Antibody specifically binding to Hb-CS protein, and antigen binding fragment and application thereof

Abstract

The invention belongs to the field of biological medicine. Specifically, the invention relates to an antibody specifically binding to Hb-CS protein, and an antigen binding fragment and application thereof. The antibody or the antigen binding fragment thereof provided by the invention can be specifically bound with Hb-CS protein instead of normal alpha globin, can be used for immunodetection of Hb-CS protein and effective recognition of Hb-CS protein positive red blood cells, and has important effect and value for development of alpha thalassemia drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antibody specifically binding to Hb-CS protein, an antigen binding fragment thereof and application thereof. Background Art

[0002] Thalassemia is a hereditary hemolytic anemia. According to genetic typing, α-thalassemia and β-thalassemia mainly appear clinically. α-thalassemia can be divided into deletion type and non-deletion type according to different mutation types. Non-deletion α-thalassemia is caused by "point mutations" in the α gene nucleotides. In α-thalassemia in Guangdong, Guangxi Zhuang Autonomous Region and Sichuan, the non-deletion type accounts for 35% to 60%. There are three common non-deletion α-thalassemias in my country: Hb-CS (CD142), Hb-QS (CD125) and Hb-WS (CD122). Among them, Hb-CS type patients have the most serious condition, low hemoglobin and low survival rate. Hemoglobin CS (Hb-CS, Haemoglobin-Constant Spring, α142, Term→Gln, TAA>CAA (α2), α cs α / ) is a non-deletion form of α-thalassemia (α-Thal), in which there is a nucleotide substitution (UAA>CAA) at the termination codon CD142 of the α2-globin gene (HBA2), which prevents the synthesis of the α-globin chain from terminating normally, but instead continues to extend, generating Hb-CS protein with an additional 31 amino acid residues. SEA ,α MED ,α THAI ), when only one normal α1-globin gene (HBA1) remains, the genotype is α CS α / --, whose clinical manifestations and blood picture are similar to those of hemoglobin H disease (HbH), is called CS type HbH disease (HbH-CS). In addition, individuals affected by HbH-CS disease usually have more severe anemia symptoms and are prone to significant hepatosplenomegaly, especially compared with individuals with triple gene deletion involving the α-globin gene (-- / -α), and HbH-CS type patients have a greater need for blood transfusion.

[0003] Hemoglobin α antibodies currently available on the market cannot distinguish between normal α-globin and α- CS Globin (i.e., Hb-CS protein), and in the development of α-thalassemia drugs, it is very important to find antibodies to Hb-CS protein. Therefore, those skilled in the art are in urgent need of developing an antibody or an antigen-binding fragment thereof that can specifically bind to Hb-CS protein. Summary of the invention

[0004] Based on this, the present invention provides an antibody that specifically binds to Hb-CS protein, an antigen-binding fragment thereof and applications thereof.

[0005] The first aspect of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to Hb-CS protein, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and light chain complementary determining regions LCDR1, LCDR2 and LCDR3;

[0006] The amino acid sequence of the HCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 1 to 3 or a variant thereof;

[0007] The amino acid sequence of the HCDR2 comprises an amino acid sequence selected from the following group or a variant thereof: X1IX2AGSSX3X4X5YX4X6X7X8X9G, wherein X1 is selected from R or I, X2 is selected from K or Y, X3 is selected from E, S or T, X4 is selected from S or A, X5 is selected from T, Y or E, X6 is selected from P or S, X7 is selected from R, W or E, X8 is selected from F or A, and X9 is selected from E or K;

[0008] The amino acid sequence of the HCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 4 to 6 or a variant thereof;

[0009] The amino acid sequence of LCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 7 to 9 or a variant thereof;

[0010] The amino acid sequence of LCDR2 comprises an amino acid sequence or a variant thereof selected from the following group: X10ASTLAX11, wherein X10 is selected from N or A, and X11 is selected from P or S;

[0011] The amino acid sequence of LCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 10 to 12 or a variant thereof;

[0012] The variant is a variant in which at least one, at least two or at least three conservative amino acid substitutions are made in the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3.

