CN112979799B

CN112979799B - Binding protein containing hemoglobin antigen structural domain

Info

Publication number: CN112979799B
Application number: CN201911311915.3A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 姜瑢瑢; 游辉; 王晨; 覃婷
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2022-11-08
Anticipated expiration: 2039-12-18
Also published as: CN112979799A

Abstract

The present invention provides a binding protein comprising an antigen binding domain that binds to hemoglobin, which is capable of efficiently binding to hemoglobin in feces and is further effectively and widely used for detection of fecal occult blood. The binding protein has strong activity and high affinity, and the preparation method is simple and the quality is controllable.

Description

Binding protein containing hemoglobin antigen structural domain

Technical Field

The invention relates to the technical field of biotechnology and medicine, in particular to a binding protein containing a hemoglobin antigen structural domain.

Background

In recent years, the types of digestive tract diseases tend to be diversified, and the incidence rate and the death rate of intestinal tumors also tend to rise year by year. The fecal occult blood test is a relatively common detection technology in clinic and has important value for diagnosis of alimentary tract hemorrhage. The patients with the early-stage gastrointestinal tumors have a high probability of appearing positive occult blood tests, and the patients with the late-stage gastrointestinal tumors have higher occult blood positive rate and are continuously positive, so the fecal occult blood test can be used as a first-choice index for screening the gastrointestinal tumors.

Fecal Occult Blood (FOB) refers to hemorrhage with digestive tract bleeding amount below 5ml/d, no visible blood color, and no erythrocyte destruction under microscope. The digestive tract hemorrhage is diagnosed by detecting the hemoglobin in the fecal occult blood. Therefore, hemoglobin (Hb) in feces is clinically recognized as one of the important tumor markers of rectal cancer and colon cancer.

The chemical method for detecting fecal occult blood is designed by utilizing the fact that heme contained in hemoglobin (Hb) has the effect similar to peroxidase. The method has more oxidation color developing agents, such as benzidine, o-toluidine, o-tolidine, guaiazulene, reduced phenolphthalein, aminopyridine, colorless malachite green, tetramethyl benzidine, diphenylamine, dimethyl benzidine, etc. Although the chemical method is simple and easy to implement and wide in application, the specificity is low and the interference factors are more. Animal blood, meat, liver and food rich in chlorophyll, iron, vitamin C, chinese medicine, etc. can all cause false positive.

In recent years, colloidal gold immunochromatography has been developed. Antihuman hemoglobin antibody is detected by using heme of hemoglobin (Hb) in blood to specifically bind with human Hb antigen. Compared with the traditional chemical method, the immunity method for detecting the hemoglobin monoclonal antibody has the advantages that the sensitivity and the specificity are greatly improved, the immunity method is not influenced by diet or related medicines, and the immunity method plays an important role in diagnosing and treating early tumors.

The method is convenient for accurately detecting occult blood, avoids the characteristics of false positive and poor specificity, and has strong practical value for producing the high-specificity anti-human hemoglobin monoclonal antibody. At present, the mouse monoclonal antibody produced by using the hybridoma technology is widely applied to scientific research, clinical diagnosis and treatment. However, as the mouse peritoneal cavity is adopted for hybridoma production, the influence of individual mice is very large, and the problems of unstable production, large batch difference, large mouse autoantibody purification difficulty and the like exist. Therefore, there is a strong need in the art for an antibody that effectively stably and specifically binds hemoglobin and detects it, while the anti-CRP antibody existing in the art cannot be well applied to the detection of CRP due to low activity and poor affinity.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to a novel binding protein containing an antigen binding domain combined with hemoglobin, and researches on the aspects of preparation, application and the like of the binding protein.

The antigen binding domain includes at least one complementarity determining region selected from the group consisting of amino acid sequences described below, or has at least 80% sequence identity with a complementarity determining region of an amino acid sequence described below and has a KD ≦ 9.43 × 10 for hemoglobin ^- ⁹ Affinity of mol/L:

CDR-VH1 is G-Y-X1-F-X2-N-Y-T-W-X3-N, wherein:

x1 is T or S, X2 is T or S, X3 is F or M;

CDR-VH2 is

I-I-Y-P-G-X1-S-D-T-X2-Y-X3-Q-K-F-X4-D, wherein:

x1 is Q, H or N, X2 is S or T, X3 is N or GG, X4 is K or R;

