody comprises antibodies 32-1,12-5 and 12-8.
31. The kit according to claim 25, wherein the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
32. The kit of claim 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the SARS-CoV-2 spike protein or functionally active fragment thereof.
33. The kit of claim 32, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
34. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
use of a kit according to claim 1 or 13.
Technical Field
The application relates to the field of biological medicine, in particular to a kit and a method for detecting SARS-CoV-2 neutralizing antibody.
Background
SARS-CoV-2 surface spike protein (spike protein) is important receptor binding site on coronavirus surface, and can bind with cell surface virus specific receptor, mediate virus outer membrane and cell fusion, virus adsorption and membrane penetration; the coronavirus S protein Receptor Binding Domain (RBD) can be combined with angiotensin-converting enzyme2 (ACE 2) on the surface of a host cell, and the virus enters the host cell.
When SARS-CoV-2 invades the body, it stimulates the body to produce neutralizing antibodies with protective action. The neutralizing antibody is a soluble protein secreted by adaptive immune response cells, and has the functions of recognizing virus surface protein and blocking the binding of the virus surface protein and a human cell surface specific receptor, thereby playing an antiviral role. The amount of the neutralizing antibody is an important index of the immune protection effect of the vaccine, and is an important basis for the evaluation and quality control of the vaccine.
The existing detection method for SARS-CoV-2 mainly comprises nucleic acid detection and antibody detection, and the main methods comprise fluorescence PCR method, enzyme-linked immunosorbent assay, colloidal gold chromatography and chemiluminescence method, wherein the nucleic acid detection has high sensitivity, strong specificity and high accuracy, but has relatively high operation requirement and long detection time. The antibody detection sensitivity is high, the specificity is strong, but IgM/IgG antibody can be generated after 7-14 days of infection. Moreover, these methods can only detect whether the patient is in the infection stage, and cannot confirm whether the patient is at risk of re-infection after healing, and whether the general public is exposed or at risk of infection. Therefore, there is an urgent need for a novel method for detecting SARS-CoV-2.
Disclosure of Invention
The present application provides a kit and method for detecting SARS-CoV-2 neutralizing antibodies having one or more of the following properties: 1) after the RBD and ACE2 receptor protein or functional active fragment thereof is fused with Fc, compared with the protein with a natural structure, the protein has better stability and reactivity; 2) the detection sensitivity is improved; 3) the clinical curative effect of the COVID-19 vaccine can be judged, and the autoimmune effect of an individual is evaluated; 4) can judge whether the risk of SARS-CoV-2 reinfection exists; and 5) the reference antibody has good neutralization effect and can well simulate the natural form of the neutralizing antibody in human bodies.
In one aspect, the present application provides a kit for detecting SARS-CoV-2 neutralizing antibodies, comprising:
1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and
2) a competitive binding agent comprising a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region, RBD, and the RBD is linked, directly or indirectly, to an Fc region.
In certain embodiments, the RBD is fused in frame with the Fc.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOS 6-11.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof is present at a concentration of from 50ng/ml to 4000 ng/ml.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof is immobilized on a solid support.
In certain embodiments, the competing binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises the extracellular domain ECD.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the competing binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs 1-5.
In certain embodiments, the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
In certain embodiments, the concentration of the competing binding agent is from 100ng/ml to 2000 ng/ml.
In certain embodiments, the kit further comprises a calibrator comprising one or more reference antibody molecules, or antigen-binding fragments thereof, that specifically recognize the SARS-CoV-2 spike protein, or functionally active fragment thereof.
In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or the VH comprises the amino acid sequence shown in SEQ ID NO 42-44 HCDR1, HCDR2 and HCDR3, and the VL comprises the amino acid sequence shown in SEQ ID NO 45-47 LCDR1, LCDR2 and LCDR 3.
In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22; the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or the VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
In certain embodiments, the reference antibody comprises antibodies 32-1,12-5 and 12-8.
In certain embodiments, the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the SARS-CoV-2 spike protein or functionally active fragment thereof.
In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
In another aspect, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising: the kit is used.
In another aspect, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
1) coating the SARS-CoV-2 spike protein or a functionally active fragment thereof in a solid support;
2) adding a sample to be detected into the SARS-CoV-2 spike protein or the functional active fragment thereof in the step 1) to react;
3) adding a competing binding agent to step 2);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the competitive binding agent comprises a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof; said SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD; the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region, RBD, and the RBD is linked, directly or indirectly, to an Fc region.
In certain embodiments, the RBD is fused in frame with the Fc.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOS 6-11.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof is present at a concentration of from 50ng/ml to 4000 ng/ml.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof is immobilized on a solid support.
In certain embodiments, the competing binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises the extracellular domain ECD.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the competing binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs 1-5.
In certain embodiments, the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
In certain embodiments, the concentration of the competing binding agent is from 100ng/ml to 2000 ng/ml.
In certain embodiments, the kit further comprises a calibrator comprising one or more reference antibody molecules, or antigen-binding fragments thereof, that specifically recognize the SARS-CoV-2 spike protein, or functionally active fragment thereof.
In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In certain embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or the VH comprises the amino acid sequence shown in SEQ ID NO 42-44 HCDR1, HCDR2 and HCDR3, and the VL comprises the amino acid sequence shown in SEQ ID NO 45-47 LCDR1, LCDR2 and LCDR 3.
In certain embodiments, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22; the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or the VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
In certain embodiments, the reference antibody comprises antibodies 32-1,12-5 and 12-8.
In certain embodiments, the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
In certain embodiments, the sample is selected from the group consisting of: urine, saliva, serum, plasma, nasopharyngeal aspirate, and bronchial lavage.
In certain embodiments, the sample is derived from a subject.
In certain embodiments, the subject is a COVID-19 convalescent patient, a healthy subject, and/or a subject who has received a COVID-19 vaccine therapy.
In another aspect, the present application is a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
1) coating an ACE2 receptor protein or a functionally active fragment thereof in a solid support;
2) mixing and incubating a sample to be tested with SARS-CoV-2 spike protein or functional active fragment thereof to obtain an incubated product;
3) adding the incubated product of step 2) into step 1);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding Region (RBD) and the RBD comprises one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the RBD.
In certain embodiments, the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
In certain embodiments, the RBD is linked directly or indirectly to the Fc region.
In certain embodiments, the RBD is fused in frame with the Fc.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 7.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 8.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 9.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 10.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 11.
In certain embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof is present at a concentration of from 50ng/ml to 4000 ng/ml.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
In certain embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 2.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 3.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 4.
In certain embodiments, the ACE2 receptor protein or functionally active fragment thereof is present at a concentration of 100ng/ml to 2000 ng/ml.
In certain embodiments, it comprises a sample diluent having a pH of 6.0 to 9.6.
In certain embodiments, the detection is obtained by comparison to a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein: the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or the VH comprises the amino acid sequence shown in SEQ ID NO 42-44 HCDR1, HCDR2 and HCDR3, and the VL comprises the amino acid sequence shown in SEQ ID NO 45-47 LCDR1, LCDR2 and LCDR 3.
In certain embodiments, the detection is obtained by comparison to a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22; the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or the VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
In certain embodiments, the reference antibody comprises antibodies 32-1,12-5 and 12-8.
In certain embodiments, the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
In certain embodiments, the sample to be tested is selected from the group consisting of: urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirates, and bronchial lavage.
In certain embodiments, the test sample is derived from a subject.
In certain embodiments, the subject is a COVID-19 convalescent patient, a healthy subject, and/or a subject who has received a COVID-19 vaccine therapy.
Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.
Drawings
The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:
FIG. 1 shows the content of neutralizing antibodies in human body, which was simulated by the calibrator described in this application.
FIG. 2 shows a ROC curve for the Cutoff values described herein.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.
Definition of terms
In the present application, the term "sample" generally refers to any substance that contains or is assumed to contain antibodies, particularly SARS-CoV-2 neutralizing antibodies. The "sample" may be of natural or synthetic origin and may be obtained by any means known to those skilled in the art. A "sample" can be a tissue or liquid sample isolated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirates, and bronchial lavage. The sample may be a research sample and a clinical sample. The sample may also be a blood sample for transfusion or therapy. The sample may also be synthetic and include, but is not limited to, in vitro cell culture components, including, but not limited to, conditioned media, recombinant cells, and cell components. In the present application, the terms "sample" and "specimen" are used interchangeably.
In this application, the term "detecting" generally refers to finding the presence or appearance of something.
In the present application, the term "antigen" generally refers to a substance that causes the production of antibodies and can elicit an immune response. They may also be used for diagnostic or patient selection or characterization purposes.
In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including, but not limited to, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, as well as any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.
In the present application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind antigen. The antigen binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen binding function of an antibody can also be achieved by: a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab 'or F (ab') 2, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab 'or F (ab') 2. (1) Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH domains; (2) a F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of the VH and CH domains; (4) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341: 544-546); (6) an isolated Complementarity Determining Region (CDR), and (7) a combination of two or more isolated CDRs which may optionally be joined by a linker. Furthermore, there may be included a monovalent single chain molecule formed by pairing VL and VH, fv (scFv) (see Bird et al (1988) Science 242: 423-. The "antigen-binding portion" may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region.