[0013] In some embodiments of the present invention, the amino acid sequence of HCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 1 to 3 or a variant thereof; and / or,

[0014] The amino acid sequence of the HCDR2 comprises one of the amino acid sequences shown in SEQ ID NOs: 13 to 15 or a variant thereof; and / or,

[0015] The amino acid sequence of the HCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 4 to 6 or a variant thereof; and / or,

[0016] The amino acid sequence of LCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 7 to 9 or a variant thereof; and / or,

[0017] The amino acid sequence of LCDR2 comprises one of the amino acid sequences shown in SEQ ID NOs: 16 to 18 or a variant thereof; and / or,

[0018] The amino acid sequence of the LCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 10 to 12 or a variant thereof.

[0019] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0020] The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 13, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 4; or

[0021] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:14, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5; or

[0022] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; or

[0023] LCDR1 having an amino acid sequence as shown in SEQ ID NO:7, LCDR2 having an amino acid sequence as shown in SEQ ID NO:16, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:10; or

[0024] LCDR1 having an amino acid sequence as shown in SEQ ID NO:8, LCDR2 having an amino acid sequence as shown in SEQ ID NO:17, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:11; or

[0025] The amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:18, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:12.

[0026] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0027] HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:13, HCDR3 with an amino acid sequence as shown in SEQ ID NO:4, LCDR1 with an amino acid sequence as shown in SEQ ID NO:7, LCDR2 with an amino acid sequence as shown in SEQ ID NO:16, LCDR3 with an amino acid sequence as shown in SEQ ID NO:10; or

[0028] HCDR1 with an amino acid sequence as shown in SEQ ID NO:2, HCDR2 with an amino acid sequence as shown in SEQ ID NO:14, HCDR3 with an amino acid sequence as shown in SEQ ID NO:5, LCDR1 with an amino acid sequence as shown in SEQ ID NO:8, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17, LCDR3 with an amino acid sequence as shown in SEQ ID NO:11; or

[0029] The amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, the amino acid sequence of HCDR3 is shown in SEQ ID NO:6, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:18, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:12.

[0030] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and / or a light chain framework region;

[0031] Preferably, the heavy chain framework region is selected from the antibody heavy chain framework region of human, mouse or rabbit origin; and / or, the light chain framework region is selected from the antibody light chain framework region of human, mouse or rabbit origin.

[0032] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; and / or,

[0033] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is any one of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 with at least 1, at least 2 or at least 3 conservative amino acid substitutions; and / or,

[0034] The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; and / or,

[0035] The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is a conservative amino acid substitution made on the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

[0036] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0037] The heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 22; or

[0038] The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:23; or

[0039] The heavy chain variable region is shown as SEQ ID NO:21 and the light chain variable region is shown as SEQ ID NO:24.

[0040] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof specifically binds to the C-terminus of the Hb-CS protein;

[0041] The C-terminus of the Hb-CS protein includes the amino acid sequence shown in SEQ ID NO:25.

[0042] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

[0043] The second aspect of the present invention is to provide a protein comprising the antibody or antigen-binding fragment thereof as described above;

[0044] Preferably, the protein further comprises other functional molecules connected to the end of the antibody or antigen-binding fragment thereof;

[0045] More preferably, the other functional molecules are selected from one or more of the following: a signal peptide or a protein tag.

[0046] The third aspect of the present invention is to provide an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described above, or the protein as described above.

[0047] The fourth aspect of the present invention is to provide a gene expression cassette, wherein the gene expression cassette comprises the nucleic acid as described above.

[0048] The fifth aspect of the present invention is to provide a vector, wherein the vector comprises the nucleic acid as described above, or the gene expression cassette as described above;

[0049] Preferably, the vector is a plasmid, cosmid, phage or viral vector.

[0050] A fifth aspect of the present invention is to provide a host cell, wherein the host cell expresses the antibody or antigen-binding fragment thereof as described above, or the protein as described above;

[0051] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell;

[0052] More preferably, the host cell is a bacterial cell or a yeast cell.

