Monoclonal antibodies against sars-cov-2 spike protein s1 and uses thereof

Abstract

The present application discloses a monoclonal antibody against SARS-CoV-2 spike protein S1 and its application. In one embodiment, the antibody comprises: a V H CDR1 having a V H CDR2 having a V H CDR3 having a V L CDR1 having a V L CDR2 having a V L CDR3. The antibody can be used for rapid detection or screening of SARS-CoV-2 infection. The antibody can also be used in the preparation of drugs for treating or preventing diseases related to SARS-CoV-2 infection.

Description

Technical Field

[0001] The present application relates to the technical field of monoclonal antibodies (mAbs) against SARS-CoV-2, and in particular to monoclonal antibodies against SARS-CoV-2 spike protein S1 and their applications. Background Art

[0002] The novel coronavirus 2 (SARS-CoV-2) utilizes its envelope spike (S) glycoprotein to mediate its attack and entry into host cells. The S glycoprotein structurally includes a subunit, S1, known as the SpikeS1 protein or S1. S1 facilitates the attachment of SARS-CoV-2 to the cell surface receptor, angiotensin-converting enzyme 2 (ACE2), via its receptor binding domain (RBD). Therefore, by blocking the interaction between SARS-CoV-2 and RBD-ACE2, SARS-CoV-2 attack and entry into host cells could be blocked or disrupted, providing potential therapeutic approaches and applications. Summary of the Invention

[0003] The inventors of this application have creatively discovered an antibody that recognizes SARS-CoV-2 and its use. This antibody can be used for rapid detection or screening of SARS-CoV-2 infection. This antibody can also be used to prepare a drug for treating or preventing diseases associated with SARS-CoV-2 infection.

[0004] In the first aspect, the present application discloses an antibody that binds to the SARS-CoV-2 spike protein S1, wherein the V H The amino acid sequence of the CDR1 region is shown in SEQ ID NO: 2. H The amino acid sequence of the CDR2 region is shown in SEQ ID NO: 5. H The amino acid sequence of the CDR3 region is shown in SEQ ID NO: 8. L The amino acid sequence of the CDR1 region is shown in SEQ ID NO: 11. L The amino acid sequence of the CDR2 region is shown in SEQ ID NO: 14. L The amino acid sequence of the CDR3 region is shown in SEQ ID NO:17.

[0005] In the second aspect, the present application discloses an antibody that binds to the SARS-CoV-2 spike protein S1, wherein the V H The amino acid sequence of the region is shown in SEQ ID NO: 20, and the V L The amino acid sequence of the region is shown in SEQ ID NO: 23.

[0006] In the third aspect, the embodiments of the present application disclose an antibody that binds to the SARS-CoV-2 spike protein S1, and the antibody includes a Fab segment having a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain is shown in SEQ ID NO: 29.

[0007] In the fourth aspect, the embodiments of the present application disclose an ELISA kit for diagnosing SARS-CoV-2 or detecting SARS-CoV-2 spike protein S1, which comprises the antibody as described in any one of aspects 1 to 3, or a combination thereof.

[0008] In aspect 5, the embodiments of the present application disclose a method for in vitro diagnosis of SARS-CoV-2 or detection of SARS-CoV-2 spike protein S1, comprising using the antibody as described in any one of aspects 1 to 3, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the structure of the rabbit monoclonal antibody against SARS-CoV-2 spike protein S1 provided in the examples of the present application.

[0010] Figure 2 The rabbit monoclonal antibodies provided in multiple embodiments of the present application are based on the results of direct antigen ELISA detection of SARS-CoV-2 spike protein S1, wherein the rabbit monoclonal antibodies provided in multiple embodiments include 1D2, 5E1 and 9A5.

[0011] Figure 3 The rabbit monoclonal antibodies provided in multiple embodiments of the present application are based on the results of direct antigen ELISA detection of the SARS-CoV-2 spike protein S1 RBD domain, wherein the rabbit monoclonal antibodies provided in multiple embodiments include 1D2, 5E1 and 9A5.

[0012] Figure 4 These are the results of detecting SARS-CoV-2 spike protein S1 using the ELISA capture method using the rabbit monoclonal antibodies provided in multiple embodiments of the present application, wherein the rabbit monoclonal antibodies provided in multiple embodiments include 1D2, 5E1, and 9A5.

[0013] Figure 5 These are the results of the ELISA capture method-based detection of the SARS-CoV-2 spike protein S1 RBD domain using the rabbit monoclonal antibodies provided in multiple embodiments of the present application, wherein the rabbit monoclonal antibodies provided in multiple embodiments include 1D2, 5E1, and 9A5.

[0014] Figure 6The neutralization ability results of the rabbit monoclonal antibody 5E1 in the pseudovirus infection test provided in multiple examples of the present application are shown. The X-axis represents the antibody concentration, and the Y-axis represents the percentage of pseudovirus infection in host cells.

[0015] Figure 7 These are the blocking activity test results of the rabbit monoclonal antibodies provided in multiple embodiments of the present application against ACE2-S1, wherein the rabbit monoclonal antibodies provided in multiple embodiments include 1H1, 9H1, 5E1 and 7G5.

[0016] Figure 8 The ELISA double antibody sandwich assay results provided in the examples of this application include 5E1 as the capture antibody and 1D2 as the detection antibody.

[0017] Figure 9 The ELISA double antibody sandwich assay results provided in the examples of this application use 9A5 as the capture antibody and 5E1 as the detection antibody.