In the present application, the term "subject" generally refers to any organism, including but not limited to mammals, such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the present application, the subject may be a human, a pet, or a livestock. In the present application, the subject may be a COVID-19 convalescent patient, a healthy subject, and/or a subject who has been treated with a COVID-19 vaccine.
In the present application, the term "RBD" refers to the receptor binding domain of the SARS-CoV-2 spike protein (S protein). In the present application, the RBD may be a mutant or a truncation of the spike protein (S protein) of SARS-CoV-2. In this application, the truncation generally refers to anything less than a whole. For example, a truncation is at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% less of the amino acid sequence of a reference sequence. In the present application, "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids. In the present application, a truncation or mutant may occur naturally or non-naturally. Non-naturally occurring truncations or mutants may be generated using techniques known in the art.
In the present application, the term "SARS-CoV-2 spike protein or functionally active fragment thereof" generally refers to the capsid surface glycoprotein of a coronavirus. SARS-COV-2 binds to the ACE2 receptor via the SARS-CoV-2 spike protein and invades the cell. The SARS-CoV-2 spike protein contains a transmembrane region comprising a fragment of the capsid surface glycoprotein from coronavirus from the N-terminus or from amino acid position 14 up to at least 1213 amino acids, or the corresponding region from other SAS viruses. In the present application, the SARS-CoV-2 spike protein can be a trimeric full-length SARS-CoV-2 spike protein. In the present application, the SARS-CoV-2 spike protein can be a full-length SARS-CoV-2 spike protein.
In the present application, the term "ACE 2 receptor protein or a functionally active fragment thereof" generally refers to a functional receptor of the coronavirus spike protein. In some embodiments, the ACE2 receptor protein may be a human ACE2 receptor protein. For example, the ACE2 receptor protein sequence can be found in Uniprot accession number Q9BYF 1.
In the present application, the term "functionally active fragment" generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same activity or ability as its larger counterpart. The level of activity of a functionally active fragment may be the same as, less than, or greater than the activity of the larger counterpart. For example, a functionally active fragment of the SARS-CoV-2 spike protein can be a polypeptide that consists of fewer amino acids than the full-length SARS-CoV-2 spike protein, but can still retain the activity of the full-length SARS-CoV-2 spike protein. For another example, a functionally active fragment of the human ACE2 receptor protein may be a polypeptide consisting of fewer amino acids than the full-length human ACE2 receptor protein, but which may retain the activity of the full-length human ACE2 receptor protein.
In the present application, the term "Fc region" or "Fc" generally refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The Fc region includes native sequence Fc regions and variant Fc regions. In some embodiments, the Fc region can be linked directly or indirectly to the SARS-CoV-2 spike protein. In some embodiments, the Fc region can be fused in frame to the SARS-CoV-2 spike protein. In some embodiments, the Fc region may be linked directly or indirectly to an ACE2 receptor protein. In some embodiments, the Fc region may be fused in frame to the ACE2 receptor protein. In the present application, an Fc region includes a human or non-human (e.g., rat, mouse, rabbit) Fc region. For example, the Fc region may include a rabbit Fc region (rFc). For example, the Fc region may include a mouse Fc region (mFc). For example, the Fc region may include a human Fc region (hFc).
In this application, "in-frame fusion" generally refers to the joining of two or more Open Reading Frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct reading frame of the original ORF. Thus, the resulting "fusion polypeptide" is a single protein comprising two or more fragments corresponding to the polypeptide encoded by the original ORF (which fragments are not normally so linked in nature). "fusion site" refers to a sequence of two or more fragments joined together. In some cases, the fusion site may be a sequence identical to a sequence in two or more fragments to be joined. In some cases, the fusion site can further comprise a gap fragment that is not identical to any of the sequences of the two or more fragments being linked.
Detailed Description
Detection kit
In one aspect, the present application provides a kit for detecting SARS-CoV-2 neutralizing antibodies, which may comprise:
1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and
2) a competitive binding agent comprising a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
In the present application, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise a receptor binding region RBD.
In the present application, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a full-length SARS-CoV-2 spike protein.
In the present application, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a trimeric full-length SARS-CoV-2 spike protein.
For example, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise the amino acid sequence shown in SEQ ID NO. 10. For example, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise the amino acid sequence shown in SEQ ID NO. 11.
In the present application, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a receptor binding region RBD, and the RBD can be linked directly or indirectly to an Fc region.
In the present application, the RBD may be fused in frame with the Fc. In the present application, the Fc may be selected from the group consisting of: rFc, mFc, hFc, and hFc.
In the present application, the RBD may be linked directly or indirectly to the Fc region. In the present application, the Fc may be rFc.
In the present application, the RBD may be linked directly or indirectly to the Fc region. In the present application, the Fc may be mFc.
In the present application, the RBD may be linked directly or indirectly to the Fc region. In the present application, the Fc may be hFc.
For example, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 7. For example, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 8. For example, the SARS-CoV-2 spike protein or a functionally active fragment thereof can comprise the amino acid sequence shown in SEQ ID NO. 9.
In the present application, the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof can be from 50ng/ml to 4000 ng/ml. For example, the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof can be 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 800ng/ml, 900ng/ml, 1000ng/ml, 1500ng/ml, 2000ng/ml, 2500ng/ml, 3000ng/ml, 3500ng/ml or 4000 ng/ml.
In the present application, the SARS-CoV-2 spike protein or functionally active fragment thereof can be immobilized on a solid support.
In the present application, the solid support may be a reaction plate or an ELISA plate (a reaction plate or an ELISA plate adapted for an ELISA assay).
In the present application, the competing binding agent may comprise an ACE2 receptor protein or a functionally active fragment thereof.
In the present application, the competing binding agent may comprise a known RBD neutralizing antibody.
In the present application, the ACE2 receptor protein may comprise the human ACE2 receptor protein or a functionally active fragment thereof.
In the present application, the ACE2 receptor protein or functionally active fragment thereof may comprise the extracellular domain ECD.
In the present application, the ACE2 receptor protein or functionally active fragment thereof may be linked directly or indirectly to an Fc region.
In the present application, the ACE2 receptor protein or a functionally active fragment thereof may be fused in frame to the Fc region.
In the present application, the Fc region may be selected from the group consisting of: rFc, mFc, hFc, and hFc.
In the present application, the Fc region may be rFc. In the present application, the Fc region may be mFc. In the present application, the Fc region may be hFc.
In the present application, the competing binding agent may comprise the amino acid sequence set forth in any one of SEQ ID NOs 1-5.
For example, the competing binding agent may comprise the amino acid sequence set forth in SEQ ID NO. 2. For example, the competing binding agent may comprise the amino acid sequence set forth in SEQ ID NO. 3. For example, the competing binding agent may comprise the amino acid sequence set forth in SEQ ID NO. 4.
In the present application, the competing binding agent may comprise one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent. For example, the label may comprise horseradish peroxidase (HRP) or an HRP-like enzyme. For example, the label may be horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase or β -Galactosidase. In some embodiments, the label can be horseradish peroxidase (HRP).
In the present application, the competitive binding agent may comprise horseradish peroxidase (HRP) or an HRP-like enzyme and one or more labels selected from the group consisting of: AVI tags, biotin, fluorescein, and streptavidin.
For example, the competing binding agent may comprise HRP, AVI tag, streptavidin.
In the present application, the concentration of the competing binding agent may be from 100ng/ml to 2000 ng/ml. For example, the concentration of the competing binding agent may be 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 1000ng/ml, 1500ng/ml, 1800ng/ml, 1900ng/ml or 2000 ng/ml.
In the present application, the kit may include a sample diluent, which may have a pH of 6.0 to 9.6. For example, the pH of the sample diluent may be 6.0, 6.1, 6.2, 6.3, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5, or 9.6.
For example, the pH of the sample diluent may be 6.0.
In the present application, the kit further comprises a calibrator that can comprise one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof.
In the present application, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In the present application, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In another aspect, the present application provides a kit for detecting a SARS-CoV-2 neutralizing antibody that differs from the aforementioned kits in that the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise one or more markers that can directly or indirectly generate a signal indicating the presence and/or amount of the competing binding agent. For example, the competing binding agent may be free of a label.
Reference antibody
In the present application, the reference antibodies may include antibodies 32-1,12-5, and 12-8.