[0053] A sixth aspect of the present invention is to provide a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned protein, the method comprising the following steps:

[0054] The host cell as described above is cultured, and the antibody or antigen-binding fragment thereof as described above, or the protein as described above is obtained from the culture.

[0055] The seventh aspect of the present invention is to provide a detection reagent, which comprises the antibody or antigen-binding fragment thereof as described above, or the protein as described above.

[0056] The eighth aspect of the present invention is to provide a method for detecting Hb-CS protein, comprising the following steps: contacting the antibody or antigen-binding fragment thereof as described above, or the protein as described above with a sample to be tested.

[0057] The antibody or antigen-binding fragment thereof provided by the present invention can specifically bind to Hb-CS protein but not to normal α-globin, and can be used for immunodetection of Hb-CS protein and effectively identify Hb-CS protein-positive red blood cells, which has an important role and value in the development of α-thalassemia drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1is an amino acid sequence alignment diagram of the wild-type α-globin and Hb-CS proteins of the present invention;

[0059] Figure 2 This is a flow cytometry test result diagram of rabbit serum and red blood cells before and after immunization in Example 3 of the present invention;

[0060] Figure 3 This is a graph showing the results of flow cytometry detection of culture supernatants of 14 monoclonal cell strains using CS-positive red blood cells in Example 4 of the present invention;

[0061] Figure 4 This is a graph showing the results of flow cytometry testing of 7 antibody strains using CS-positive red blood cells and CS-negative red blood cells in Example 4 of the present invention;

[0062] Figure 5 This is a result diagram of flow cytometry detection of culture supernatants of clones 13#, 16# and 17# using peripheral blood from patients in Example 4 of the present invention;

[0063] Figure 6 This is a graph showing the results of Western Blotting of proteins extracted from peripheral blood of Hb-CS patients and healthy subjects using monoclonal antibodies in Example 5 of the present invention;

[0064] Figure 7 This is a graph showing the results of flow cytometry testing of peripheral blood of Hb-CS patients and healthy subjects using monoclonal antibodies in Example 6 of the present invention. DETAILED DESCRIPTION

[0065] Definition of terms:

[0066] The term "Hb-CS protein" used in this article refers to hemoglobin Constant Spring, which is an abnormal hemoglobin. Hb-CS protein is caused by a mutation in the α-globin gene. In normal hemoglobin, the 142nd codon of the α-globin chain is the termination codon UAA, while in Hb-CS patients, this codon mutates to CAA, so that the synthesis of the α-globin chain cannot be terminated normally, but continues to extend, producing Hb-CS protein with an additional 31 amino acid residues. "Hb-CS protein" is different from "CS type α-globin", α CS Globin is used interchangeably.

[0067] As used herein, the terms "heavy chain variable region" and "VH" and "HCVR" are used interchangeably, and "light chain variable region" and "VL" and "LCVR" are used interchangeably. The variable domains of the heavy chain and light chain of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein, usually referring to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL), which is also called the complementary determining region because it can form precise complementarity with the antigen epitope in spatial structure, wherein the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR.

[0068] The term "complementarity determining region" or "CDR region" or "CDR" as used herein is a region of an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes and are numbered sequentially from the N-terminus and include CDR1, CDR2, and CDR3. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. It is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0069] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, IMGT, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.

[0070] As used herein, "percent (%) sequence identity" and "sequence identity" of amino acid sequences have a definition recognized in the art, which refers to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). It can be determined using methods known to those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.

[0071] The term "antibody" herein is used in the broadest sense and refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the heavy chain of an immunoglobulin and / or sufficient sequence from the variable region of the light chain of an immunoglobulin, so as to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only complete antibodies, but also antigen-binding fragments of antibodies. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53 (1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0072] The "antigen-binding fragment" herein does not have the entire structure of a complete antibody, but only contains a portion or a partial variant of the complete antibody, and the portion or partial variant has the ability to bind to the antigen. For example, the "antigen-binding fragment" herein includes but is not limited to Fab, F(ab') 2 , Fab', Fab'-SH, Fd, Fv, scFv, diabody and single domain antibody. The terms "singledomain antibody (sdAb)", "VHH domain" and "nanobody" herein have the same meaning and can be used interchangeably, referring to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Usually, a heavy chain antibody that naturally lacks a light chain and a heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region. It is necessary to explain that nanobodies can be used to form other forms of antibodies, such as antibodies that can contain VH-CH2-CH3 from the N-terminus to the C-terminus, or contain VH-CH1-CH2-CH3; homodimers can be formed, such as heavy chain dimer antibodies without light chains.