[0018] Figure 10 The rabbit monoclonal antibodies provided in multiple embodiments of the present application are specific to SARS-CoV-2 spike protein S1, SARS protein S1, SARS protein S2, MERS-CoV spike protein, HKU1 protein S1, HKU1 protein S2, HCoV-NL63 protein S, H CoV-OC43 protein S and HCoV-229E protein S. Among them, the rabbit monoclonal antibodies provided in multiple embodiments include 1D2, 5E1 and 9A5. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0020] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0021] The terms "a" or "an" encompass both singular and plural references unless the context dictates otherwise. The terms "including," "having," "having," and "comprising" are open-ended terms meaning "including, but not limited to," unless otherwise noted.

[0022] This application mainly discloses antibodies against SARS-CoV-2. Specifically, this application discloses rabbit monoclonal antibodies (mAbs) against SARS-CoV-2 spike protein S1 and their applications.

[0023] In this application, the term "antibody" should be interpreted in the broadest sense, including various antibody structures, including but not limited to Y-shaped antibodies, so-called full-length antibodies, antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications thereof. Among them, "antigen-binding portion" refers to one or more parts or fragments of a Y-shaped antibody that can retain the ability of the antibody to specifically bind to SARS-CoV-2 S1.

[0024] In this application, the term "monoclonal antibody" (mAb) includes a highly homogeneous antibody population with substantially the same antigenic determinant. That is, in this antibody population, the individual antibodies are substantially the same, except for a small amount of mutations that may occur naturally. Monoclonal antibodies can show a single binding specificity and affinity for a specific epitope on an antigen. Compared to polyclonal antibodies that typically contain antibodies directed against different epitopes, each monoclonal antibody can be directed against the same or substantially the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population and should not be interpreted as an antibody that needs to be made by any specific method. The antibody can be prepared by a variety of methods, including but not limited to hybridoma method, recombinant DNA method, phage antibody library and similar methods.

[0025] In this application, the terms "anti-SARS-CoV-2 S1 monoclonal antibody", "anti-SARS-CoV-2 spike protein S1 monoclonal antibody" and "anti-S1 monoclonal antibody" are used interchangeably to refer to monoclonal antibodies that specifically bind to the S1 protein of SARS-CoV-2 with sufficient affinity so that they can be used to prepare SARS-CoV-2 detection, diagnostic agents, therapeutic agents and / or drugs. The term "affinity" refers to the binding strength of all non-covalent intermolecular interactions between a single molecule (e.g., an antibody) and a single binding site of its binder (e.g., an antigen). Among them, "intermolecular interactions" can include hydrogen bonding, electrostatic interactions, hydrophobic interactions and van der Waals forces.

[0026] In this application, the term "rabbit antibody" or "anti-SARS-CoV-2 S1 rabbit monoclonal antibody" or the modifier "rabbit" in similar terms indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 may comprise the CDRs and framework regions (FRs) of an antibody derived from a rabbit immunoglobulin sequence. In one embodiment, the rabbit antibody or rabbit monoclonal antibody against the spike protein S1 of SARS-CoV-2 may comprise the CDRs of an antibody derived from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 may be an antibody in which the CDR region is derived from a rabbit immunoglobulin sequence and the FRs are derived from the germline immunoglobulin sequence of other mammals (such as mouse or human). The term "rabbit monoclonal antibody against SARS-CoV-2S1" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo. However, the term “rabbit monoclonal antibody against SARS-CoV-2 S1” does not include antibodies whose CDR regions are derived from the germline of other mammals (such as mice).

[0027] In the examples of the present application, the rabbit monoclonal antibody against SARS-CoV-2S1 may have a Y-shaped molecular structure (e.g. Figure 1 See Figure 1 , which details the Y-shaped structure of a specific rabbit monoclonal antibody against SARS-CoV-2 S1. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2 S1 may include a pair of heavy chains 2 and a pair of light chains 3. The heavy chain 2 may include a heavy chain variable region (V H ) and one or more heavy chain constant regions (C HS In one embodiment, heavy chain 2 may include a V H and the three Cs HS (named C H1. C H 2 and C H 1) With the three Cs HS In comparison, V H Closer to the N-terminus of the heavy chain. HS In comparison, V H Shows higher polymorphism in amino acid sequence. H Can vary between different antibodies and confers specificity to each antibody. HS The amino acid sequence of a heavy chain can be identical among all antibodies of the same type (class), or it can differ between different types of antibodies. The term "isotype" refers to antibodies of the same type (e.g., all IgG) encoded by the heavy chain constant region gene. Mammalian antibodies generally contain five types of heavy chains: γ, δ, α, μ, and ε. The corresponding antibodies are called IgG, Ig D, IgA, IgM, and IgE.

[0028] Light chain 3 can be a smaller polypeptide subunit than heavy chain 2. Light chain 3 can include a light chain variable region (V L ) and a light chain constant region (C L ). V L It is usually the N-terminal part of light chain 3 and shows higher variability in amino acid sequence. L Has a specific amino acid sequence.

[0029] In one embodiment, the heavy chain variable region V H and light chain variable region V L Can be used to recognize and bind S1 protein. In one embodiment, C HS and C L Does not bind to residues of spike protein S1.

[0030] above Figure 1 A pair of heavy chains 2 and a light chain 3 in an antibody can form a Y-shaped structure. This "Y-shaped structure" includes two Fab segments 7 (antigen-binding fragments), an Fc segment 8 (labelable fragment), and a hinge region 10. The two Fab segments 7 are similar to the two arms of the "Y" structure, while the Fc segment 8 is similar to the base of the "Y" structure. The hinge region 10 connects the Fc segment 8 and the two Fab segments 7.