For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain variable region VH comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region VL comprising LCDR1, LCDR2, and LCDR 3. Wherein the HCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 30 or a variant thereof; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 31 or a variant thereof; the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 32 or a variant thereof; the LCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 33 or a variant thereof; the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 34 or a variant thereof; the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 35 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof can include antibody 32-1 or an antibody having the same HCDR1-3 and LCDR 1-3. In the present application, the VH may comprise the amino acid sequence shown as SEQ ID NO 21 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO. 22 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO 12; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO. 13. For example, the reference antibody or antigen-binding fragment thereof can include antibody 32-1 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may comprise the amino acid sequence shown in SEQ ID NO. 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO. 23 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may comprise the sequence shown in SEQ ID NO. 20; and the nucleic acid sequence encoding CL may comprise the sequence set forth in SEQ ID NO. 14. For example, the reference antibody or antigen-binding fragment thereof can include antibody 32-1 or an antibody having the same heavy and light chain constant regions.
In some embodiments, the antibody described herein can be 32-1. The HCDR1, HCDR2, and HCDR3 of antibody 32-1 can comprise the amino acid sequences shown in SEQ ID NO 30, SEQ ID NO 31, and SEQ ID NO 32, respectively; the VH may comprise the amino acid sequence shown as SEQ ID NO 21; LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO 33, SEQ ID NO 34 and SEQ ID NO 35, respectively; VL may comprise the amino acid sequence shown in SEQ ID NO. 22.
For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain variable region VH, which can comprise HCDR1, HCDR2, and HCDR3, and a light chain variable region VL, which can comprise LCDR1, LCDR2, and LCDR 3. Wherein the HCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 36 or a variant thereof; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 37 or a variant thereof; the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 38 or a variant thereof; the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:39 or a variant thereof; the LCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 40 or a variant thereof; the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 41 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof can comprise antibody 12-5 or an antibody having the same HCDR1-3 and LCDR1-3 as it does. In the present application, the VH may comprise the amino acid sequence shown as SEQ ID NO 24 or a variant thereof; and the VL may comprise the amino acid sequence shown as SEQ ID NO. 25 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO. 15; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO 16. For example, the reference antibody or antigen-binding fragment thereof can include antibody 12-5 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may comprise the amino acid sequence shown in SEQ ID NO. 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO. 26 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may comprise the sequence shown in SEQ ID NO. 20; and the nucleic acid sequence encoding CL may comprise the sequence set forth in SEQ ID NO. 17. For example, the reference antibody or antigen-binding fragment thereof can include antibody 12-5 or an antibody having the same heavy and light chain constant regions.
In some embodiments, the antibody described herein can be 12-5. The HCDR1, HCDR2, and HCDR3 of antibodies 12-5 can comprise the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; the VH may comprise the amino acid sequence shown as SEQ ID NO. 24; LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO 39, SEQ ID NO 40 and SEQ ID NO 41, respectively; VL may comprise the amino acid sequence shown in SEQ ID NO. 25.
For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain variable region VH comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region VL comprising LCDR1, LCDR2, and LCDR 3. Wherein the HCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 42 or a variant thereof; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 43 or a variant thereof; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 44 or a variant thereof; the LCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 45 or a variant thereof; the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 46 or a variant thereof; the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 47 or a variant thereof. For example, the reference antibody or antigen-binding fragment thereof can comprise antibody 12-8 or an antibody having the same HCDR1-3 and LCDR1-3 as it does. In the present application, the VH may comprise the amino acid sequence shown as SEQ ID NO 27 or a variant thereof; and the VL may comprise the amino acid sequence shown as SEQ ID NO 28 or a variant thereof. In the present application, the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO 18; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO. 19. For example, the reference antibody or antigen-binding fragment thereof can include antibodies 12-8 or antibodies having the same light chain variable region and heavy chain variable region as the reference antibody. For example, a reference antibody or antigen-binding fragment thereof described herein can comprise a heavy chain constant region CH and a light chain constant region CL. In the present application, the CH may comprise the amino acid sequence shown in SEQ ID NO. 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO. 23 or a variant thereof. In the present application, the nucleic acid sequence encoding CH may comprise the sequence shown in SEQ ID NO. 20; and the nucleic acid sequence encoding CL may comprise the sequence set forth in SEQ ID NO. 14. For example, the reference antibody or antigen-binding fragment thereof can include antibodies 12-8 or antibodies having the same heavy and light chain constant regions.
In some embodiments, the antibodies described herein can be 12-8. The HCDR1, HCDR2, and HCDR3 of antibodies 12-8 may comprise the amino acid sequences shown in SEQ ID NO 42, SEQ ID NO 43, and SEQ ID NO 44, respectively; the VH may comprise the amino acid sequence shown as SEQ ID NO 27; LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47, respectively; VL may comprise the amino acid sequence shown in SEQ ID NO 28.
In the present application, the concentration of the reference antibody may be between 50ng/ml and 1000 ng/ml. For example, the concentration of the reference antibody may be 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml and 1000 ng/ml.
Detection method
In another aspect, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which can comprise:
the kit is used.
In another aspect, the present application provides a method of detecting SARS-CoV-2 neutralizing antibodies in a sample, which can comprise:
1) coating the SARS-CoV-2 spike protein or a functionally active fragment thereof in a solid support;
2) adding a sample to be detected into the SARS-CoV-2 spike protein or the functional active fragment thereof in the step 1) to react;
3) adding a competing binding agent to step 2);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the competitive binding agent can comprise a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof; the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise a receptor binding region RBD; the competing binding agent may comprise an ACE2 receptor protein or a functionally active fragment thereof.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a full-length SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a trimeric full-length SARS-COV-2 spike protein.
In some embodiments, the RBD can be linked directly or indirectly to the Fc region. In some embodiments, the RBD can be fused in frame with the Fc.
In some embodiments, the Fc region may be selected from the group consisting of: rFc, mFc, hFc, and hFc. For example, the Fc region can be rFc. For example, the Fc region may be mFc. For example, the Fc region may be hFc.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 7.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 8.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 9.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 10.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 11.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can be present at a concentration of 50ng/ml to 4000 ng/ml. For example, it may be 50ng/ml to 3500ng/ml, 50ng/ml to 3000ng/ml, 50ng/ml to 2000ng/ml, 50ng/ml to 1500ng/ml, 50ng/ml to 1000ng/ml, 50ng/ml to 900ng/ml or 50ng/ml to 800 ng/ml. For example, the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof can be 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 800ng/ml, 900ng/ml, 1000ng/ml, 1500ng/ml, 2000ng/ml, 2500ng/ml, 3000ng/ml, 3500ng/ml or 4000 ng/ml.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise a human ACE2 receptor protein or functionally active fragment thereof.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the extracellular domain ECD.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may be linked directly or indirectly to an Fc region.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may be fused in frame to the Fc region.
In some embodiments, the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc. For example, rFc. For example, mFc. For example, it is hFc.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 2.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 3.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 4.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the ACE2 receptor protein or functionally active fragment thereof.
In some embodiments, the label can include horseradish peroxidase (HRP) or an HRP-like enzyme.
In some embodiments, the reactive concentration of the ACE2 receptor protein or functionally active fragment thereof may be between 100ng/ml and 2000 ng/ml. For example, it may be 100ng/ml to 1500ng/ml, 100ng/ml to 1000ng/ml, 100ng/ml to 800ng/ml, 500ng/ml to 2000ng/ml, 500ng/ml to 1500ng/ml, 500ng/ml to 1000ng/ml or 500ng/ml to 800 ng/ml. For example, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml, 1000ng/ml, 1500ng/ml or 2000ng/ml may be used.
In some embodiments, the coating concentration of the RBD can be 50ng/ml to 4000 ng/ml. For example, it may be 50ng/ml to 3500ng/ml, 50ng/ml to 3000ng/ml, 50ng/ml to 2000ng/ml, 50ng/ml to 1500ng/ml, 50ng/ml to 1000ng/ml, 50ng/ml to 900ng/ml or 50ng/ml to 800 ng/ml. For example, it may be 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml or 1000 ng/ml.
In some embodiments, the reactive concentration of the ACE2 receptor protein or functionally active fragment thereof may be 500ng/ml to 1500ng/ml, while the coating concentration of the RBD may be 50ng/ml to 4000 ng/ml.
In some embodiments, it may comprise a sample diluent, which may have a pH of 6.0-9.6.
In some embodiments, it may further comprise a calibrator that may comprise one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the reference antibody can include antibodies 32-1,12-5 and 12-8.
In some embodiments, the concentration of the reference antibody can be between 50ng/ml and 1000 ng/ml.
In another aspect, the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which can comprise:
1) coating an ACE2 receptor protein or a functionally active fragment thereof in a solid support;
2) mixing and incubating a sample to be tested with SARS-CoV-2 spike protein or functional active fragment thereof to obtain an incubated product;
3) adding the incubated product of step 2) into step 1);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise a receptor binding Region (RBD), and the RBD can comprise one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the RBD.
In some embodiments, the label can include horseradish peroxidase (HRP) or an HRP-like enzyme.
In some embodiments, the RBD can be linked directly or indirectly to the Fc region.
In some embodiments, the RBD can be fused in frame with the Fc.
In some embodiments, the Fc region may be selected from the group consisting of: rFc, mFc, hFc, and hFc.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 7.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 8.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 9.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO 10.