CDR-VH3 is T-X1-D-G-Y-Y-X2-A-W-X3, wherein:

x1 is K or R, X2 is P or A, X3 is F, A or G;

the complementarity determining region CDR-VL1 is S-Q-X1-L-L-X2-S-N-X3-Q-K-X4-Y-L, wherein:

x1 is T or S, X2 is T or S, X3 is I, V or L, X4 is Q or N;

CDR-VL2 is W-X1-S-T-R-X2-S, wherein:

x1 is A or P, X2 is E or D;

CDR-VL3 is Q-X1-Y-Y-X2-Y-P, wherein:

x1 is N, Q or H, X2 is S or T;

the binding protein provided by the invention has strong activity and high affinity with hemoglobin, can be effectively used for clinical detection of fecal occult blood, and particularly can be used as an index for screening digestive tract tumors. Meanwhile, the binding protein obtained by the invention in a recombination mode has small individual difference, small batch difference and stable quality. Is more beneficial to the quality control and the detection stability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an electrophoretogram of a recombinant antibody against hemoglobin in example 1 of the present invention.

Detailed Description

The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

In order that the present invention may be more readily understood, selected terms are defined below.

The term "amino acid" denotes a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used in this application may include both naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: A1a, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1N, Q), glutamic acid (G1 u, E), glycine (G1Y, G), histidine (His, H), isoleucine (I1E, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Va 1, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

The terms "isolated binding protein comprising an antigen binding domain", "isolated binding protein", "binding protein" generally refer to all proteins/protein fragments comprising CDR regions, including Fab, F (ab') 2, fd, fv, scFv, diabodies, minimal recognition units for antibodies, and antibodies, as well as single chain derivatives of such antibodies and fragments.

The term "antibody" includes polyclonal antibodies, monoclonal antibodies, and antigenic compound-binding fragments of these antibodies. The type of antibody can be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isoforms (antibodies). The term "antibody" is used interchangeably with "immunoglobulin".

An "antibody fragment" or "antibody fragments" is only a portion of an antibody, wherein the portion retains at least one, and preferably many or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2, and Fv fragments; diabodies (diabodies); a linear antibody; single chain antibody molecules, including single chain Fv (scFv) antibody molecules; a multivalent antibody formed from multiple copies of the same antibody fragment with the same specificity; and multispecific antibodies formed from antibody fragments.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that several residues are added at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is herein denoted as such a Fab': wherein the cysteine residues of the constant domains carry a free thiol group. F (ab ') 2 antibody fragments were originally produced as Fab' fragment pairs, which have a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

"Fv" refers to antibody fragments that contain an intact antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain, either tightly non-covalently or covalently bound (disulfide-linked Fv has been described in the art, reiter et al (1996)). In this configuration, the 3 CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The combination of one or more CDRs from each VH and VL chain together confers antibody antigen binding specificity.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region is made up of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

The term "vector" refers to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e. a vector for transferring genetic information into a cell, which may be propagated and in which the presence or absence of said genetic information may be selected) or an expression vector (i.e. a vector comprising the necessary genetic elements to allow expression of the genetic information of said vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The term "framework", "framework" or "FR" region means the regions of the antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs. Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The term "affinity" denotes the equilibrium constant for reversible binding of two reagents and is denoted as K _D . The affinity of a binding protein for a ligand, such as the affinity of an antibody for an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomolar (fM). The term "avidity" as used herein means the resistance of a complex of 2 or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art.

The invention also provides a recombinant host cell containing the recombinant vector. Particularly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest, and minimal contamination from host proteins. Mammalian cell lines useful as hosts for expression are well known in the art and include many immortalized cell lines such as, but not limited to, COS-7 cells, chinese Hamster Ovary (CHO) cells, baby Hamster Kidney (BHK) cells, and many others, including cell lines of lymphoid origin such as lymphoma, myeloma, or hybridoma cells. Preferred host cells for transformation vectors and expression of the antibodies of the invention are mammalian cells, such as NSO cells (non-secretory (0) mouse myeloma cells), human Embryonic Kidney (HEK) 293, SP20 and Chinese Hamster Ovary (CHO) cells and other cell lines of lymphoid origin, such as lymphoma, myeloma or hybridoma cells. The antibodies of the invention may be expressed in cell lines other than hybridomas. Other eukaryotic hosts, such as yeast, may alternatively be used. Antibodies and more specifically antigen-binding fragments thereof can also be produced from prokaryotic cells such as e. Nucleic acids comprising sequences encoding antibodies according to the invention can be used to transform suitable mammalian host cells.