[0031] Each Fab segment 7 may contain a heavy chain variable region V H , heavy chain constant region C from heavy chain 2 H 1. A light chain variable region V L and light chain constant region C from light chain 3 L Fab segment 7 contains a light chain variable region V L and heavy chain variable region V HThe variable segment (Fv) is formed. Fv segment 9 houses the antigen binding site, also known as the antigen ligand. The antigen ligand can be located at the top of the arm of the Y-shaped structure of the rabbit monoclonal antibody.

[0032] Each variable region (V H and V L ), which may include complementarity determining regions (CDRs) and framework regions (FRs). CDRs determine the specificity and affinity of Y-shaped rabbit monoclonal antibodies. CDRs contain residues that bind to the antigen and have the function of recognizing and contacting S1 protein. Y-shaped rabbit monoclonal antibodies may include 6 CDRs, 3 of which are located in V H Middle, that is, V H CDR1, V H CDR2 and V H CD R3, the other 3 are located at V L Middle, that is, V L CDR1, V L CDR2 and V L CDR3.

[0033] In some embodiments, the V H and V L The CDRs in the region can be separated from each other by FRs. FR is a conserved region in the sequence structure. FR can usually serve as a scaffold to enable CDR to form a three-dimensional structure that can specifically bind to an antigen (such as SARS-CoV-2 spike protein S1). The three-dimensional structure of FR can be conserved in different antibodies. The CDRs of the Y-shaped rabbit monoclonal antibody can be transplanted between the FRs of another antibody from other species while retaining its ability to bind to the SARS-CoV-2 spike protein S1 to form a fusion antibody. In one embodiment, the CDRs of the Y-shaped rabbit monoclonal antibody are transplanted between the FRs of a human antibody to form a humanized antibody against the SARS-CoV-2 spike protein S1.

[0034] In some embodiments, the Fc segment 8 can be composed of C from different heavy chains 2. H 2 and C H3. In one embodiment, the Fc segment 8 may comprise three constant regions. Since the Fc segment 8 may be composed of constant regions from the heavy chain, the Fc segment 8 can be used to classify antibodies. The Fc segment 8 of the Y-shaped rabbit monoclonal antibody 1 is generally not involved in binding to an antigen. In one embodiment, the Fc segment 8 may play a role in regulating immune cell activity, for example, by binding to specific Fc receptors or other immune molecules such as complement proteins to achieve immune regulation. In one embodiment, when the CDRs bind to an antigen, the Fc segment 8 may play a role in generating an appropriate immune response. In some embodiments, the Fc segment 8 may mediate various physiological responses; these physiological responses include, but are not limited to, mediating recognition of opsonin particles when bound to FcγRs, mediating degranulation of mast cells, basophils, and eosinophils when bound to Fcε receptors, cytolysis or complement-dependent cytotoxicity, antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and slowing antibody degradation and extending the antibody half-life by reacting with the neonatal Fc receptor (FcRn).

[0035] The rabbit monoclonal antibodies against SARS-CoV-2 spike protein S1 disclosed in the examples of the present application include 1D2, 5E1 and 9A5. The V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, V L CDR3, V H 、V L The amino acid sequences contained or possessed by the heavy chain Fab segment and the light chain Fab segment are shown in Table 1.

[0036] Table 1 Amino acid sequences of relevant regions of rabbit monoclonal antibodies against SARS-CoV-2 spike protein S1

[0037] area 1D2 5E1 9A5 <![CDATA[V H CDR1]]> SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 <![CDATA[V H CDR2]]> SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 <![CDATA[V H CDR3]]> SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 <![CDATA[V L CDR1]]> As shown in SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 <![CDATA[V L CDR2]]> SEQ ID NO: 13 SEQ ID NO: 14 As shown in SEQ ID NO: 15 <![CDATA[V L CDR3]]> SEQ ID NO: 16 SEQ ID NO: 17 As shown in SEQ ID NO: 18 <![CDATA[V H ]]> SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 V L ]]> SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 Heavy chain Fab segment SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 Light chain Fab segment SEQ ID NO: 28 SEQ ID NO: 29 As shown in SEQ ID NO:30

[0038] The rabbit monoclonal antibody against SARS-CoV-2 S1 provided in the present application can also be the antigen-binding portion of the Y-shaped antibody disclosed in the above embodiment. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2 S1 can be formed by a Fab segment 7, consisting of V H 、V L 、C H1 and C LIn one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 can be formed by a F(ab')2 segment, which is formed by two subunits connected by a link (e.g., a disulfide bond connected by the hinge region 10), wherein each subunit is a Fab segment 7. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 can be formed by a V H and C H1 In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 can be V L and V H The Fv fragment formed by the structural domain 9. In one embodiment, the rabbit monoclonal antibody against SARSCoV-2S1 can be an isolated complementary determining region.