In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 11.
In some embodiments, the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof can be from 50ng/ml to 4000 ng/ml.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may be linked directly or indirectly to an Fc region.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may be fused in frame to the Fc region.
In some embodiments, the Fc region may be selected from the group consisting of: rFc, mFc, hFc, and hFc.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 2.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 3.
In some embodiments, the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence set forth in SEQ ID No. 4.
In some embodiments, the coating concentration of the ACE2 receptor protein or functionally active fragment thereof may be from 1 μ g/ml to 20 μ g/ml. For example, it may be 1. mu.g/ml to 15. mu.g/ml, 1. mu.g/ml to 10. mu.g/ml, 1. mu.g/ml to 8. mu.g/ml, 1. mu.g/ml to 7. mu.g/ml, 1. mu.g/ml to 6. mu.g/ml or 1. mu.g/ml to 5. mu.g/ml. For example, it may be 2. mu.g/ml-10. mu.g/ml, 2. mu.g/ml-8. mu.g/ml, 2. mu.g/ml-6. mu.g/ml, 3. mu.g/ml-10. mu.g/ml, 3. mu.g/ml-8. mu.g/ml or 3. mu.g/ml-6. mu.g/ml. For example, it may be 1. mu.g/ml, 2. mu.g/ml, 3. mu.g/ml, 4. mu.g/ml, 5. mu.g/ml, 6. mu.g/ml, 7. mu.g/ml, 8. mu.g/ml, 9. mu.g/ml, 10. mu.g/ml, 15. mu.g/ml or 20. mu.g/ml.
In some embodiments, the reaction concentration of the RBD can be from 10ng/ml to 200 ng/ml. For example, it may be 10ng/ml to 150ng/ml, 10ng/ml to 100ng/ml, 10ng/ml to 80ng/ml, 10ng/ml to 50ng/ml, 10ng/ml to 40ng/ml, 10ng/ml to 30ng/ml or 10ng/ml to 20 ng/ml. For example, it may be 20ng/ml to 150ng/ml, 20ng/ml to 100ng/ml, 20ng/ml to 80ng/ml, 20ng/ml to 50ng/ml, 20ng/ml to 40ng/ml or 20ng/ml to 30 ng/ml. For example, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 400ng/ml, 90ng/ml or 100 ng/ml.
In some embodiments, when the coating concentration of the ACE2 receptor protein or functionally active fragment thereof may be 1. mu.g/ml to 10. mu.g/ml, the reaction concentration of the RBD may be 20ng/ml to 150 ng/ml.
In some embodiments, when the coating concentration of the ACE2 receptor protein or functionally active fragment thereof may be 3 μ g/ml to 6 μ g/ml, the reaction concentration of the RBD may be 20ng/ml to 40 ng/ml.
In some embodiments, it may comprise a sample diluent, which may have a pH of 6.0-9.6.
In some embodiments, it may further comprise a calibrator that may comprise one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
In some embodiments, the reference antibody can include antibodies 32-1,12-5 and 12-8.
In some embodiments, the concentration of the reference antibody can be between 50ng/ml and 1000 ng/ml.
In the present application, the sample may be of natural or synthetic origin and may be obtained by any means known to the person skilled in the art. For example, the sample can be a tissue or liquid sample isolated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage. For example, the sample may be a research sample and a clinical sample. For example, the sample may also be a blood sample for transfusion or therapy. For example, the sample may also be synthetic and include, but is not limited to, in vitro cell culture components, including, but not limited to, conditioned medium, recombinant cells, and cell components. In the present application, the terms "sample" and "specimen" are used interchangeably.
In the present application, the sample is derived from a subject. In the present application, the subject generally refers to any organism, including but not limited to mammals, such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the present application, the subject may be a human, a pet, or a livestock. In the present application, the subject may be a COVID-19 convalescent patient, a healthy subject, and/or a subject who has been treated with a COVID-19 vaccine.
In some embodiments, the method may comprise the steps of:
step one, coating antigen: diluting SARS-CoV-2 spike protein or its functional active fragment with coating liquid, and adding the dilution liquid of SARS-CoV-2 spike protein or its functional active fragment into the enzyme-labeled hole of reaction plate.
Step two, the sample to be tested is added and incubated for 30-60 minutes (e.g., 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes).
Step three, adding the competitive binding agent, and incubating for 30-60 minutes (e.g., 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes).
And step four, developing color and stopping reading.
In some embodiments, the test sample can be incubated at 35 ℃ to 40 ℃ (e.g., incubation can be at 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 36.6 ℃, 36.7 ℃, 36.8 ℃, 36.9 ℃, 37 ℃, 37.1 ℃, 37.2 ℃, 37.3 ℃, 37.4 ℃, 37.5 ℃, 37.6 ℃, 37.7 ℃, 37.8 ℃, 37.9 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃ or 40 ℃).
In some embodiments, the competitive binding agent can be incubated at 35 ℃ to 40 ℃ (e.g., incubation temperature can be 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 36.6 ℃, 36.7 ℃, 36.8 ℃, 36.9 ℃, 37 ℃, 37.1 ℃, 37.2 ℃, 37.3 ℃, 37.4 ℃, 37.5 ℃, 37.6 ℃, 37.7 ℃, 37.8 ℃, 37.9 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃ or 40 ℃).
In some embodiments, the color development process can be in 35-40 degrees C environment (for example, can be 35 degrees, 35.5 degrees, 36 degrees, 36.5 degrees, 36.6 degrees, 36.7 degrees, 36.8 degrees, 36.9 degrees, 37 degrees, 37.1 degrees, 37.2 degrees, 37.3 degrees, 37.4 degrees, 37.5 degrees, 37.6 degrees, 37.7 degrees, 37.8 degrees, 37.9 degrees, 38 degrees, 38.5 degrees, 39 degrees, 39.5 degrees or 40 degrees C).
Some embodiments of the invention:
1. a detection kit for SARS-CoV-2 neutralizing antibodies comprising:
1) SARS-CoV-2 spike protein or a functionally active fragment thereof; and
2) a competitive binding agent comprising a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
2. The kit of embodiment 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region, RBD.
3. The kit of any of embodiments 1-2, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
4. The kit of any of embodiments 1-3, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-COV-2 spike protein.
5. The kit of any one of embodiments 2-4, wherein the RBD is linked directly or indirectly to an Fc.
6. The kit of embodiment 5, wherein the RBD is fused in frame to the Fc.
7. The kit according to any one of embodiments 5-6, wherein said Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
8. The kit of embodiment 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 7.
9. The kit of embodiment 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 8.
10. The kit of embodiment 7, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 9.
11. The kit of embodiment 3, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 10.
12. The kit of embodiment 4, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 11.
13. The kit according to any one of embodiments 1-12, wherein the competing binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
14. The kit of embodiment 13, wherein the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
15. The kit according to any one of embodiments 13-14, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the extracellular domain ECD.
16. The kit according to any one of embodiments 13-15, wherein the ACE2 receptor protein or a functionally active fragment thereof is linked directly or indirectly to an Fc region.
17. The kit of embodiment 16, wherein the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
18. The kit according to any one of embodiments 16-17, wherein said Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
19. The kit of embodiment 18, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 2.
20. The kit of embodiment 18, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 3.
21. The kit of embodiment 18, wherein the competing binding agent comprises the amino acid sequence set forth in SEQ ID No. 4.
22. The kit of embodiment 13, wherein the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
23. The kit of embodiment 22, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
24. The kit of any one of embodiments 1-23, comprising a sample diluent having a pH of 6.0-9.6.
25. The kit of any of embodiments 1-24, further comprising a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof.
26. The kit of embodiment 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
27. The kit of embodiment 25, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
28. The kit of any one of embodiments 25-27, the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35;
the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or
The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO 45-47.
29. The kit of any one of embodiments 25-28, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22;
the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or
The VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
30. The kit according to any one of embodiments 25-29, wherein the reference antibody comprises antibodies 32-1,12-5 and 12-8.
31. The kit according to any one of embodiments 25-30, wherein the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
32. The kit of any of embodiments 1-31, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the SARS-CoV-2 spike protein or functionally active fragment thereof.
33. The kit of embodiment 32, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
34. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
the kit of embodiment 1 or 13 is used.
35. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
1) coating the SARS-CoV-2 spike protein or a functionally active fragment thereof in a solid support;
2) adding a sample to be detected into the SARS-CoV-2 spike protein or the functional active fragment thereof in the step 1) to react;
3) adding a competing binding agent to step 2);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the competitive binding agent comprises a substance that is capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof;
said SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD; the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
36. The method of embodiment 35, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
37. The method of any of embodiments 35-36, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-CoV-2 spike protein.
38. The method of any one of embodiments 35-37, wherein the RBD is linked directly or indirectly to an Fc region.
39. The method of embodiment 38, wherein the RBD is fused in frame with the Fc.
40. The method according to any one of embodiments 38-39, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
41. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 7.
42. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 8.