[0073] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0074] Herein, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0075] The term "conservative amino acids" herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, main-chain conformation, and rigidity).

[0076] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0077] 1) Alanine (A), serine (S), threonine (T);

[0078] 2) Aspartic acid (D), glutamic acid (E);

[0079] 3) Asparagine (N), glutamine (Q);

[0080] 4) Arginine (R), Lysine (K), Histidine (H);

[0081] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0082] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0083] The terms "framework region" or "FR region" are interchangeable and refer to those amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR.

[0084] The term "heavy chain constant region" herein refers to the carboxyl terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interactions with Fc receptors, which have a more conservative amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment", the former having a structure substantially similar to that of a natural antibody constant region, while the latter only includes "a portion of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

[0085] The term "isolated" as used herein refers to something obtained artificially from a natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called "isolated". The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0086] As used herein, "vector" means a construct that can deliver one or more genes or sequences of interest into a host cell and preferably express the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. The term "regulatory element" as used herein includes promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoters or 35S enhanced promoters), internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). In some cases, regulatory elements include those sequences that direct the constitutive expression of a nucleotide sequence in many types of cells and those sequences that direct the nucleotide sequence to be expressed only in certain cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific.

[0087] The term "host cell" as used herein refers to a cell that can be used to introduce a vector, as a model industrial cell for bioengineering, for expressing antibodies or antigen-binding fragments thereof in large quantities, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0088] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0089] Example 1 provides Hb-CS protein specific sequence

[0090] The Hb-CS mutation prevents the timely termination of HBA translation, resulting in an extension of the CDS region of the Hb-CS protein by 93 nt ( Caagctggagcctcggtagccgttcctcctgcccgctgggcctcccaacgggccctcctcccctccttgcaccggcccttcctggtctttgaa, SEQ ID NO: 26; the bold underlined C is the CS mutation site), that is, the peptide chain is extended by 31 amino acids (QAGASVAVPPARWASQRALLPSLHRPFLVFE, SEQ ID NO: 25). This sequence was subsequently used as an antigen to screen monoclonal antibodies after rabbit immunization. Please refer to Figure 1 , is an amino acid sequence alignment diagram of the wild-type α-globin and Hb-CS proteins of the present invention.

[0091] Example 2 In vitro differentiation to obtain red blood cells

[0092] Bone marrow samples from Hb-CS mutation carriers or healthy donors were obtained from the 923rd Hospital, and hematopoietic stem cells were sorted using human CD34 nanomagnetic beads (Miltenyi Biotec, catalog number #130-046-702). Hematopoietic stem cells from Hb-CS mutation carriers or healthy donors were induced to undergo erythroid differentiation in vitro. The specific erythroid differentiation induction process is as follows:

[0093] Thaw SFEMII medium (StemSpan, catalog number #09605) at room temperature, aliquot and store in a -20°C refrigerator. Mix 100× Erythroid Expansion Supplement (StemSpan, catalog number #02692) and SFEMII medium at a ratio of 1:99 to prepare complete differentiation medium. On day 0 of differentiation induction, use complete differentiation medium at 37°C and 5% CO 2 Hematopoietic stem cells are cultured in an incubator, and half of the medium is replaced with fresh complete differentiation medium every 3-4 days, and the differentiation is induced for 14 days. After culture, the red blood cells differentiated from the hematopoietic stem cells of Hb-CS mutation carriers are Hb-CS protein positive red blood cells (referred to as CS positive red blood cells or CS+ cells), and the red blood cells differentiated from the hematopoietic stem cells of healthy donors are Hb-CS protein negative red blood cells (referred to as CS negative red blood cells or CS- cells).