[0039] The rabbit monoclonal antibodies against SARS-CoV-2 S1 provided in the examples of this application may also include antigen-binding portions thereof derived from the structures provided in the above examples or obtained through genetic modification. In some embodiments, the rabbit monoclonal antibodies against SARS-CoV-2 S1 may have different transgenic antibody structures, including but not limited to humanized antibodies and chimeric antibodies. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2 S1 may be a humanized antibody whose protein sequence has a high degree of homology with naturally occurring variant antibodies adapted to the human body. The protein sequence of a "humanized antibody" may be substantially identical to that of a human variant antibody, while maintaining the ability of its rabbit-derived CDR regions to bind to the SARS-CoV-2 spike protein S1. In one embodiment, the "humanized antibody" may be created by inserting the CDR regions of a non-human antibody, for example, by inserting the CDR regions of a rabbit antibody into a human antibody scaffold to produce a humanized antibody. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2 S1 may be a chimeric antibody. In one embodiment, the chimeric antibody can be an antibody made by transplanting the variable regions of the heavy and light chains of Y-shaped antibodies from different sources into the constant region of another animal (such as the human body). In one embodiment, the chimeric antibody is formed by fusing the Fab segment of the rabbit monoclonal antibody against SARS-CoV-2S1 disclosed in the present application with the human Fc segment. In one embodiment, the rabbit monoclonal antibody against SARS-CoV-2S1 can be a single-chain Fv (scFv). Although the two domains of the Fv segment, namely V L and V H, is encoded by two separate genes, but a linker can be formed by connecting the two separate encoding genes through recombinant methods to encode and express scFv. In one embodiment, relevant gene modification and transgenic manipulation can be performed according to methods well known to those skilled in the art, and transgenic antibody structures can be screened in the same manner as full-length antibodies.

[0040] The rabbit monoclonal antibodies against SARS-CoV-2S1 provided in the embodiments of the present application may also have structures derived from the antibodies provided in the above embodiments and antigen-binding portions thereof produced by chemical modification. In one embodiment, the chemical modification may be chemical cross-linking. In one embodiment, one or more conjugates may be covalently attached to the antibody or non-covalently attached to the antibody. In one embodiment, the conjugate may be a molecular label covalently attached to the antibody to facilitate detection of its antigen. The conjugate may be any suitable small molecule. The small molecule may include, but is not limited to, for example, biotin, streptavidin, and / or a fluorescent dye. The fluorescent dye may be any suitable fluorescent dye, including but not limited to Alexa Flour dye, aminocoumarin (AMCA), Atto dye, cyanine dye, DyLight dye, FITC, fluorescent probe 647H, rhodamine, and Texas Red. The Alexa Flour dye includes but is not limited to Alexa Flour 488, Alexa Flour 555, Alexa Flour 568, Alexa Flour 594, Alexa Flour 647, and Alexa Flour 700. The Atto dyes may include but are not limited to Atto390, Atto488, Atto565, Atto633 and Atto700. The cyanine dyes may include but are not limited to Cy3, Cy5 and Cy5.5. The DyLight dyes may include but are not limited to DyLight350, DyLight405, DyLight488, DyLight550, DyLight594, DyLight633, DyLight650, DyLight680, DyLight755 and DyLight800. In one embodiment, the conjugate can be a tandem dye having two covalently linked fluorescent molecules. In an embodiment, one fluorescent molecule acts as a donor and the other as an acceptor. In one embodiment, the donor has donor excitation characteristics and the acceptor has acceptor emission characteristics, and the two can perform unique fluorescence excitation and emission reactions. The tandem dyes may include, but are not limited to, allophycocyanin-Cy5.5, allophycocyanin-Cy7, PE-Atto594, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, PE-AlexaFluor647, PE-AlexaFluor700, PE-AlexaFluor750, APC-AlexaFluor750, and PerCP-Cy5.5.

[0041] The conjugate in the above embodiment can also be a macromolecule. In one embodiment, the macromolecule can be an enzyme. The enzyme may include but is not limited to alkaline phosphatase (AP), glucose oxidase (Gox), horseradish peroxidase (HRP). In one embodiment, the macromolecule can be a fluorescent protein. The fluorescent protein may include but is not limited to allophycocyanin (APC), B-phycoerythrin (BPER-phycoerythrin (R-PE), PerCP and R-phycocyanin (RPC). In one embodiment, the macromolecule can also be an antibody with different anti-SARS-CoV-2 S1 rabbit monoclonal antibody specificities, forming a multivalent antibody with multiple specificities.

[0042] The rabbit monoclonal antibodies against SARS-CoV-2S1 provided in the embodiments of the present application have uses in vivo and in vitro. Such uses include, but are not limited to, the preparation of immunoassay kits, the preparation of immunostaining kits, the preparation of immunochemical kits, the preparation of diagnostic kits for SARS-CoV-2 virus infection, the preparation of immuno-tumor therapeutic drugs, and the preparation of therapeutic drugs for some infectious diseases caused by SARS-CoV-2, as well as in vitro immunoassays, immunostaining, immunochemical reactions, and SARS-CoV-2 virus infection diagnosis. Among them, the immunoassay method may include an enzyme-linked immunosorbent assay (ELISA), and the monoclonal antibodies against SARS-CoV-2S1 provided in the embodiments of the present application can be used for different forms of ELISA. In one embodiment, the disclosed rabbit monoclonal antibodies against SARS-CoV-2S1 can be used for direct ELISA. The direct ELISA can be a plate-based immunosorbent assay for detecting and quantifying specific antigens from or within complex biological samples, and direct ELISA can be implemented using a variety of methods. In one embodiment, the antigen, for example, the spike protein S1 of SARS-CoV-2 can be immobilized or adsorbed on the surface of a plastic plate. In one embodiment, the plastic plate can be a multi-well microtiter plate. In one embodiment, the multi-well microtiter surface can be a 96-well polystyrene plate. In one embodiment, an excess blocking protein can be added to the surface to block all other binding sites. In one embodiment, the blocking protein is bovine serum albumin. In one embodiment, an antibody against an antigen (e.g., the SARS-CoV-2 spike protein S1) can form a complex with the antigen conjugated to the surface. In one embodiment, the antibody can be conjugated to an enzyme. In one embodiment, the enzyme can be HRP. After the excess conjugated antibody is washed away, the conjugated antibody bound to the antigen remains on the surface. In one embodiment, the conjugated antibody catalyzes a reaction with an added substrate to produce a visible colorimetric output that can be measured by a spectrophotometer or absorbance microplate reader. Direct ELISA testing uses only one antibody, resulting in fewer detection steps and higher detection efficiency compared to other forms of ELISA testing. In one embodiment, direct ELISA can test for specific antibody-antigen reactions and help eliminate cross-reactivity with other antibodies. Direct ELISA is suitable for qualitative and quantitative applications in target samples for antigen detection, antibody screening, and epitope mapping.