The expression vectors of the invention are useful for transforming host cells. Such transformed cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the polypeptides of the invention. The transformed cells of the present invention include microorganisms such as bacteria (e.g., escherichia coli, bacillus spp., etc.). The transformed cells are capable of replicating the nucleic acid fragments of the invention. When the peptide combination of the present invention is recombinantly produced, the expression product may be exported into the culture medium or carried on the surface of the transformed cell.

The present invention provides an article of manufacture (e.g., a kit) comprising materials useful for detecting digestive tract bleeding. The article includes a container and a label or package insert on or with the container. Suitable containers include, for example, bottles or syringes, and the like. The container may be made of various materials such as glass or plastic. The container contains a composition, either alone or in combination with another composition effective for diagnosing fecal occult blood. At least one active agent in the composition is a binding protein provided by the invention.

The term "functional fragment" as used herein refers in particular to an antibody fragment having the same specificity for haemoglobin as the parent antibody. In addition to the above functional fragments, any fragment having an increased half-life is also included.

The invention states that "operably linked" means that the coding sequence is linked to the regulatory sequence in a manner that allows for expression of the coding sequence. Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell and include promoters, enhancers and other expression control elements.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention

The present invention provides an isolated binding protein comprising an antigen binding domain comprising at least one complementarity determining region selected from the group consisting of amino acid sequences set forth below, or having at least 80% sequence identity with a complementarity determining region of an amino acid sequence set forth below and having a K with hemoglobin _D ≤9.43×10 ^-9 (ii) a mol/L affinity;

CDR-VH1 is G-Y-X1-F-X2-N-Y-T-W-X3-N, wherein:

x1 is T or S, X2 is T or S, X3 is F or M;

CDR-VH2 is I-I-Y-P-G-X1-S-D-T-X2-Y-X3-Q-K-F-X4-D, wherein:

x1 is Q, H or N, X2 is S or T, X3 is N or GG, X4 is K or R;

CDR-VH3 is T-X1-D-G-Y-Y-X2-A-W-X3, wherein:

x1 is K or R, X2 is P or A, X3 is F, A or G;

CDR-VL1 is S-Q-X1-L-L-X2-S-N-X3-Q-K-X4-Y-L, wherein:

x1 is T or S, X2 is T or S, X3 is I, V or L, X4 is Q or N;

CDR-VL2 is W-X1-S-T-R-X2-S, wherein:

x1 is A or P, X2 is E or D;

CDR-VL3 is Q-X1-Y-Y-X2-Y-P, wherein:

x1 is N, Q or H, X2 is S or T;

in some embodiments, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to a complementarity determining region of an amino acid sequence having a K with hemoglobin _D ≤9.43×10 ^-9 mol/L，K _D The value can also be selected to be 1 × 10 ^-10 mol/L、2×10 ^-10 mol/L、3×10 ^-10 mol/L、4×10 ^-10 mol/L、5×10 ^-10 mol/L、6×10 ^-10 mol/L、7×10 ^-10 mol/L、8×10 ^-10 mol/L、9×10 ^-10 mol/L、1×10 ^-9 mol/L、2×10 ^-9 mol/L、3×10 ^-9 mol/L、4×10 ^-9 mol/L、5×10 ^-9 mol/L、6×10 ^-9 mol/L、7×10 ^-9 mol/L、8×10 ^-9 mol/L, or 9X 10 ^-9 mol/L; or 1.34X 10 ^- ¹⁰ mol/L≤K _D ≤9.43×10 ^-9 mol/L。

Wherein the affinity is determined according to the method in the examples of the present invention.