[0094] Example 3 Animal immunization and monoclonal cell screening

[0095] 1. Antigen preparation and animal immunization

[0096] The polypeptide antigen was synthesized according to the amino acid sequence (SEQ ID NO: 25) and coupled with OVA. Two rabbits were immunized with the prepared antigen, and the antibody titer in the rabbit serum was detected by indirect ELISA after immunization. After detection, significant antibody expression was detected in the rabbit serum after 4 immunizations.

[0097] 2. Rabbit serum antibody detection

[0098] Take 5x 10 each 6 445 μl PBS (Gibco, catalog number C10010500BT) was added to each of the CS-positive and CS-negative erythrocytes obtained by the in vitro differentiation method of Example 2, and then 50 μl of 0.5% glutaraldehyde (Sangon Biotechnology, catalog number A600875-0100) was added to each tube, mixed, and fixed at room temperature in the dark for 10 min; after fixation, 500 μl PBS was added to wash, centrifuged at 500×g for 5 min, and the supernatant was removed; 450 μl of PBS after ice bath was added to each tube to resuspend the cells, and then 50 μl of 1% Trition X-100 (Sangon Biotechnology, catalog number A110694-0500) was added to each tube, mixed, and permeabilized at room temperature in the dark for 5 min; after permeabilization, 500 μl PBS was added to wash, centrifuged at 500×g for 5 min, and the supernatant was removed; 100 μl Resuspend in PBS, add 0.5 μl rabbit serum before / after immunization, incubate at 4°C for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend cells in 50 μl PBS, add 0.5 μl fluorescently labeled anti-rabbit secondary antibody (Cell Signaling Technology, catalog number #4414), incubate at 4°C for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend cells in 200 μl PBS and test on the machine. Use flow cytometer (Beckman, model A00-1-1102) to detect indicators such as the proportion of positive cells and the grouping of the spectrum, and then screen and determine whether the immune serum can specifically identify CS-positive red blood cells.

[0099] See also Figure 2 , which is a flow cytometry result diagram of rabbit serum and red blood cells before and after immunization in Example 3 of the present invention. The results show that a large number of nonspecific antibodies bind to red blood cells in the rabbit serum before and after immunization. Since the specific antibodies against Hb-CS antigen in the rabbit serum after immunization increase, its recognition level for CS-positive red blood cells increases, and its recognition ability for CS-negative red blood cells weakens. Since the recognition ability of the immunized serum of rabbit No. 2 is slightly stronger than that of the immunized serum of rabbit No. 1, rabbit No. 2 was selected for monoclonal antibody screening.

[0100] 3. Monoclonal serum screening

[0101] According to the above results, 2# rabbit spleen cells were sorted, and a total of about 960 cells were sorted. After the cells were cultured, the supernatant was taken for ELISA detection, and a total of about 200 positive clones that can identify CS-positive red blood cells were obtained. The top 30 cells with higher signal values ​​were taken for antibody sequence amplification, and a total of 14 positive clones were obtained after excluding repeated sequences. The culture supernatant of the 14 cells was taken, and the antibody expression level in the supernatant was repeatedly detected by ELISA. The test results are shown in Table 1.

[0102] Table 1 shows the ELISA test results of 14 monoclonal cell supernatants. The supernatants of 14 antibodies were repeatedly tested, namely GR1-GR14. The negative control cell culture supernatant and blank culture medium are blank controls, which are used to exclude non-specific binding in the data. The ELISA plates were coated with free peptide (SinoA11815) and OVA-coupled peptide (OVA-SMCC-SinoA11815), and three concentration gradients of 0.1μg / ml, 1μg / ml, and 5μg / ml were made to verify the specificity of the antibody. The results showed that the 14 monoclonal cells screened can secrete antibodies that specifically bind to the peptide.