[0043] Table 2 shows the binding kinetic parameters of multiple rabbit monoclonal antibodies provided in the Examples of this application to SARS-CoV-2 S, including 1D2, 5E1, and 9A5. As shown in Table 2, 1D2, 5E1, and 9A5 exhibit high affinity and specificity for SARS-CoV-2 S1.

[0044] Table 2 Kinetic parameters of binding to SARS-CoV-2S

[0045] clone <![CDATA[K off (1 / s)]]> K on (1 / Ms) <![CDATA[K D (M)]]> 1D2 2.59E-04 7.29E+04 3.55E-09 5E1 1.00E-05 7.23E+04 1.38E-10 9A5 1.15E-04 1.32E+05 8.70E-10

[0046] See Figure 2 , which shows the curves of the anti-SARS-CoV-2 S1 rabbit monoclonal antibodies provided in multiple embodiments for detecting SARS-CoV-2 S1 antigen based on direct ELISA, wherein the multiple rabbit monoclonal antibodies include 1D2, 5E1 and 9A5. In the figure, the X-axis represents the antibody concentration in ng / ml units, and the Y-axis represents the optical density at a wavelength of 450nm (OD450). Figure 2 As shown, all rabbit monoclonal antibodies specifically bind to SARS-CoV-2 S1, with binding curves exhibiting a near-S-shaped pattern across the antibody concentration range from approximately 1 ng / ml to approximately 1000 ng / ml. A negative control group was included in the assay. Following the same assay steps as for the anti-SARS-CoV-2 S1 rabbit monoclonal antibody, blank buffer was used instead of the anti-SARS-CoV-2 S1 rabbit monoclonal antibody. Blank buffer is the buffer used to dilute the rabbit monoclonal antibody. Compared to the negative control, which exhibited almost no OD450 absorbance, 1D2, 5E1, and 9A5 exhibited good detection signals, demonstrating that their absorbance at OD450 is capable of detecting a wide range of SARS-CoV-2 S1 concentrations.

[0047] See Figure 3 , which shows the detection curves of the RBD of SARS-CoV-2 S1 based on the direct ELISA method of the anti-SARS-CoV-2 S1 rabbit monoclonal antibodies provided by multiple embodiments, wherein the multiple rabbit monoclonal antibodies include 1D2, 5E1 and 9A5. In the figure, the X-axis represents the antibody concentration in ng / ml, and the Y-axis represents the optical density at 450nm (OD450). The negative control was carried out in the same steps as the rabbit monoclonal antibody against SARS-CoV-2S1, using blank buffer instead of the rabbit monoclonal antibody against SARS-CoV-2S1. Blank buffer is a buffer used to dilute rabbit monoclonal antibodies. The OD450 value of the negative control group is close to zero, and there is almost no detection signal. Compared with the negative control, 1H1, 5E1, 7G5, 9A5 and 9H1 can produce significant OD450 values. By Figure 3It can be seen that the binding curves formed by 1H1, 5E1, 7G5, 9A5, and 9H1 in the antibody concentration range of 0.5ng / ml to 1000ng / ml are approximately "S" shaped, indicating that 1H1, 5E1, 7G5, 9A5, and 9H1 can specifically bind to the RBD of SARS-CoV-2 S1. Compared with 1H1, 5E1, 7G5, 9A5, and 9H1, the OD450 detection values ​​of 1A3 and 1D2 are close to zero, indicating that 1A3 and 1D2 cannot specifically bind to the RBD of SARS-CoV-2 S1.

[0048] To verify that the rabbit monoclonal antibodies provided in the examples of this application can bind to the S1 protein in its native state, this application also conducted a detection test based on capture ELISA. In this test, the rabbit monoclonal antibodies were captured by the Fc coated on the plate, and then the native S1 or RBD was added to the plate.

[0049] Figure 4 The capture ELISA results of 1D2, 5E1 and 9A5 binding to S1 are shown. Figure 4 As shown, except 1D2 and 1A3, 1H1, 5E1, 7G5, 9A5 and 9H1 can all bind to S1 in their natural state. Figure 5 , which shows the capture ELISA results of 1D2, 5E1 and 9A5 binding to RBD in their natural state. Figure 5 As shown, except for 1D2 and 1A3, 1H1, 5E1, 7G5, 9A5 and 9H1 can all bind to RBD in their native state.