In some embodiments:

in the CDR-VH1, X3 is M;

in the CDR-VH2, X4 is K;

in the CDR-VH3, X1 is R;

in the complementarity determining region CDR-VL1, X4 is N;

in the complementarity determining region CDR-VL2, X1 is A;

in the complementarity determining region CDR-VL3, X2 is S;

optionally:

in the CDR-VH1, X3 is F;

in the CDR-VH2, X4 is R;

in the CDR-VH3, X1 is K;

in the complementarity determining region CDR-VL1, X4 is Q;

in the complementarity determining region CDR-VL2, X1 is P;

in the complementarity determining region CDR-VL3, X2 is T;

preferably, in the complementarity determining region CDR-VH1, X1 is T;

preferably, in the complementarity determining region CDR-VH1, X1 is S;

preferably, in the complementarity determining region CDR-VH1, X2 is T;

preferably, in the complementarity determining region CDR-VH1, X2 is S;

preferably, in the complementarity determining region CDR-VH2, X1 is Q;

preferably, in the complementarity determining region CDR-VH2, X1 is H;

preferably, in the complementarity determining region CDR-VH2, X1 is N;

preferably, in the complementarity determining region CDR-VH2, X2 is S;

preferably, in the complementarity determining region CDR-VH2, X2 is T;

preferably, in the complementarity determining region CDR-VH2, X3 is N;

preferably, in the complementarity determining region CDR-VH2, X3 is GG;

preferably, in the complementarity determining region CDR-VH3, X2 is P;

preferably, in the complementarity determining region CDR-VH3, X2 is a;

preferably, in the complementarity determining region CDR-VH3, X3 is F;

preferably, in the complementarity determining region CDR-VH3, X3 is a;

preferably, in the complementarity determining region CDR-VH3, X3 is G;

preferably, in the complementarity determining region CDR-VL1, X1 is T;

preferably, in the complementarity determining region CDR-VL1, X1 is S;

preferably, in the complementarity determining region CDR-VL1, X2 is T;

preferably, in the complementarity determining region CDR-VL1, X2 is S;

preferably, in the complementarity determining region CDR-VL1, X3 is I;

preferably, in the complementarity determining region CDR-VL1, X3 is V;

preferably, in the complementarity determining region CDR-VL1, X3 is L;

preferably, in the complementarity determining region CDR-VL2, X2 is E;

preferably, in the complementarity determining region CDR-VL2, X2 is D;

preferably, in the complementarity determining region CDR-VL3, X1 is N;

preferably, in the complementarity determining region CDR-VL3, X1 is Q;

preferably, in the complementarity determining region CDR-VL3, X1 is H;

in some embodiments, the mutation site of each complementarity determining region is selected from any one of the following sequences:

in some embodiments, at least 3 CDRs are included in the binding protein; alternatively, the binding protein comprises at least 6 CDRs, which may alternatively be any combination selected from the group consisting of heavy chain CDR regions and/or light chain CDR regions.

In some embodiments, 3 CDRs, 4 CDRs, 5 CDRs, 6 CDRs may be included in the binding proteins described herein.

In some embodiments, the binding protein is a nanobody, F (ab') ₂ Any one of Fab', fab, fv, scFv, diabody and antibody minimal recognition unit.

In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having the sequences shown in SEQ ID NOS: 1-4 in order, and/or heavy chain framework regions FRH1, FR-H2, FR-H3 and FR-H4 having the sequences shown in SEQ ID NOS: 5-8 in order.

In some embodiments, the framework regions can be of human species origin to constitute humanized antibodies.

In some embodiments, the binding protein further comprises an antibody constant region sequence.

In some embodiments, the constant region sequence is selected from the group consisting of sequences of any one of the constant regions IgG1, igG2, igG3, igG4, igA, igM, igE, igD.

In some embodiments, the species of the constant region is from a cow, horse, dairy cow, pig, sheep, goat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, chicken, or human.

In some embodiments, the constant region is derived from a mouse;

in other embodiments, the:

the light chain constant region sequence is shown as SEQ ID NO. 9;

the heavy chain constant region sequence is shown in SEQ ID NO 10.

In another aspect of the invention, the constant regions of the invention may be derived from human, to constitute humanized antibodies.

In some embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

According to one aspect of the invention, the invention also relates to an isolated nucleic acid molecule, which is DNA or RNA, encoding a binding protein as described above. The nucleic acids of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragments of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the present invention are capable of autonomous replication and therefore are capable of providing high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a signal peptide for secretion or integration of the peptide expression product into a membrane, a nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In some embodiments, the host cell can be a eukaryotic cell, such as a mammalian cell.

In some embodiments, the host cell is an NSO cell (mouse myeloma cell), a Human Embryonic Kidney (HEK) cell, a human osteosarcoma cell (SP 20), a Chinese Hamster Ovary (CHO) cell, or other cell line of lymphoid origin, such as a lymphoma, myeloma, or hybridoma cell.

