Single-domain antibody of human D-dimer and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a single-domain antibody of a human D-dimer (D-dimer) and application of the single-domain antibody. Specifically, the invention provides a single-domain antibody combined with D-dimer and an application of the D-dimer single-domain antibody. Furthermore, the single-domain antibody disclosed by the invention is specifically combined with the D-dimer and is not combined with fibrinogen, so that the single-domain antibody is suitable for constructing a method for qualitatively and quantitatively detecting the human D-dimer based on an antigen-antibody recognition principle. The single-domain antibody is obtained through camel immunization, immune library construction, phage panning and the like, and compared with a conventional monoclonal antibody, the single-domain antibody has the advantages of being small in molecular weight, high in stability, good in water solubility, high in expression quantity, easy to modify and produce and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-human D-dimer (D-dimer) single-domain antibody and applications thereof. Background Art

[0002] Venous thromboembolism (VTE) is the third leading cause of death from vascular diseases after myocardial infarction and stroke worldwide. Its clinical manifestations are non-specific, the course of the disease is insidious, and it is easy to be missed. D-dimer is a soluble degradation product of cross-linked fibrin produced by plasmin. The increase in its mass concentration reflects the hypercoagulable state and secondary hyperfibrinolysis in the body. At present, the detection of D-dimer has been recognized as the first step of screening and diagnostic test for VTE, and its negative result can exclude VTE nearly 100%. However, positive results cannot exclude non-thrombotic diseases such as liver and kidney diseases, severe infections, malignant tumors, and physiological causes such as aging and pregnancy. It has the characteristics of high sensitivity and low specificity. In addition to being used for clinical screening of VTE, D-dimer is also used to evaluate the optimal anticoagulation time of VTE patients, diagnose and monitor disseminated intravascular coagulation, and identify patients at high risk of VTE.

[0003] Single-domain antibody (SdAb) originated from the heavy-chain antibody (HcAb) with naturally missing light chains that was first isolated from camel serum by the Hamers-Casterman team in 1993. Single-domain antibodies are the variable structural region on the heavy chain of traditional monoclonal antibodies. They are prepared from camel heavy chain antibodies through genetic engineering. Their molecular weight is only one-tenth of that of traditional monoclonal antibodies, about 15kDa, and they are also called nanobodies. Single-domain antibodies are the smallest active antigen-binding fragments, which maintain the same specificity as monoclonal antibodies. At the same time, they have the characteristics of weak immunogenicity, low production cost, good water solubility, non-aggregation, good tissue permeability, higher stability and affinity, and easy transformation and modification. At present, single-domain antibodies have been incorporated into biomaterials and used in molecular recognition, drug delivery systems, disease diagnosis and treatment, etc.

[0004] Clinically, the negative diagnosis value of D-dimer is high, while the positive diagnosis requires comprehensive diagnosis of multiple thrombosis-related indicators at the same time. However, the D-dimer antibodies currently used in the immunoturbidimetry, lateral flow analysis, ELISA and other methods for D-dimer detection are not ideal in terms of sensitivity, detection limit and other indicators. In addition, monoclonal antibodies have strict storage conditions, are easy to deteriorate, have high production costs, are expensive, and are difficult to transform and modify. Scientific research institutions, medical institutions and third-party testing institutions are in urgent need of D-dimer antibodies that are both high-quality and low-priced. Summary of the invention

[0005] The purpose of the present invention is to provide a single domain antibody capable of specifically binding to human D-dimer and its application.

[0006] In a first aspect of the present invention, an anti-D-dimer single domain antibody is provided, wherein the complementary determining region (CDR) region of the VHH chain in the single domain antibody is as follows: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0007] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain;

[0008] In another preferred embodiment, the VHH chain further comprises a framework region FR, and the framework region FR is as follows: FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7;

[0009] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to SEQ ID NO: 1-3;

[0010] In another preferred embodiment, the amino acid sequence of the CDR region of the single-domain antibody VHH chain comprises one or more amino acid substitutions compared with SEQ ID NO: 1-3, preferably conservative amino acid substitutions;

[0011] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence which is optionally subjected to addition, deletion, modification and / or substitution of at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to D-dimer;

[0012] In another preferred embodiment, the single domain antibody can specifically bind to D-dimer;

[0013] In another preferred embodiment, the D-dimer is human D-dimer.