[0050] Figure 10 In the figure, the Y-axis shows the optical density of the direct ELISA at 450 nm. Figure 10 In the figure, each column contains 7 groups, which are 1A3, 1, 1D2, 5E1 and 9A5 along the X-axis, showing the specificity for SARS-CoV-2S1, SARS S1, MERS S1 and HCoV-NL63S1 respectively. Figure 10As shown, the antibodies provided in the examples of this application exhibit the highest specificity for SARS-CoV-2 S1. Among the antibodies provided in the examples of this application, 1A3, 1H1, 7G5, and 9A5 have unique specificity for SARS-CoV-2 S1, but have no significant specificity for SARS protein S1 and SARS protein S2, MERS-CoV spike protein, HKU1 protein S1, HKU1 protein S2, HCoV-NL63 protein S, HCoV-OC43 protein S, and HCoV-229E protein S. In comparison, 1D2, 5E1, and 9H1 exhibit higher specificity for SARS-CoV-2 protein S1, SARS protein S1, SARS protein S2, and MERS-CoV spike protein, but lower specificity for KU1 protein S1 and HKU1 protein S2. However, 1D2, 5E1, and 9H1 have no specificity for HCoV-NL63 protein S, HCoV-OC43 protein S, and HCoV-229E protein S.

[0051] In order to evaluate the neutralization ability of 5E1, pseudovirus neutralization test and live virus neutralization test were performed. Figure 6 As shown, 5E1 can neutralize SARS-CoV-2 wild-type pseudovirus, and the IC50 (μg / mL) against SARS-CoV-2 live virus is 0.512 μg / mL.

[0052] In order to evaluate whether 1H1, 9H1, 5E1 and 7G5 can block the binding of S1 to ACE2, the present application further conducted a blocking experiment. Recombinant ACE2 was coated on an ELISA plate, and the different rabbit monoclonal antibodies provided in the examples of the present application were pre-incubated with different concentrations of RBD domain proteins to form antibody-RBD mixtures, which were then loaded onto the ACE2-coated ELISA plate. The results are shown in Figure 2. Figure 7 As shown, 5E1 can block the binding of RBD domain to ACE2. Figure 8 The following figure shows the test results of a double antibody sandwich ELISA using 5E1 as the capture antibody and 1D2 as the detection antibody. Figure 8 As shown, 1D2 can bind to or detect S1 captured by 5E1. Figure 9 Figure 2 shows the test results of a double antibody sandwich ELISA using 9A5 as the capture antibody and 5E1 as the detection antibody. Figure 9 As shown, 5E1 can bind to or detect S1 captured by 9A5.

[0053] method

[0054] 1. Preparation, Isolation, and Purification of Rabbit Monoclonal Antibodies Against SARS-CoV-2 S1

[0055] Rabbit monoclonal antibodies against SARS-CoV-2 S1 can be produced by a variety of techniques, including monoclonal antibody preparation methods, such as somatic cell hybridization technology and other technologies, including but not limited to B lymphocyte hybridoma technology. In one embodiment, recombinant rabbit monoclonal antibodies are produced based on B cells.

[0056] In one embodiment, the RBD region corresponding to genomic position 22,553-23,312 bp of SARS-CoV-2 (GenBank: MN908947.3) was codon-optimized and cloned into the pcDNA3.4 expression vector to construct an expression vector. The expression construct was transformed into a competent Escherichia coli DH5a strain, cultured, and positive clones were screened. The expression vector was extracted using the Qiagen Plasmid Mega kit (Cat NO: 10023). Gold nanoparticles (AlphaAesar, Catalog NO: 14817) were pre-coated with 100 mg / mL spermidine (Sigma, Catalog NO: S2626), and then 36 μg of the purified expression vector was coated with 100 μL of 100 mg / mL gold powder. The gold nanoparticles encapsulated with the expression vector were washed several times with anhydrous ethanol and transferred to the pellet tube of a bullet maker (Scientz Scientific). 200 μL of 2.5 M CaCl2 solution was slowly dripped into the pellet tube to promote the binding of DNA and gold nanoparticles. The pellet tube loaded with gold powder including the expression vector was slowly dried under a 0.1 MPa N2 flow for 10 minutes, and then the dried pellet tube was cut into DNA pellets.

[0057] The SARS-CoV-2 RBD DNA pellets prepared above were loaded into the magazine of an SJ-500 gene gun (Scientz Scientific). The DNA pellets in the SJ-500 gene gun were fired with 4MPa helium and injected into the abdominal skin of New Zealand white rabbits (4-6 weeks old) (36μg / each immunization). Each rabbit was immunized with DNA three times on days 0, 7, and 21. On days 35 and 49, an emulsified preparation of SARSCoV-2 S1 protein prepared with incomplete Freund's adjuvant was injected intramuscularly twice for booster immunization. Two weeks later, 200μg of S1 protein was injected subcutaneously into the rabbit again for booster immunization. Pre- and post-immunization sera were collected on days 0, 14, 28, 42, and 69, respectively.

[0058] Fresh single splenocytes were isolated from the spleen of the immunized rabbit and cultured overnight in B cell culture medium (e.g., Unisys Biotech). Fresh single cell suspension was prepared by diluting the splenocytes with PBS containing 2% fetal bovine serum and 1 mM EDTA in oxygenated water.

[0059] use The single B cells in the single-cell suspension were separated using a FACS Aria II (BD Biosciences, USA) platform. The single B cells were then sorted using a FACS Aria II (BD Biosciences, USA) and placed in each well of a 96-well plate. Primary B cells with S1 specificity were added to complete B cell culture medium (e.g., a rabbit B cell culture medium, Unisys Biosciences) and cultured at 37°C and 5% CO2 for 10 to 14 days. At the end of the primary B cell culture, the primary B cell culture supernatant was screened for S1 by direct ELISA to identify S1-specific B cell positive clones. Generally speaking, the OD450nm value of the B cell positive clone is more than 5 times the background noise. The variable regions of the IgG heavy and light chains in the positive clones at the top of the primary B cell supernatant were detected by RT-PCR. The full-length IgG heavy and light chains of each clone were co-transfected into HEK293T cells. The supernatant containing the rabbit IgG recombinant protein transfected into HEK293T cells was screened for its specificity to S1 by ELISA.