According to one aspect of the invention, the invention also relates to a method for producing a binding protein as described above, said method comprising the steps of:

the host cells as described above are cultured in a medium and under suitable culture conditions, and the binding protein so produced is recovered from the medium or from the cultured host cells.

According to one aspect of the invention, the invention also relates to the use of a binding protein as described above for the preparation of a stool hemoglobin detector.

In some embodiments, the tumor of the digestive tract comprises rectal cancer, colon cancer.

According to one aspect of the invention, the invention also relates to a method of detecting hemoglobin in a test sample, comprising:

a) Contacting the binding protein as described above with a hemoglobin antigen in a test sample under suitable reaction conditions;

b) Detecting the immune complex for indicating the presence of hemoglobin in the test sample.

In this embodiment, the binding protein may be labeled with an indicator that indicates the strength of the signal, so that the complex is readily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the binding protein.

In some embodiments, in step a), a second antibody is further included in the immune complex, which second antibody binds to hemoglobin in the test sample;

in this embodiment, the binding protein is in the form of a first antibody that forms a partner antibody with the second antibody for binding to a different epitope of hemoglobin;

the second antibody may be labeled with an indicator showing the intensity of the signal so that the complex is easily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to hemoglobin antigen in the stool;

in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator that indicates the strength of the signal to allow the complex to be readily detected.

In the above embodiments, the test sample is human stool.

In some embodiments, the indicator that indicates signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In some embodiments, the fluorescent species comprise Alexa 350, alexa 405, alexa 430, alexa488, alexa555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, rhodamine, and mixtures thereof azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate or chelate, diamine, bispyanine glycoside, la Jolla Blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.

In some embodiments, the radioisotope comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ⁹⁴ mTc、 ⁹⁴ Tc、 ⁹⁹ mTc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ¹⁵⁴ -158Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ⁵² mMn、 ⁵⁵ Co、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ⁸² mRb and ⁸³ any one of Sr.

In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In some embodiments, the fluorescent microsphere is: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.

According to one aspect of the invention, the invention also relates to a reagent or kit comprising a binding protein as described above.

Further, the reagent or the kit further comprises one or more of a buffering agent, a stabilizing agent and a diluting agent.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. The SMARTERTM RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation. The hybridoma cell strain secreting Anti-FOB7E2 monoclonal antibody is an existing hybridoma cell strain in the laboratory of the applicant, and is recovered for later use.

1. Primer and method for producing the same

Amplifying Heavy Chain and Light Chain 5' RACE primers:

SMARTER II A Oligonucleotide:

5’>AAGCAGTGGTATCAACGCAGAGTACXXXXX<3’

5'-RACE CDS Primer(5'-CDS)：

5’>(T)25VN<3’(N＝A,C,G,orT；V＝A,G,orC)；

Universal Primer A Mix(UPM):5’>

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT<3’

Nested Universal Primer A(NUP):

5’>AAGCAGTGGTATCAACGCAGAGT<3’

mIgG CKR：5’>CTAACACTCATTCCTGTTGAAGCTCTTGACAAT<3’

mIgG CHR：5’>TCATTTACCAGGAGAGTGGGAGAGGC<3’

2. antibody variable region gene cloning and sequencing

RNA is extracted from hybridoma cell strains secreting Anti-FOB7E2 monoclonal antibodies, first strand cDNA synthesis is carried out by a SMARTERTM RACECDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and obtained first strand cDNA products are used as PCR Amplification templates. The Light Chain gene was amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP), and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP), and mIgG CHR primers. The primer pair of Light Chain can amplify target band about 0.75KB, and the primer pair of Heavy Chain can amplify target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was inserted into pMD-18T vector after A-addition reaction with rTaq DNA polymerase, transformed into DH 5. Alpha. Competent cells, and after colonies were grown, 4 clones of the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen corporation for sequencing.