[0103] Table 1. ELISA test results of 14 monoclonal cell supernatants

[0104]

[0105]

[0106] Example 4 Specific detection of monoclonal cell supernatant (antibodies can effectively distinguish CS-positive red blood cells from CS-negative red blood cells)

[0107] Take 5x 10 each 6445 μl PBS (Gibco, catalog number C10010500BT) was added to dilute the CS-positive erythrocytes and CS-negative erythrocytes obtained by the in vitro differentiation method of Example 2, and then 50 μl of 0.5% glutaraldehyde (Sangon Biotechnology, catalog number A600875-0100) was added to each tube, mixed well, and fixed at room temperature in the dark for 10 min; after fixation, 500 μl PBS was added to wash, centrifuged at 500×g for 5 min, and the supernatant was removed; 450 μl of PBS after ice bath was added to each tube to resuspend the cells, and then 50 μl of 1% Trition X-100 (Sangon Biotechnology, catalog number A110694-0500) was added to each tube, mixed well, and permeabilized at room temperature in the dark for 5 min; after permeabilization, 500 μl PBS was added to wash, centrifuged at 500×g for 5 min, and the supernatant was removed; 100 μl Resuspend in PBS, add 0.5 μl monoclonal cell supernatant (the supernatant contains Hb-CS antibody secreted by cells), incubate at 4°C for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend cells in 50 μl PBS, add 0.5 μl secondary antibody (Cell Signaling Technology, catalog number #4414), incubate at 4°C for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend cells in 200 μl PBS and detect on the machine; use flow cytometer (Beckman, model A00-1-1102) to detect indicators such as the proportion of positive cells and the clustering situation, and then screen and determine whether the antibody can specifically recognize CS-positive red blood cells.

[0108] Please refer to the results Figure 3 , is a result diagram of flow cytometry detection of 14 monoclonal cell culture supernatants using CS-positive red blood cells in Example 4 of the present invention. The results show that 1#, 6#, 10#, 13#, 16#, 17# and 25# can all identify CS-positive red blood cells.

[0109] In order to fully verify the specificity of the antibodies, the CS-positive erythrocytes and CS-negative erythrocytes obtained by the in vitro differentiation method in Example 2 were used to repeat the functional identification of the above 7 antibodies. Figure 4 , which is a result diagram of flow cytometry detection of 7 antibodies using CS-positive erythrocytes and CS-negative erythrocytes in Example 4 of the present invention. The results show that the cell supernatants derived from clones 13#, 16# and 17# can specifically identify CS-positive erythrocytes.

[0110] In order to detect the detection efficiency of Hb-CS antibodies in cell supernatants on human red blood cells, the applicant collected peripheral blood from Hb-CS patients from the 923rd Hospital and used the blood samples to perform repeated functional verification on the culture supernatants of clones 13#, 16# and 17#. For the results, please refer to Figure 5, which is a result diagram of flow cytometry detection of culture supernatants of clones 13#, 16# and 17# using peripheral blood from patients in Example 4 of the present invention. The results show that in the peripheral blood samples from patients, culture supernatants of clones 13#, 16# and 17# all have strong positive signals, and the groups are obvious.

[0111] Example 5 Monoclonal Antibody Preparation and Immunogenicity Testing

[0112] 1. Monoclonal Antibody Preparation

[0113] Antibody sequence expression vectors were constructed based on the antibody sequences of 13#, 16# and 17#, antibodies were expressed using 293T cells, and monoclonal antibodies were obtained through protein purification. In this embodiment, the antibody constant region adopts the wild-type constant region of rabbit, and the sequence information of the variable regions HCDR1-HCDR3 and LCDR1-LCDR3 of monoclonal antibodies #13, #16 and #17 is shown in Table 2. The HCVR amino acid sequence of monoclonal antibody #13 is SEQ ID NO:19, and the HCVR nucleotide sequence is SEQ ID NO:27; the LCVR amino acid sequence is SEQ ID NO:22, and the LCVR nucleotide sequence is SEQ ID NO:28. The HCVR amino acid sequence of monoclonal antibody #16 is SEQ ID NO:20, and the HCVR nucleotide sequence is SEQ ID NO:29; the LCVR amino acid sequence is SEQ ID NO:23, and the LCVR nucleotide sequence is SEQ ID NO:30. The HCVR amino acid sequence of monoclonal antibody #17 is SEQ ID NO:21, and the HCVR nucleotide sequence is SEQ ID NO:31; the LCVR amino acid sequence is SEQ ID NO:24, and the LCVR nucleotide sequence is SEQ ID NO:32.