[0060] In the examples of the present application, the variable region PCR fragments of the selected clones were cloned into the pcDNA3.4 vector, and the antibodies were expressed in HEK293F cells.

[0061] In the examples of the present application, rabbit monoclonal antibodies can be isolated and purified by conventional methods well known to those skilled in the art.

[0062] In one embodiment, rabbit monoclonal antibodies can be isolated from the culture supernatant of mammalian cells transfected with rabbit antibody genes and fully purified by protein A affinity chromatography. The purity and function of the purified rabbit monoclonal antibodies can be verified by SDS-PAGE and ELISA, respectively.

[0063] 2. Preparation of rabbit monoclonal conjugated antibodies against SARS-CoV-2 S1

[0064] Rabbit monoclonal antibodies were biotinylated using Pierce EZ-Link Sulfo-NHS-Biotin according to the manufacturer's instructions. Briefly, the anti-SARS-CoV-2 S1 rabbit monoclonal antibody provided in the examples of this application was mixed with sulfo-NHS-biotin in a 1:1 dilution volume of PBS and incubated at room temperature for 30 minutes.

[0065] 3. ELISA identification of immune rabbit serum and monoclonal antibodies against SARS-CoV-2 S1

[0066] (1) SARS-CoV-2 protein S1 or S1 protein from other viruses was coated onto an ELISA plate (e.g., Corning, Cat. NO: 4018) as an antigen and incubated overnight at 4°C in 1× PBS, pH 7.4.

[0067] (2) The coated plate was washed three times with washing buffer (1×PBS supplemented with 0.5% (V / V) Tween-20 (Sigma, Cat. NO: P96416)) and then blocked with blocking buffer (1×PBS supplemented with 5% (W / V) skim milk).

[0068] (3) After blocking, serially diluted rabbit serum samples or monoclonal antibodies were added to the wells and incubated at room temperature for 1 h, then washed 5 times with washing buffer, and then incubated with goat anti-rabbit IgG antibody conjugated with HRP (e.g., HRP from Jackson Immuno Research, Cat. NO: 111-035-045) diluted 1:5000 in blocking buffer.

[0069] (4) After washing the plate 5 times with washing buffer, 25 μL of TMB substrate (MossINS, Cat. NO: TMBHK-1000) was added and placed in the dark at room temperature for 3 minutes.

[0070] (5) The colorimetric reaction of TMB substrate was then stopped with 20 μL of 1 M sulfuric acid. Optical density (OD) values ​​at 450 nm and 630 nm were measured using an Epoch microplate spectrophotometer (Biotek, USA). The final value was calculated by subtracting OD630 from OD450. The serum titer was calculated as the highest dilution at which the OD450 reading of the serum was 2-fold or greater than that of the control sample.

[0071] 4. Rabbit monoclonal antibody used in capture ELISA to detect SARS-CoV-2S1

[0072] The rabbit monoclonal antibody capture ELISA method for detecting SARS-CoV-2S1 involved in this application generally includes the following steps:

[0073] Coating: Add anti-rabbit IgG Fc antibody to the wells of a high-binding ELISA plate and coat overnight at 4°C in 1-fold diluted volume of pH 7.4 PBS.

[0074] Blocking: The coated plate was washed with washing buffer (1×PBS supplemented with 0.5% (V / V) Tween-20) and blocked with blocking buffer (1×PBS supplemented with 5% (W / V) skim milk).

[0075] Incubation: Add rabbit antibody against SARS-CoV-2 protein S1 to the blocked wells, incubate at room temperature for 1 hour, wash, and then incubate with 3-fold diluted biotinylated SARS-CoV-2 S1 protein. Finally, add HRP-conjugated streptavidin for incubation.

[0076] Colorimetric: After washing the incubated plate, use an HRP-catalyzed colorimetric reaction to detect whether the monoclonal antibody can capture S1.

[0077] It should be understood by those skilled in the art that the capture ELISA step described above may be performed using steps, reagents, and experimental parameters different from those described above.

[0078] 5. Rabbit monoclonal antibody used in double antibody sandwich ELISA to detect SARS-CoV-2S1

[0079] The double antibody sandwich ELISA detection steps of the rabbit monoclonal antibody against SARS-CoV-2S1 involved in this application generally include:

[0080] Coating: Capture antibody was added to the wells of a high binding ELISA plate and coated overnight at 4°C in 0.02 M bicarbonate buffer, pH 9.4.

[0081] Blocking: The coated plate was washed with washing buffer (1×PBS supplemented with 0.5% (V / V) Tween-20) and blocked with blocking buffer (1×PBS supplemented with 5% (W / V) skim milk).

[0082] Incubation: Add SARS-CoV-2 protein S1 to the blocked plate, incubate at room temperature for 1 hour, wash, and then add biotinylated monoclonal antibody against SARS-CoV-2 S1. Add HRP-conjugated streptavidin to the plate and incubate.

[0083] Colorimetric: After washing the incubated plate, an HRP-catalyzed colorimetric reaction is used to observe whether the monoclonal antibodies used for capture and detection can bind to SARS-CoV-2S1 simultaneously or not.

[0084] It should be understood by those skilled in the art that the capture ELISA step described above may be performed using steps, reagents, and experimental parameters different from those described above.