Sequence analysis of Anti-FOB7E2 antibody variable region Gene

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 342bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

4. Construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the Anti-FOB7E2 antibody are designed, the two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:

FOB-7E2-HF：

5’>CCCAAGCTTGCCACCATGGAATGGAGCTGGGTCTTTC<3’；

FOB-7E2-HR:

5’>CCGAATTCTCATTATTTACCAGGAGAGTGGGAGAGGCTCTTCTC<3’；

FOB-7E2-LF:

5’>CCCAAGCTTGCCACCATGGATTCACAGGCCCAGGTTCTTA<3’；

FOB-7E2-LR:5’>CCCGAATTCTCATTAACACTCATTCCTGTTGAAGCTCTTGACAA<3’；

a0.75 KB Light Chain gene fragment and a 1.4KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the gene of the Heavy Chain and the gene of the Light Chain are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

5. Screening for Stable cell lines

5.1 transient transfection of recombinant antibody expression plasmids into CHO cells, determination of expression plasmid Activity

Plasmid-use supersetDiluting with pure water to 400ng/ml, regulating CHO cells to 1.43 × 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

The coating solution dilutes human hemoglobin to 3 mug/ml, each well is 100 mug, and the temperature is kept overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; add blocking solution (20% BSA +80% PBS) 120 μ l per well, 37 ℃,1h, patted dry; adding diluted cell supernatant at 100 μ l/well, 37 deg.C for 30min (partial supernatant for 1 h); washing with the washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results show that the reaction OD after the cell supernatant is diluted 1000 times is still larger than 1.0, and the reaction OD of the wells without the cell supernatant is smaller than 0.1, which indicates that the anti-human hemoglobin produced after the plasmid is transiently transformed has the in vivo pertinence.

5.2 linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mu g DNA/tube, 10 mul PuvI enzyme and sterile water to 500 mul, and carrying out enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25; precipitating with 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

5.3 Stable transfection of recombinant antibody expression plasmids, pressurized selection of Stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; preserving seeds after 3 daysBatch culture, adjusting cell density to 0.5 × 10 ⁶ cells/ml,2.2ml, cell density 0.3X 10 ⁶ cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter, transferring the cell strains to TPP (thermoplastic vulcanizate) for seed preservation and passage.

6. Recombinant antibody production

6.1 cell expansion culture

After the cell recovery, the cells were first cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at 100% volume, and placed in a shaker at a rotation speed of 120r/min, a temperature of 37 ℃ and a carbon dioxide content of 8%. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/ml, and calculating the expanding volume according to production requirements, wherein the culture medium accounts for 100 percent. Then carrying out propagation every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing manner: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of an external control antibody was used as a control, and the electrophoretogram was shown in the figure. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (heavy chain) and 28kD of Mr (light chain).

Example 2

The antibody obtained in example 1 was analyzed to have a light chain having a sequence shown in SEQ ID NO. 11 and a heavy chain having a sequence shown in SEQ ID NO. 12. The antibody has certain hemoglobin binding capacity and good affinity, and in order to further screen an antibody with better affinity, the applicant performs mutation on the light chain CDR and the heavy chain CDR of the antibody.

Upon analysis, the complementarity determining region (WT) of the heavy chain:

CDR-VH1 is G-Y-T (X1) -F-S (X2) -N-Y-T-W-F (X3) -N;

CDR-VH2 is I-I-Y-P-G-H (X1) -S-D-T-T (X2) -Y-GG (X3) -Q-K-F-R (X4) -D;

CDR-VH3 is T-K (X1) -D-G-Y-Y-P (X2) -A-W-G (X3);

complementarity determining regions of the light chain:

CDR-VL1 is S-Q-S (X1) -L-L-T (X2) -S-N-V (X3) -Q-K-Q (X4) -Y-L;

CDR-VL2 is W-P (X1) -S-T-R-E (X2) -S;

CDR-VL3 is Q-N (X1) -Y-Y-T (X2) -Y-P;

wherein, X1, X2, X3 and X4 are all mutation sites.

TABLE 1 mutant sites associated with antibody Activity

The inventors performed the above-described mutation of the CDR sites in WT to obtain a more active antibody.

Diluting human hemoglobin to 3 mug/ml by coating liquid, coating the human hemoglobin by a micropore plate with 100 mul per hole, and standing overnight at 4 ℃; on the next day, washing with the washing solution for 2 times, and patting to dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding diluted human hemoglobin monoclonal antibody, 100 μ l/well, 37 deg.C, 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. Some of the results are as follows:

TABLE 2 antibody Activity assay data

As is clear from the above table, since the activity of mutation 1 was most effective, mutation sites having a high potency were selected using mutation 1 as a backbone sequence, and some results are as follows.