43. The method of embodiment 40, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 9.
44. The method of embodiment 36, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 10.
45. The method of embodiment 37, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 11.
46. The method of any of embodiments 35-45, wherein the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof is from 50ng/ml to 4000 ng/ml.
47. The method according to any one of embodiments 35 to 46, wherein the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
48. The method according to embodiment 47, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the extracellular domain ECD.
49. The method according to any one of embodiments 47 to 48, wherein the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
50. The method of embodiment 49, wherein the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
51. The method according to any one of embodiments 49-50, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
52. The method according to embodiment 51, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown as SEQ ID NO 2.
53. The method according to embodiment 51, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown as SEQ ID NO 3.
54. The method according to embodiment 51, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown as SEQ ID NO 4.
55. The method according to any one of embodiments 35 to 54, wherein the ACE2 receptor protein or functionally active fragment thereof comprises one or more labels capable of directly or indirectly generating a signal indicating the presence and/or amount of the ACE2 receptor protein or functionally active fragment thereof.
56. The method of embodiment 55, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
57. The method according to any one of embodiments 35 to 56, wherein the concentration of the ACE2 receptor protein or functionally active fragment thereof is 100ng/ml to 2000 ng/ml.
58. The method of any one of embodiments 35-57, comprising a sample diluent having a pH of 6.0-9.6.
59. The method of any of embodiments 35-58, further comprising a calibrator comprising one or more reference antibody molecules, or antigen-binding fragments thereof, that specifically recognize the SARS-CoV-2 spike protein, or functionally active fragment thereof.
60. The method of embodiment 59, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
61. The method of embodiment 59, wherein the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof.
62. The method according to any one of embodiments 59-61, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35;
the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or
The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO 45-47.
63. The method according to any one of embodiments 59-62, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22;
the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or
The VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
64. The method according to any one of embodiments 59-63, wherein the reference antibody comprises antibodies 32-1,12-5 and 12-8.
65. The method according to any one of embodiments 59-64, wherein the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
66. The method of embodiment 35, wherein the sample is selected from the group consisting of: urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirates, and bronchial lavage.
67. The method of embodiment 66, wherein the sample is derived from a subject.
68. The method of embodiment 67, wherein the subject is a COVID-19 convalescent patient, a healthy subject, and/or a subject who has received a COVID-19 vaccine treatment.
69. A method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
1) coating an ACE2 receptor protein or a functionally active fragment thereof in a solid support;
2) mixing and incubating a sample to be tested with SARS-CoV-2 spike protein or functional active fragment thereof to obtain an incubated product;
3) adding the incubated product of step 2) into step 1);
4) detecting SARS-CoV-2 neutralizing antibody in the sample to be detected;
wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding Region (RBD) and the RBD comprises one or more markers capable of directly or indirectly generating a signal indicative of the presence and/or amount of the RBD.
70. The method of embodiment 69, wherein the label comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
71. The method of any one of embodiments 69-70, wherein said RBD is directly or indirectly linked to an Fc region.
72. The method of embodiment 71, wherein the RBD is fused in frame with the Fc.
73. The method according to any one of embodiments 71-72, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
74. The method of embodiment 73, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 7.
75. The method of embodiment 73, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 8.
76. The method of embodiment 73, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 9.
77. The method of embodiment 69, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 10.
78. The method of embodiment 69, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO 11.
79. The method of any of embodiments 69-78, wherein the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof is from 50ng/ml to 4000 ng/ml.
80. The method according to embodiments 69-79, wherein the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to an Fc region.
81. The method of embodiment 80, wherein the ACE2 receptor protein or functionally active fragment thereof is fused in frame to the Fc region.
82. The method according to any one of embodiments 80-81, wherein the Fc region is selected from the group consisting of: rFc, mFc, hFc, and hFc.
83. The method according to embodiment 82, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO 2.
84. The method according to embodiment 82, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown as SEQ ID NO 3.
85. The method according to embodiment 82, wherein the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown as SEQ ID NO 4.
86. The method according to any one of embodiments 69 to 85, wherein the concentration of the ACE2 receptor protein or functionally active fragment thereof is 100ng/ml to 2000 ng/ml.
87. The method of any one of embodiments 69-86, comprising a sample diluent having a pH of 6.0-9.6.
88. The method of any of embodiments 69-87, wherein said detecting is obtained by comparison to a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize said SARS-CoV-2 spike protein or functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein:
the VH comprises HCDR1, HCDR2 and HCDR3 of amino acid sequences shown as SEQ ID NO. 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of amino acid sequences shown as SEQ ID NO. 33-35;
the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO. 36-38, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO. 39-41; or
The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO 42-44, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO 45-47.
89. The method of embodiment 88, wherein said detecting is obtained by comparison to a calibrator comprising one or more reference antibody molecules or antigen-binding fragments thereof that specifically recognize said SARS-CoV-2 spike protein or functionally active fragment thereof; the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein:
the VH comprises an amino acid sequence shown as SEQ ID NO. 21, and the VL comprises an amino acid sequence shown as SEQ ID NO. 22;
the VH comprises an amino acid sequence shown as SEQ ID NO. 24, and the VL comprises an amino acid sequence shown as SEQ ID NO. 25; or
The VH comprises an amino acid sequence shown as SEQ ID NO. 27, and the VL comprises an amino acid sequence shown as SEQ ID NO. 28.
90. The method according to any one of embodiments 88-89, wherein the reference antibody comprises antibodies 32-1,12-5 and 12-8.
91. The method according to any one of embodiments 88-90, wherein the concentration of the reference antibody is between 50ng/ml and 1000 ng/ml.
92. The method of embodiment 69, wherein the sample to be tested is selected from the group consisting of: urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirates, and bronchial lavage.
93. The method of embodiment 92, wherein the test sample is derived from a subject.
94. The method of embodiment 93, wherein the subject is a COVID-19 convalescent patient, a healthy subject, and/or a subject who has received a COVID-19 vaccine treatment.
Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.
Examples
EXAMPLE 1 ELISA assay for SARS-CoV-2 neutralizing antibodies
1.1 activation of Horse Radish Peroxidase (HRP): dissolving horseradish peroxidase (HRP) powder in ddH2O10 mg/ml horseradish peroxidase (HRP) solution, sodium periodate powder dissolved in ddH2O to prepare a 0.1M sodium periodate solution, and mixing a horseradish peroxidase (HRP) solution with a sodium periodate solution in a volume ratio of 1: 0.51, mixing evenly, and keeping out of the sun for 1 hour at the temperature of 2-8 ℃; adding 0.26 volume of glycol of horseradish peroxidase (HRP) solution, and keeping away from light at 2-8 deg.C for 20 min; 50mL of an ultrafiltration tube 6000-7500rpm for 10-20 minutes; 1mM acetic acid buffer solution, centrifuging for 3-5 times; storing at-80 deg.C.
1.2 Horse Radish Peroxidase (HRP) labeled human ACE2 receptor protein: according to the mass ratio of 1: 4, adding human ACE2 receptor protein and activated Horse Radish Peroxidase (HRP), adding sodium carbonate buffer (pH9.6) with the volume total amount of human ACE2 receptor protein and Horse Radish Peroxidase (HRP) being 1/5, and placing for 1 hour at room temperature in a dark place; adding a reducing agent with the volume of 1/10 horseradish peroxidase (HRP), and standing for 1 hour at room temperature in a dark place; adding terminator in the volume of 1/10 horseradish peroxidase (HRP), and standing at room temperature for 30 minutes in the dark, wherein the solution can be used or stored at 4 ℃.
1.3 coating antigen: 1 Xcoating solution (10 Xcoating solution is 15.9g/L Na)2CO3+29.3g/L NaHCO3By ddH2O diluted to 1x for use) to dilute the RBD antigen to 0.3 μ g/mL and mix well, add the RBD antigen dilution to the enzyme-labeled wells of the reaction plate, 100 μ L per well, cover the reaction plate and place it overnight at 4 ℃ to coat, the upper layer is liquid, and the coated RBD antigen is adsorbed on the reaction plate.
1.4 sealing: washing with 1 Xwashing solution (20 Xwashing solution is 2.4g/L KH)2PO4+29g/L NaHPO4·12H2O +160g/L NaCl +4g/L KCl +10mL/L Tween-20+2.24g/L P300 using ddH2O diluted to 1x use) wash the plate once per well at 350 μ LRemoving the coating solution, drying the washing solution in the reaction plate on absorbent paper, quickly adding 100-200 mu L of confining liquid (104.5 g/L10 x PBS +10g/L Casein +50g/L sucrose +2.24g/L P300) into each hole, placing the hole in an incubator at 37 ℃ and incubating for 2 hours; and pouring the confining liquid, and spin-drying by a plate-throwing machine or beating the confining liquid on absorbent paper to obtain the reaction plate, wherein if the reaction plate is not used immediately, the reaction plate is placed in a sealing bag and added with a drying bag to be stored at 2-8 ℃, and the drying bag is required to be positioned at the bottom of the reaction plate and cannot contact with the holes of the reaction plate.