[0085] 6. Binding kinetics of rabbit monoclonal antibodies against SARS-CoV-2 protein S1

[0086] The binding kinetics of rabbit mAbs against SARS-CoV-2 S1 were analyzed by surface plasmon resonance (SPR) method using a Biacore instrument with a protein A sensor chip (GE Health, USA). All experiments were performed at 25 °C with a flow rate of 40 μL / min. PBS with 0.005% Tween-20 was used as running buffer. Channel 1 was loaded with a reference antibody that did not bind specifically to SARS-CoV-2 protein S1, and channels 2, 3 and 4 were loaded with candidate antibodies, respectively.

[0087] In general, during the detection process, 2 μg / mL of antibody was used and a rapid injection of 20-30 seconds was performed to load the channel, so that the detection results could produce 150-250 reaction units (RU) and have high reproducibility. Therefore, after the 2 μg / mL antibody was rapidly injected into the channel at a speed of 20-30 s, the antigen was again injected into the surface of all channels for 5 min to facilitate the binding of the antibody, and then the injection buffer was flushed for 10 min to enter the dissociation stage.

[0088] Multiple binding / dissociation cycles were performed using an antigen dilution gradient in the range of 1.2-100 nM and a blank buffer. At the end of each cycle, the channel was regenerated by a 30 s injection of glycine buffer (pH 2.0, 10 mM) to load the antibody again in each channel. The kinetic curves were analyzed using BIA evaluation 3.2 software and a 1:1 Langmuir model with a double coefficient curve, and the association rate constant, dissociation rate constant and affinity constant were calculated.

[0089] 7. Neutralization test of rabbit mAbs against SARS-CoV-2 S1

[0090] Neutralization activity against SARS-CoV-2 was performed in a certified biosafety level III laboratory. Live SARS-CoV-2 isolates were isolated from nasopharyngeal swabs of an infected patient in Jiangsu Province, China. The neutralization assay procedure involved seeding Vero cells in 24-well plates (200,000 cells / well) and incubating for approximately 16 hours until they reached 90% to 100% confluency. Antibodies were diluted 1:1 and 9:13 in DMEM containing 2% fetal bovine serum and mixed with virus at a 1:1 (vol / vol) ratio to generate a mixture containing 100 focus-forming units (PFU) / ml of virus, which was then incubated at 37°C for 1 hour. The mAb-virus complex was then added repeatedly to Vero cell monolayers in 24-well plates and incubated at 37°C for 1 hour. The mixture was removed and the cells were overlaid with DMEM containing 1% low-melting-point agarose (Promega) and 2% fetal bovine serum. After incubation at 37°C for 3 days, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution (Sigma). The number of plaques visualized cellular foci infected with SARS-CoV-2. The 50% inhibitory concentration (half-inhibitory concentration) of the monoclonal antibody was defined as the antibody concentration (μg / mL) corresponding to 50% of the total number of plaques in the absence of antibody.

[0091] 8. ELISA test for ACE2 receptor blocking

[0092] ELISA plates were coated with 1 μg / mL recombinant ACE2 (Kactus Biosystems, Cat. No. ACE-HM501). Antibodies were preincubated with RBD domain proteins at varying dilutions for 1 hour at room temperature. The antibody-RBD complexes were then deposited onto the ACE2-coated ELISA plates and incubated at room temperature for 1 hour.

[0093] The above description is merely a preferred embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, further modifications and equivalents of the disclosure disclosed in the art may occur to those with ordinary skills using no more than routine experiments. These modifications and equivalents of the present application include nucleic acid sequences encoding the disclosed amino acid sequences.

Claims

1. An antibody that binds to the SARS-CoV-2 spike protein S1, wherein: The V of the antibody H The amino acid sequence of the CDR1 region is shown in SEQ ID NO:

2. H The amino acid sequence of the CDR2 region is shown in SEQ ID NO:

5. H The amino acid sequence of the CDR3 region is shown in SEQ ID NO:

8. L The amino acid sequence of the CDR1 region is shown in SEQ ID NO:

11. L The amino acid sequence of the CDR2 region is shown in SEQ ID NO:

14. L The amino acid sequence of the CDR3 region is shown in SEQ ID NO:

17.

2. An antibody that binds to the SARS-CoV-2 spike protein S1, wherein: The V of the antibody H The amino acid sequence of the region is shown in SEQ ID NO: 20, and the V L The amino acid sequence of the region is shown in SEQ ID NO:

23.

3. An antibody that binds to SARS-CoV-2 spike protein S1, the antibody comprising a Fab segment having a heavy chain and a light chain, the heavy chain amino acid sequence being as shown in SEQ ID NO: 26, and the light chain amino acid sequence being as shown in SEQ ID NO:

29. The antibody according to claim 1 , which is a humanized or chimeric antibody.

5. The antibody according to any one of claims 1 to 4, further comprising a covalently or non-covalently linked conjugate, wherein the conjugate is an enzyme, a fluorophore, biotin, streptavidin, or a combination thereof.

6. An ELISA kit for in vitro diagnosis of SARS-CoV-2 or detection of SARS-CoV-2 spike protein S1, comprising the antibody according to any one of claims 1 to 4, or a combination thereof.

7. Use of the antibody according to any one of claims 1 to 4 in the preparation of a kit for in vitro diagnosis of SARS-CoV-2 or detection of SARS-CoV-2 spike protein S1.

8. The use according to claim 7, wherein the kit is selected from the group consisting of direct ELISA, capture ELISA and double antibody sandwich ELISA.

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