[0114] 2. Western Blotting to detect antibody specificity

[0115] Western Blotting was performed on proteins extracted from peripheral blood samples of Hb-CS patients and healthy subjects (from the 923rd Hospital) using monoclonal antibodies 13#, 16# and 17#. For the results, please refer to Figure 6 , which is the result of Western Blotting test of proteins extracted from peripheral blood of Hb-CS patients and healthy people using monoclonal antibodies in Example 5 of the present invention. The results show that wild-type α-globin antibodies can recognize α CS globin (Hb-CS protein) and α-globin, and its CS The recognition ability of globin is weaker. However, monoclonal antibodies 13#, 16# and 17# can specifically recognize α CS Globin.

[0116] Table 2 Sequence information of monoclonal antibody variable regions (HCDR1-HCDR3, LCDR1-LCDR3)

[0117]

[0118] Example 6 Monoclonal antibodies can effectively identify patients' CS-positive red blood cells

[0119] In this example, red blood cells from Hb-CS patients and healthy subjects (from the 923rd Hospital) were used to detect the specificity of the monoclonal antibody.

[0120] 5 μl of peripheral blood samples from Hb-CS patients and healthy subjects were respectively placed in two EP tubes, diluted with 445 μl PBS (Gibco, catalog number C10010500BT), and then 50 μl of 0.5% glutaraldehyde (Sangon Biotechnology, catalog number A600875-0100) was added to each tube, mixed well, and fixed at room temperature for 10 min; after fixation, 500 μl PBS was added for washing, centrifuged at 500×g for 5 min, and the supernatant was removed; 450 μl of PBS after ice bath was added to each tube to resuspend the cells, and then 50 μl of 1% TritionX-100 (Sangon Biotechnology, catalog number A110694-0500) was added to each tube, mixed well, and permeabilized at room temperature for 5 min; after permeabilization, 500 μl PBS was added for washing, centrifuged at 500×g for 5 min, and the supernatant was removed; 100 μl Resuspend in PBS, divide each tube of cells into three equal parts, add 0.5 μl of 13#, 16# and 17# monoclonal antibodies respectively, incubate at 4℃ for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend the cells in 50 μl PBS, add 0.5 μl secondary antibody (Cell Signaling Technology, catalog number #4414); incubate at 4℃ for 30 min, wash with 500 μl PBS, centrifuge at 500×g for 5 min, and remove the supernatant; resuspend the cells in 200 μl PBS and detect on the machine.