1.5 adding a sample to be tested: all components including the serum to be tested were equilibrated to room temperature in advance, the reaction plate was equilibrated to room temperature and then removed from the sealed bag, 80. mu.L of sample diluent (0.02M PBS + 0.9% NaCl + 0.5% BSA + 0.1% Tween-20+ 0.2% P300, pH7.4) was added to each well, 20. mu.L of calibrator (calibrator was antibody of different concentrations, 1000ng/mL, 800ng/mL, 400ng/mL, 200ng/mL, 100ng/mL and 50ng/mL, respectively, as described in example 6) and 20. mu.L of serum to be tested were added to the corresponding well to which the sample diluent had been added, the plate was attached, the wells were mixed well by shaking on a micro-shaker for 60 seconds, placed in an incubator at 37 ℃ and incubated for 0.5 hours.
1.6 addition of enzyme-labeled conjugates: using a sample diluent at 1: dilution of ACE2-HRP (initial concentration 5mg/mL) was performed at 3000, the patch was carefully removed, 100. mu.L of diluted ACE2-HRP was added to each well, the patch was replaced and attached, the wells were mixed well by shaking on a micro shaker for 60 seconds, placed in a 37 ℃ incubator and incubated for 0.5 hours.
1.7 plate washing: carefully remove the plate, wash the plate six times with 1 Xwashing solution (recommended plate washer, 350. mu.L of solution per well), and finally dry on absorbent paper or spin-dry with a plate-throwing machine.
1.8 color development: the color solution A (2.1g/L citric acid monohydrate +8.448g/L sodium acetate +0.5g/L carbamide peroxide +0.08g/L EDTA +2mL/L P300) and solution B (10 mL/L100 Xbasic buffer (105.6g/L citric acid monohydrate +10.3g/L sodium acetate +2mL/L P300) +100mL/L glycerin +0.5g/L TMB hydrochloride +1.67mL/L Na2S2O3(20mg/mL) +2mL/L P300) according to the volume ratio of 1:1 to obtain a color development working solution, adding 100 μ L of color development working solution into each hole, replacing the plate and sticking, and reacting at 37 deg.C in the dark for 1And 0 minute.
1.9 reading:
carefully uncovering the plate, adding 50 mu L of stop solution (252.5g/L citric acid monohydrate +26.6mL/L concentrated sulfuric acid) into each hole to stop the reaction, detecting with 450nm single wavelength of a microplate reader, and measuring the light absorption value of each hole.
1.10. Critical value determination: the method is used for detecting the serum to be detected, the sample is judged to be positive when the sample inhibition rate is greater than 15%, the sample is judged to be negative when the sample inhibition rate is less than 15%, the inhibition rate is (1-absorbance of the sample to be detected/absorbance of negative control) x 100%, and the inhibition rate reflects the neutralization efficiency of the antibody in the serum to be detected.
EXAMPLE 2 ELISA assay for SARS-CoV-2 neutralizing antibodies
The steps and reagents corresponding thereto which are not specifically described in this example are the same as those in example 1.
1.1 activation of Horse Radish Peroxidase (HRP): dissolving horseradish peroxidase (HRP) powder in ddH2O10 mg/ml horseradish peroxidase (HRP) solution, sodium periodate powder dissolved in ddH2O to prepare a 0.1M sodium periodate solution, and mixing a horseradish peroxidase (HRP) solution with a sodium periodate solution in a volume ratio of 1: mixing at 0.51 deg.c and heliophobic at 2-8 deg.c for 1 hr; adding 0.26 volume of glycol of horseradish peroxidase (HRP) solution, and keeping away from light at 2-8 deg.C for 20 min; 50ml of an ultrafiltration tube 6000-7500rpm for 10-20 minutes; 1mM acetic acid buffer solution, centrifuging for 3-5 times; storing at-80 deg.C.
1.2 horseradish peroxidase (HRP) labeled RBD protein: according to the mass ratio of 1: 4 adding RBD protein and activated Horse Radish Peroxidase (HRP), adding sodium carbonate buffer solution (pH9.6) with the volume total amount of the RBD protein and the Horse Radish Peroxidase (HRP) being 1/5, and placing for 1 hour at room temperature in a dark place; adding a reducing agent with the volume of 1/10 horseradish peroxidase (HRP), and standing for 1 hour at room temperature in a dark place; adding terminator in the volume of 1/10 horseradish peroxidase (HRP), and standing at room temperature for 30 minutes in the dark, wherein the solution can be used or stored at 4 ℃.
1.3 coating protein: diluting ACE2 protein to 0.3 μ g/mL with 1 Xcoating solution, mixing well, adding ACE2 protein diluent into enzyme-labeled wells of a reaction plate at a volume of 100 μ L per well, covering the reaction plate, placing the reaction plate at 4 ℃ for overnight coating, and adsorbing the coated ACE2 protein on the reaction plate.
1.4 sealing: washing the reaction plate once by using 350 mu L of washing liquid per hole of 1x to wash out the coating liquid, drying the washing liquid in the reaction plate on absorbent paper, quickly adding 100-200 mu L of confining liquid per hole, placing the reaction plate in an incubator at 37 ℃, and incubating for 2 hours; and pouring the confining liquid, and spin-drying by a plate-throwing machine or beating the confining liquid on absorbent paper to obtain the reaction plate, wherein if the reaction plate is not used immediately, the reaction plate is placed in a sealing bag and added with a drying bag to be stored at 2-8 ℃, and the drying bag is required to be positioned at the bottom of the reaction plate and cannot contact with the holes of the reaction plate.
1.5 adding a sample to be tested: all components including the sample are taken out to be balanced to the room temperature in advance, the reaction plate is taken out from the sealed bag after being balanced to the room temperature, after the sample is diluted by the sample diluent in a two-time gradient manner, 10 mu L of positive control, negative control and a series of samples with different dilution times are respectively added into each hole, and 90 mu L of sample diluent is added.
1.6 addition of enzyme-labeled conjugates: RBD-HRP was diluted to the desired concentration (initial concentration 5mg/mL) using the sample diluent, 100. mu.L of diluted RBD-HRP was added to each well, the plate was attached, the wells were mixed well by shaking on a micro-shaker for 60 seconds, placed in an incubator at 37 ℃ and incubated for 0.5 h.
1.7 transfer: carefully uncover the plate, transfer 100 μ L of sample/control reaction solution from a 96-well PCR plate to the corresponding well of the reaction plate, attach the plate, place in a 37 ℃ incubator, incubate for 0.5 h.
1.8 plate washing: carefully remove the plate, wash the plate six times with 1 Xwashing solution (recommended plate washer, 350. mu.L of solution per well), and finally dry on absorbent paper or spin-dry with a plate-throwing machine.
1.9 color development: and uniformly mixing the color development solution A and the color development solution B according to the volume ratio of 1:1 to obtain color development working solution, adding 100 mu L of the color development working solution into each hole, replacing the plates, sticking the plates, and reacting for 10 minutes at 37 ℃ in a dark place.
1.10 reading: carefully uncovering the plate, adding 50 mu L of stop solution into each hole to stop the reaction, detecting with 450nm single wavelength of an enzyme-labeling instrument, and measuring the light absorption value of each hole.
1.11 titration value determination: the sample is tested using the method described above, and when the inhibition of this sample is > 15% at a dilution factor and < 15% at the next dilution gradient at that dilution factor, then the dilution is the titer of this sample. The titer is the maximum dilution that still produces a positive result, reflecting the titer of the antibody.
Example 3 Effect of different pH sample dilutions on assay results
Nonspecific binding of serum samples was reduced by changing the pH of the sample dilutions. As shown in Table 1, the test procedure of example 1 was followed using RBD-mFc coated plates and horseradish peroxidase (HRP) labeled ACE2-mFc to test the serum samples of non-SARS-CoV-2 infected human, i.e., negative sera, using sample dilutions at pH6.0, pH7.8, and pH9.6, respectively, and the inhibition rates of the positive sera and the negative sera diluted with sample dilutions at different pH were compared. The result shows that the universal sample diluent with pH6.0 can reduce the influence of non-specific binding in negative serum on the reaction and can not influence the detection of positive samples.
TABLE 1 Effect of different pH sample dilutions on assay results
Example 4 Effect of different molecular formats of RBD and ACE2 on assay results
According to the established ELISA detection method (the operation steps are the same as those of example 1 or example 2), horseradish peroxidase (HRP) -labeled RBD-His and RBD-mFc are added on an ACE2-mFc coated plate, and horseradish peroxidase (HRP) -labeled ACE2-His, ACE2-hFc, ACE2-mFc and ACE2-rFc are added on the RBD-His coated plate. Two replicates per sample were tested for 0. mu.g/mL RBD neutralizing antibody and 1. mu.g/mL RBD neutralizing antibody, respectively. The results are shown in table 2, and the signal-to-noise ratio and stability of the selected Fc-tagged RBD and ACE2 are significantly improved compared with those of His-tagged RBD and ACE 2.