TABLE 3 mutation sites related to antibody affinity

Affinity assay

Using AMC sensors, purified antibodies were diluted to 10 μ g/ml with PBST, and human hemoglobin was diluted with PBST in a gradient: 2400nmol/ml, 2000nmol/ml, 1600nmol/ml, 1200nmol/ml, 800nmol/ml, 0nmol/ml; the operation flow is as follows: equilibrating for 60s in buffer 1 (PBST), immobilizing antibody in antibody solution for 300s, incubating in buffer 2 (PBST) for 180s, binding for 420s in antigen solution, dissociating for 1200s in buffer 2, regenerating the sensor with 10mM pH 1.69GLY solution and buffer 3, and outputting data. K is _D Indicating the equilibrium dissociation constant, i.e. affinity.

Table 4 affinity assay data

As can be seen from Table 4, the mutated sequences in Table 3 all have better affinity and can effectively bind to hemoglobin.

To verify the above results, the above experiment was repeated using WT as a backbone sequence, and affinity verification of the mutation site was performed, and some results are as follows.

TABLE 5 mutations with WT as backbone

Table 6 affinity assay data

Site of the body	KD(M)	kon(1/Ms)	kdis(1/s)
				WT	8.26E-09	3.17E+03	2.62E-05
WT 1-1	5.00E-09	3.42E+03	1.71E-05
				WT 1-2	5.36E-09	3.64E+03	1.95E-05
WT 1-3	3.55E-09	3.01E+03	1.07E-05
				WT 1-4	5.88E-09	3.01E+03	1.77E-05
WT 1-5	7.66E-09	3.72E+03	2.85E-05
				WT 1-6	4.72E-09	3.09E+03	1.46E-05
WT 1-7	5.78E-09	3.93E+03	2.27E-05
				WT 1-8	2.88E-09	4.00E+03	1.15E-05
WT 1-9	5.65E-09	3.77E+03	2.13E-05
				WT 1-10	9.43E-09	3.00E+03	2.83E-05

From the analyses in tables 5 and 6, the sequences obtained by mutation based on WT had a certain affinity.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Dongguan City Peng Zhi Biotech Co., ltd

<120> a binding protein comprising an antigenic domain of hemoglobin

<130> 2019.12.16

<160> 12

<170> PatentIn version 3.5

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Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

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Ser Val Lys Met Ser Cys Lys Ala Ser

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Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

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Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala His Met Glu

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Leu Ser Ser Leu Thr Asn Glu Glu Ser Ala Val Tyr Tyr Cys

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

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

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Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

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Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

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Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

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His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

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Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

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

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

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Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

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Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

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Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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Ser Pro Gly Lys

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

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Asn Val Gln Lys Gln Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Ser Pro Lys Leu Leu Ile Tyr Trp Pro Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

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Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

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Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

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

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Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

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Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

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Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

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

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Trp Phe Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Ile Ile Tyr Pro Gly His Ser Asp Thr Thr Tyr Gly Gly Gln Lys

50 55 60

Phe Arg Asp Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala

65 70 75 80

His Met Glu Leu Ser Ser Leu Thr Asn Glu Glu Ser Ala Val Tyr Tyr

85 90 95

Cys Thr Lys Asp Gly Tyr Tyr Pro Ala Trp Gly Ser Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

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Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

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

290 295 300

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

305 310 315 320

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

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Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

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His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

Claims

1. A binding protein comprising a hemoglobin antigen-binding domain, wherein the antigen-binding domain comprises complementarity determining region CDR-VH1, complementarity determining region CDR-VH2, complementarity determining region CDR-VH3, complementarity determining region CDR-VL1, complementarity determining region CDR-VL2, and complementarity determining region CDR-VL3:

CDR-VH1 is G-Y-X1-F-X2-N-Y-T-W-X3-N, wherein X3 is M;

CDR-VH2 is I-I-Y-P-G-X1-S-D-T-X2-Y-X3-Q-K-F-X4-D, wherein X4 is K;

CDR-VH3 is T-X1-D-G-Y-Y-X2-A-W-X3, wherein X1 is R;

CDR-VL1 is S-Q-X1-L-L-X2-S-N-X3-Q-K-X4-Y-L, wherein X4 is N;

CDR-VL2 is W-X1-S-T-R-X2-S, wherein X1 is A;

CDR-VL3 is Q-X1-Y-Y-X2-Y-P, wherein X2 is S;

the mutation site of each complementarity determining region is selected from any one of the following sequences:

。

2. a binding protein comprising a hemoglobin antigen-binding domain, wherein the antigen-binding domain comprises complementarity determining region CDR-VH1, complementarity determining region CDR-VH2, complementarity determining region CDR-VH3, complementarity determining region CDR-VL1, complementarity determining region CDR-VL2, and complementarity determining region CDR-VL3:

CDR-VH1 is G-Y-X1-F-X2-N-Y-T-W-X3-N, wherein X3 is F;

CDR-VH2 is I-I-Y-P-G-X1-S-D-T-X2-Y-X3-Q-K-F-X4-D, wherein X4 is R;

CDR-VH3 is T-X1-D-G-Y-Y-X2-A-W-X3, wherein X1 is K;

CDR-VL1 is S-Q-X1-L-L-X2-S-N-X3-Q-K-X4-Y-L, wherein X4 is Q;

CDR-VL2 is W-X1-S-T-R-X2-S, wherein X1 is P;

CDR-VL3 is Q-X1-Y-Y-X2-Y-P, wherein X2 is T;

the mutation site of each complementarity determining region is selected from any one of the following mutations:

site of the body CDR-VH1 X1/X2 CDR-VH2 X1/X2/X3 CDR-VH3 X2/X3 CDR-VL1 X1/X2/X3 CDR-VL2 X2 CDR-VL3 X1 WT T/S H/T/GG P/G S/T/V E N WT 1-1 V/T N/S/N N/I D/S/V Q H WT 1-2 I/S Q/T/N N/V D/S/L H N WT 1-3 L/S N/T/GG GG/I D/T/I N Q WT 1-4 I/T N/S/GG N/V E/T/I N N WT 1-5 L/T H/S/N N/L D/S/V H H WT 1-6 V/T N/T/GG N/L D/S/V Q H WT 1-7 L/S Q/T/N GG/V E/T/L N Q WT 1-8 L/T N/S/N N/I D/S/L Q Q WT 1-9 V/T Q/T/GG GG/V E/S/I H N WT-10 T/S H/S/N P/F T/T/I E N

。

3. The binding protein comprising a hemoglobin antigen-binding domain of any one of claims 1 to 2, wherein the binding protein is F (ab') ₂ Fab', fab, fv, scFv and diabody.

4. The binding protein according to any one of claims 1 to 2, wherein the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 with the sequences shown in SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 with the sequences shown in SEQ ID NO. 5-8 in sequence.

5. The binding protein comprising a hemoglobin antigen-binding domain according to any one of claims 1 to 2, wherein the binding protein further comprises an antibody constant region sequence.

6. The binding protein of claim 5, wherein said constant region sequence is selected from the group consisting of sequences of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD constant regions.

7. The binding protein of claim 5, wherein the species of said constant region is derived from a cow, horse, pig, sheep, goat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

8. The binding protein of claim 7, wherein the species source of said constant region is a bovine.

9. The binding protein of claim 7, wherein said species source of the constant region is turkey or turkey.

10. The binding protein according to claim 7, wherein said constant region is murine.

11. The binding protein according to claim 10, wherein the light chain constant region sequence is set forth in SEQ ID NO 9;

the heavy chain constant region sequence is shown in SEQ ID NO. 10.

12. An isolated nucleic acid molecule which is DNA or RNA and which encodes a binding protein according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13.

15. A method of producing the binding protein of any one of claims 1 to 11, comprising the steps of:

culturing the host cell of claim 14 under suitable culture conditions and recovering the binding protein so produced from the culture medium or from the cultured host cell.

16. Use of the binding protein of any one of claims 1 to 11 in the preparation of a hemoglobin detection reagent.

17. Use of a binding protein according to any one of claims 1 to 11 in the preparation of a kit for detecting hemoglobin in a test sample, comprising:

a) Contacting the binding protein of any one of claims 1-11 with a hemoglobin antigen in a test sample under suitable reaction conditions to form an immune complex;

b) Detecting the immune complex.

18. The use of claim 17, wherein in step a) the immune complex further comprises a second antibody, wherein the second antibody binds to the binding protein.

19. The use of claim 17, wherein in step a) the immune complex further comprises a second antibody, said second antibody binding to said hemoglobin.

20. A kit comprising the binding protein according to any one of claims 1 to 11.

21. The kit of claim 20, further comprising one or more of a buffer, a stabilizer, a diluent.