[0121] Please refer to the test results Figure 7 , which is the result of flow cytometry detection of peripheral blood of Hb-CS patients and healthy people using monoclonal antibodies in Example 6 of the present invention. The results show that monoclonal antibodies No. 13#, No. 16# and No. 17# can effectively identify Hb-CS protein positive red blood cells, and the detection ratio is in line with expectations, that is, the proportion of CS positive red blood cells in the peripheral blood of Hb-CS patients who have been transfused for two weeks is close to 50%, while there are no CS positive red blood cells in the peripheral blood of healthy donors.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Hb-CS protein, characterized in that: The antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and light chain complementary determining regions LCDR1, LCDR2 and LCDR3; The amino acid sequence of the HCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 1 to 3 or a variant thereof; The amino acid sequence of the HCDR2 comprises an amino acid sequence selected from the following group or a variant thereof: X1IX2AGSSX3X4X5YX4X6X7X8X9G, wherein X1 is selected from R or I, X2 is selected from K or Y, X3 is selected from E, S or T, X4 is selected from S or A, X5 is selected from T, Y or E, X6 is selected from P or S, X7 is selected from R, W or E, X8 is selected from F or A, and X9 is selected from E or K; The amino acid sequence of the HCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 4 to 6 or a variant thereof; The amino acid sequence of LCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 7 to 9 or a variant thereof; The amino acid sequence of the LCDR2 comprises an amino acid sequence or a variant thereof selected from the following group: X 10 ASTLAX 11 , where X 10 Select from N or A, X 11 Selected from P or S; The amino acid sequence of LCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 10 to 12 or a variant thereof; The variant is a variant in which at least one, at least two or at least three conservative amino acid substitutions are made in the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the HCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 1 to 3 or a variant thereof; and / or, The amino acid sequence of the HCDR2 comprises one of the amino acid sequences shown in SEQ ID NOs: 13 to 15 or a variant thereof; and / or, The amino acid sequence of the HCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 4 to 6 or a variant thereof; and / or, The amino acid sequence of LCDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 7 to 9 or a variant thereof; and / or, The amino acid sequence of LCDR2 comprises one of the amino acid sequences shown in SEQ ID NOs: 16 to 18 or a variant thereof; and / or, The amino acid sequence of the LCDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 10 to 12 or a variant thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises: The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 13, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 4; or The amino acid sequence of HCDR1 is as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:14, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5; or The amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:15, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; or LCDR1 having an amino acid sequence as shown in SEQ ID NO:7, LCDR2 having an amino acid sequence as shown in SEQ ID NO:16, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:10; or LCDR1 having an amino acid sequence as shown in SEQ ID NO:8, LCDR2 having an amino acid sequence as shown in SEQ ID NO:17, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:11; or The amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:18, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody or antigen-binding fragment thereof comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:13, HCDR3 with an amino acid sequence as shown in SEQ ID NO:4, LCDR1 with an amino acid sequence as shown in SEQ ID NO:7, LCDR2 with an amino acid sequence as shown in SEQ ID NO:16, LCDR3 with an amino acid sequence as shown in SEQ ID NO:10; or HCDR1 with an amino acid sequence as shown in SEQ ID NO:2, HCDR2 with an amino acid sequence as shown in SEQ ID NO:14, HCDR3 with an amino acid sequence as shown in SEQ ID NO:5, LCDR1 with an amino acid sequence as shown in SEQ ID NO:8, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17, LCDR3 with an amino acid sequence as shown in SEQ ID NO:11; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:15, the amino acid sequence of HCDR3 is shown in SEQ ID NO:6, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:18, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

12.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and / or a light chain framework region; Preferably, the heavy chain framework region is selected from the antibody heavy chain framework region of human, mouse or rabbit origin; and / or, the light chain framework region is selected from the antibody light chain framework region of human, mouse or rabbit origin.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; and / or, The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is any one of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 with at least 1, at least 2 or at least 3 conservative amino acid substitutions; and / or, The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; and / or, The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is a conservative amino acid substitution made on the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:

24.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein: The antibody or antigen-binding fragment thereof comprises: The heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 22; or The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:23; or The heavy chain variable region is shown as SEQ ID NO:21 and the light chain variable region is shown as SEQ ID NO:

24.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein: The antibody or antigen-binding fragment thereof specifically binds to the C-terminus of the Hb-CS protein; The C-terminus of the Hb-CS protein includes the amino acid sequence shown in SEQ ID NO:

25.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

10. A protein, characterized in that The protein comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the protein further comprises other functional molecules connected to the end of the antibody or antigen-binding fragment thereof; More preferably, the other functional molecules are selected from one or more of the following: a signal peptide or a protein tag.

11. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10.

12. A gene expression cassette, characterized in that: The gene expression cassette comprises the nucleic acid of claim 11 or 12.

13. A carrier, characterized in that The vector comprises the nucleic acid according to claim 11 or 12, or the gene expression cassette according to claim 13; Preferably, the vector is a plasmid, cosmid, phage or viral vector.

14. A host cell, characterized in that The host cell expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is a bacterial cell or a yeast cell.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10, characterized in that: The method comprises the following steps: Cultivate the host cell according to claim 15, and obtain the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10 from the culture.

16. A detection reagent, characterized in that: The detection reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10.

17. A method for detecting Hb-CS protein, characterized in that: The following steps are involved: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the protein according to claim 10 is contacted with a sample to be tested.

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