TABLE 2 Effect of differently labeled RBD and ACE2 on test results
Example 5 Effect of RBD and ACE2 amounts on test results
5.1 reaction plates with five different concentrations of RBD-mFc were used, using a 1: 2000 and 1: ACE2-mFc-HRP was diluted at 4000 two dilution ratios, and 25 covd-19 vaccine sera, i.e. positive sera and 25 negative sera, were tested according to the procedure of example 1, comparing sensitivity, specificity and linear range at different RBD-mFc coating concentrations and ACE2-mFc-HRP dilution ratios.
5.2 Using five reaction plates with different full-length S protein concentrations, the procedure is as in 5.1, comparing sensitivity, specificity and linear range for different full-length S protein coating concentrations and dilution ratio of ACE 2-mFc-HRP.
5.3 Using five reaction plates of different trimer S protein concentrations, the procedure is as in 5.1, comparing the sensitivity, specificity and linear range of different trimer S protein coating concentrations and the dilution ratio of ACE 2-mFc-HRP.
The results show that the detection sensitivity can be significantly improved by optimizing the plate-coating concentration of RBD-mFc or full-length S protein or trimer S protein, as shown in tables 3-5.
TABLE 3 RBD-mFc and ACE2-mFc-HRP dosage optimization results
TABLE 4 full-Length S protein and ACE2-mFc-HRP dosage optimization results
TABLE 5 trimer S protein and ACE2-mFc-HRP dosage optimization results
Optimizing ACE2-mFc coating amount and RBD-mFc-HRP dosage
RBD-mFc-HRP was diluted with five dilution ratios of 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml and 30ng/ml using reaction plates of three different ACE2-mFc concentrations, and the detection line differences at different ACE2-mFc coating concentrations and RBD-mFc-HRP dilution ratios were compared by detecting antibodies at the gradient dilution concentrations according to the procedure of example 2.
The result shows that the detection sensitivity can be remarkably improved by optimizing the plate-wrapping concentration of ACE2-mFc and the use concentration of RBD-mFc-HRP, and the result is shown in Table 6.
TABLE 6 ACE2-mFc coating amount and RBD-HRP dosage optimization
EXAMPLE 6 selection of calibrator
A plurality of strains of natural humanized neutralizing antibodies are screened out through single B cells, and the natural form of the neutralizing antibodies in human bodies can be well simulated through true virus verification, wherein the neutralizing antibodies have good neutralizing effect. 3 strains of antibodies with different epitopes are screened out from the test sample and mixed to prepare a standard substance, and six calibration substances with antibody concentrations of C.1-C.6 are prepared, wherein the antibody concentrations of C.1-C.6 are respectively 1000ng/mL, 800ng/mL, 400ng/mL, 200ng/mL, 100ng/mL and 50 ng/mL. The absorbance at 450nm of these six antibody concentration standards was measured according to the procedure of example 1. The antibody concentration and absorbance of the calibrator were plotted using Graphpad version 6.02 software. The reactivity (reaction slope) is estimated to be closer to a real sample according to the result, and the content change trend of the neutralizing antibody in the human body can be simulated more truly, as shown in fig. 1.
EXAMPLE 7 determination of the Cutoff value
The Cut-off value is the amount of sample examined and is used to determine whether the result is above or below a clinical or analytical decision point. Setting the cut-off value gives a brief combination of sensitivity and specificity. The Cutoff value was determined from the evaluation of 27 sera from convalescent patients, 1452 serum samples from non-neocoronal infected human, 72 sera from vaccine (one third placebo) by SPSS software analysis, based on which our kit had a sensitivity of > 90% and a specificity of > 99% following the procedure of example 1. The evaluation of vaccine effect is 95% consistent with the results of the euvirus experiment. Sensitivity: i.e., sensitivity, indicates the detection rate of positive samples. For example, 0.977 indicates that 99.7% of positive samples can be correctly judged as positive results. Specificity: indicating the detection rate of negative samples. 99.1% of negative samples can be correctly judged as negative results as shown in 0.991, the detection variable results are shown in table 7, the receiver operating characteristic curve (or ROC curve) is shown in fig. 2, and the curve coordinates and the detection results are shown in table 8.
TABLE 7 test variable results
TABLE 8 Curve coordinates and test variable results Table
Sequence listing
<110> Nanjing Novozan Biotechnology GmbH
<120> a kit and method for detecting SARS-CoV-2 neutralizing antibody
<130> 0129-PA-003
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 723
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ACE2 amino acid sequence
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Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
595 600 605
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
610 615 620
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
625 630 635 640
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
660 665 670
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
675 680 685
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
690 695 700
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
705 710 715 720
Pro Val Ser
<210> 2
<211> 954
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ACE2-mFc (mIgG 1-CH) amino acid sequence
<400> 2
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
595 600 605
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
610 615 620
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
625 630 635 640
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
660 665 670
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
675 680 685
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
690 695 700
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
705 710 715 720
Pro Val Ser Gly Gly Gly Ser Val Pro Arg Asp Cys Gly Cys Lys Pro
725 730 735
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
740 745 750
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
755 760 765
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
770 775 780
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
785 790 795 800
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
805 810 815
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
820 825 830
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
835 840 845
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
850 855 860
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
865 870 875 880
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
885 890 895
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
900 905 910
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
915 920 925
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
930 935 940
Glu Lys Ser Leu Ser His Ser Pro Gly Lys
945 950
<210> 3
<211> 955
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ACE2-rFc amino acid sequence
<400> 3
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
595 600 605
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
610 615 620
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
625 630 635 640
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
660 665 670
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
675 680 685
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
690 695 700
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
705 710 715 720
Pro Val Ser Gly Gly Gly Ser Ala Pro Ser Thr Cys Ser Lys Pro Thr
725 730 735
Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro
740 745 750
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
755 760 765
Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
770 775 780
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
785 790 795 800
Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
805 810 815
Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
820 825 830
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
835 840 845
Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
850 855 860
Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
865 870 875 880
Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
885 890 895
Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr
900 905 910
Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
915 920 925
Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
930 935 940
Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
945 950 955
<210> 4
<211> 959
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ACE2-hFc (HuIgG 1-CH) amino acid sequence
<400> 4
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
595 600 605
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
610 615 620
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
625 630 635 640
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
660 665 670
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
675 680 685
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
690 695 700
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
705 710 715 720
Pro Val Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His
725 730 735
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
740 745 750
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
755 760 765
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
770 775 780
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
785 790 795 800
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
805 810 815
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
820 825 830
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
835 840 845
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
850 855 860
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
865 870 875 880
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
885 890 895
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
900 905 910
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
915 920 925
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
930 935 940
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
945 950 955
<210> 5
<211> 740
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ACE2-AVI amino acid sequence
<400> 5
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys
595 600 605
Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr
610 615 620
Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys
625 630 635 640
Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655
Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys
660 665 670
Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg
675 680 685
Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser
690 695 700
Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro
705 710 715 720
Pro Val Ser Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile
725 730 735
Glu Trp His Glu
740
<210> 6
<211> 223
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> RBD amino acid sequence
<400> 6
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 7
<211> 455
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> RBD-mFc (mIgG 1-CH) amino acid sequence
<400> 7
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly
210 215 220
Gly Gly Gly Ser Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
225 230 235 240
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
245 250 255
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
260 265 270
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
275 280 285
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
290 295 300
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
340 345 350
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
355 360 365
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
370 375 380
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
385 390 395 400
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
405 410 415
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
420 425 430
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
435 440 445
Leu Ser His Ser Pro Gly Lys
450 455
<210> 8
<211> 456
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> RBD-rFc amino acid sequence
<400> 8
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly
210 215 220
Gly Gly Gly Ser Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro
225 230 235 240
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile
275 280 285
Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln
290 295 300
Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln
305 310 315 320
Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro
340 345 350
Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser
355 360 365
Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr
385 390 395 400
Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe
420 425 430
Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Arg Ser Pro Gly Lys
450 455
<210> 9
<211> 460
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> RBD-hFc (HuIgG 1-CH) amino acid sequence
<400> 9
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly
210 215 220
Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
225 230 235 240
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
245 250 255
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
260 265 270
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
275 280 285
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
290 295 300
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
305 310 315 320
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
325 330 335
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
340 345 350
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
355 360 365
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
370 375 380
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
385 390 395 400
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
405 410 415
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
420 425 430
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
435 440 445
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 10
<211> 1195
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> S protein amino acid sequence
<400> 10
Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr
1 5 10 15
Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser
20 25 30
Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn
35 40 45
Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
50 55 60
Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
65 70 75 80
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
85 90 95
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
100 105 110
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
115 120 125
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
130 135 140
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
145 150 155 160
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
165 170 175
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
180 185 190
Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
195 200 205
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
210 215 220
Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp
225 230 235 240
Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr
245 250 255
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
260 265 270
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
275 280 285
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
290 295 300
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
305 310 315 320
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
325 330 335
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
340 345 350
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
355 360 365
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
370 375 380
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
385 390 395 400
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
405 410 415
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
420 425 430
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
435 440 445
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
450 455 460
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
465 470 475 480
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
485 490 495
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
500 505 510
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
530 535 540
Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr
545 550 555 560
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
565 570 575
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
580 585 590
Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro
595 600 605
Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser
610 615 620
Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala
625 630 635 640
Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
645 650 655
Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg
660 665 670
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
675 680 685
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
690 695 700
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
705 710 715 720
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
725 730 735
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
740 745 750
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
755 760 765
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
770 775 780
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
785 790 795 800
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
805 810 815
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
820 825 830
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
835 840 845
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
850 855 860
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
865 870 875 880
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
885 890 895
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
900 905 910
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
915 920 925
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
930 935 940
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
945 950 955 960
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
965 970 975
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
980 985 990
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
995 1000 1005
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
1010 1015 1020
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1025 1030 1035 1040
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1045 1050 1055
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
1060 1065 1070
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
1075 1080 1085
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
1090 1095 1100
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
1105 1110 1115 1120
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
1125 1130 1135
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
1140 1145 1150
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
1155 1160 1165
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
1170 1175 1180
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln
1185 1190 1195
<210> 11
<211> 1224
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> trimeric amino acid sequence of S protein
<400> 11
Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr
1 5 10 15
Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser
20 25 30
Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn
35 40 45
Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
50 55 60
Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
65 70 75 80
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
85 90 95
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
100 105 110
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
115 120 125
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
130 135 140
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
145 150 155 160
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
165 170 175
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
180 185 190
Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
195 200 205
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
210 215 220
Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp
225 230 235 240
Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr
245 250 255
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
260 265 270
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
275 280 285
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
290 295 300
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
305 310 315 320
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
325 330 335
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
340 345 350
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
355 360 365
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
370 375 380
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
385 390 395 400
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
405 410 415
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
420 425 430
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
435 440 445
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
450 455 460
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
465 470 475 480
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
485 490 495
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
500 505 510
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
530 535 540
Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr
545 550 555 560
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
565 570 575
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
580 585 590
Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro
595 600 605
Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser
610 615 620
Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala
625 630 635 640
Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
645 650 655
Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg
660 665 670
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
675 680 685
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
690 695 700
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
705 710 715 720
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
725 730 735
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
740 745 750
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
755 760 765
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
770 775 780
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
785 790 795 800
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
805 810 815
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
820 825 830
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
835 840 845
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
850 855 860
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
865 870 875 880
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
885 890 895
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
900 905 910
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
915 920 925
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
930 935 940
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
945 950 955 960
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
965 970 975
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
980 985 990
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
995 1000 1005
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
1010 1015 1020
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1025 1030 1035 1040
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1045 1050 1055
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
1060 1065 1070
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
1075 1080 1085
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
1090 1095 1100
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
1105 1110 1115 1120
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
1125 1130 1135
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
1140 1145 1150
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
1155 1160 1165
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
1170 1175 1180
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile
1185 1190 1195 1200
Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu
1205 1210 1215
Trp Val Leu Leu Ser Thr Phe Leu
1220
<210> 12
<211> 345
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 32-1 VH
<400> 12
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtacag cctctgaaat caacgtcagt agcaactaca tgaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagtt ctttatgccg gtggtaccac atactacgca 180
gactccgtga aggacaggtt caccatctcc agagacaatt ccaagaacac gctgtatctt 240
caaatgaaca acctgagagc cgaggacacg gctttctatt actgtgcgag agaggcctat 300
gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345
<210> 13
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 32-1 VL
<400> 13
gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaactg cttaatagtt accctccgag caccttcggc 300
caagggacac gactggagat taaa 324
<210> 14
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 32-1 CL/nucleic acid sequence encoding 12-8 CL
<400> 14
cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaaca ggggagagtg t 321
<210> 15
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 12-5 VH
<400> 15
gaggtgcaac tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttggt gattatgcca tgagctgggt ccgccaagct 120
ccagagaagg ggctggagtg ggtctctggt attaattgga atggtggtag gacaggttat 180
gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240
ctgcaaatga acagtctgag agccgaggac acggccttgt attactgtgc gagatatctt 300
cagcactcat attttgactt gttattacct aatgatgctt ttgatatctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 16
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 12-5 VL
<400> 16
gaaattgtgg tgacgcagtc tccaggcacc ctgtctttgt ctccggggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agcaactact tagcctggta ccagcagaaa 120
cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag aagactggag 240
gccgaagatt ttgcagtgta ttactgtcaa cagtatggga gctcaccgct cactttcggc 300
ggagggacca aggtggagat caaa 324
<210> 17
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 12-5 CL
<400> 17
cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaac tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaaca ggggagagtg t 321
<210> 18
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 12-8 VH
<400> 18
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120
ccggggaggg gactggagtg gattgggtat atccattaca gtgggagcac caactacaac 180
ccctccctca ggagtcgact caccatatca gtagacacgt ccaagaacca gttctccctg 240
aagctgagct ccgtgaccgc tgcggacacg gccgtgtatt attgtgcgag agatcagggg 300
tatagcagtg gctggcctga tgcttttgat atctggggcc aagggacaat ggtcaccatc 360
tcttca 366
<210> 19
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleic acid sequence encoding 12-8 VL
<400> 19
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactga catcccagcc 180
aggttcagtg acagtgggtc tgggacagac ttcactctca ccatcaccag cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctgggac gttcggccaa 300
gggaccaagg tggaaatcaa a 321
<210> 20
<211> 990
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CH (Universal, IgG1 constant region) -encoding nucleic acid sequence
<400> 20
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210> 21
<211> 115
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 VH
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asn Val Ser Ser Asn
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Leu Tyr Ala Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Phe Tyr Tyr Cys Ala
85 90 95
Arg Glu Ala Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
100 105 110
Val Ser Ser
115
<210> 22
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 VL
<400> 22
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Leu Asn Ser Tyr Pro Pro
85 90 95
Ser Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 23
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 CL/12-8CL
<400> 23
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 24
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 VH
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Asn Trp Asn Gly Gly Arg Thr Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Tyr Leu Gln His Ser Tyr Phe Asp Leu Leu Leu Pro Asn Asp
100 105 110
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 25
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 VL
<400> 25
Glu Ile Val Val Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Arg Leu Glu
65 70 75 80
Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 26
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 CL
<400> 26
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 27
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 VH
<400> 27
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile His Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg
50 55 60
Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Gln Gly Tyr Ser Ser Gly Trp Pro Asp Ala Phe Asp Ile Trp
100 105 110
Gly Gln Gly Thr Met Val Thr Ile Ser Ser
115 120
<210> 28
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 VL
<400> 28
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Asp
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Gly
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 29
<211> 330
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CH (Universal, IgG1 constant region)
<400> 29
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 HCDR1
<400> 30
Glu Ile Asn Val Ser Ser Asn Tyr
1 5
<210> 31
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 HCDR2
<400> 31
Leu Tyr Ala Gly Gly Thr Thr
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 HCDR3
<400> 32
Ala Arg Glu Ala Tyr Ala Phe Asp Ile
1 5
<210> 33
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 LCDR1
<400> 33
Gln Gly Ile Ser Ser Tyr
1 5
<210> 34
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 LCDR2
<400> 34
Ala Ala Ser
1
<210> 35
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 32-1 LCDR3
<400> 35
Gln Leu Leu Asn Ser Tyr Pro Pro Ser Thr
1 5 10
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 HCDR1
<400> 36
Gly Phe Thr Phe Gly Asp Tyr Ala
1 5
<210> 37
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 HCDR2
<400> 37
Ile Asn Trp Asn Gly Gly Arg Thr
1 5
<210> 38
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 HCDR3
<400> 38
Ala Arg Tyr Leu Gln His Ser Tyr Phe Asp Leu Leu Leu Pro Asn Asp
1 5 10 15
Ala Phe Asp Ile
20
<210> 39
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 LCDR1
<400> 39
Gln Ser Val Ser Ser Asn Tyr
1 5
<210> 40
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 LCDR2
<400> 40
Gly Ala Ser
1
<210> 41
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-5 LCDR3
<400> 41
Gln Gln Tyr Gly Ser Ser Pro Leu Thr
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 HCDR1
<400> 42
Gly Gly Ser Ile Ser Ser Tyr Tyr
1 5
<210> 43
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 HCDR2
<400> 43
Ile His Tyr Ser Gly Ser Thr
1 5
<210> 44
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 HCDR3
<400> 44
Ala Arg Asp Gln Gly Tyr Ser Ser Gly Trp Pro Asp Ala Phe Asp Ile
1 5 10 15
<210> 45
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 LCDR1
<400> 45
Gln Ser Val Ser Ser Tyr
1 5
<210> 46
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 LCDR2
<400> 46
Asp Ala Ser
1
<210> 47
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 12-8 LCDR3
<400> 47
Gln Gln Arg Ser Asn Trp Pro Gly Thr
1 5
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