[0014] The second aspect of the present invention provides an anti-D-dimer antibody, which is an antibody against D-dimer and has the anti-D-dimer single domain antibody described in the first aspect of the present invention;

[0015] In another preferred embodiment, the anti-D-dimer antibody comprises one or more anti-D-dimer single domain antibodies;

[0016] In another preferred embodiment, the anti-D-dimer antibody includes monomers, divalent bodies (divalent antibodies), tetravalent bodies (tetravalent antibodies), and / or multivalent bodies (multivalent antibodies);

[0017] In another preferred embodiment, the anti-D-dimer antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 8;

[0018] In another preferred embodiment, the anti-D-dimer antibody comprises two VHH chains having an amino acid sequence as shown in SEQ ID NO: 8;

[0019] In another preferred embodiment, the antibody can specifically bind to D-dimer;

[0020] In another preferred embodiment, the two VHH chains having the amino acid sequence shown in SEQ ID NO: 8 are connected via a linker;

[0021] In another preferred embodiment, the linker is a peptide linker;

[0022] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids;

[0023] In another preferred embodiment, the linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5;

[0024] In another preferred embodiment, the sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS;

[0025] In another preferred example, the anti-human D-dimer antibody has an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:10.

[0026] The third aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the anti-human D-dimer single domain antibody described in the first aspect of the present invention or the anti-human D-dimer antibody described in the second aspect of the present invention;

[0027] In another preferred embodiment, the polynucleotide is in a combined form;

[0028] In another preferred embodiment, the polynucleotide sequence comprises one or more sequences shown in SEQ ID NO: 9;

[0029] In another preferred embodiment, the polynucleotide includes DNA or RNA.

[0030] The fourth aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide according to the third aspect of the present invention;

[0031] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof;

[0032] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus;

[0033] The fifth aspect of the present invention provides a host cell, the host cell contains the expression vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into its genome;

[0034] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell;

[0035] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof;

[0036] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof;

[0037] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof;

[0038] In another preferred embodiment, the host cell is Escherichia coli.

[0039] The sixth aspect of the present invention provides a method for producing an anti-D-dimer single domain antibody, comprising the steps of:

[0040] (1) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-D-dimer single domain antibody;

[0041] (2) isolating or recovering the anti-D-dimer single domain antibody from the culture;

[0042] (3) Optionally, purifying and / or modifying the anti-D-dimer single domain antibody obtained in step (b).

[0043] The seventh aspect of the present invention provides the use of the anti-human D-dimer single domain antibody as described in the first aspect of the present invention or the anti-human D-dimer antibody as described in the second aspect of the present invention for preparing a reagent, a detection plate or a kit;

[0044] Wherein, the reagent, detection plate or kit is used for: detecting human D-dimer protein in a sample;

[0045] In another preferred embodiment, the detection includes immunoturbidimetry, colloidal gold, enzyme-linked immunosorbent assay (ELISA), lateral flow chromatography, flow cytometry, and cell immunofluorescence detection.

[0046] The eighth aspect of the present invention provides a method for detecting human D-dimer protein in a sample, the method comprising the steps of:

[0047] (1) contacting a sample with the anti-human D-dimer single domain antibody described in the first aspect of the present invention or the anti-human D-dimer antibody described in the second aspect of the present invention;

[0048] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of human D-dimer in the sample.

[0049] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0050] The ninth aspect of the present invention provides a human D-dimer detection reagent, the detection reagent comprising:

[0051] (1) the anti-human D-dimer single domain antibody according to the first aspect of the present invention, or the anti-human D-dimer antibody according to the second aspect of the present invention; and

[0052] (2) A carrier that is acceptable for testing;

[0053] In another preferred embodiment, the test-acceptable carrier is a non-toxic, inert aqueous carrier medium;

[0054] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: enzyme, isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nanomagnetic particle and imaging agent;

[0055] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet).

[0056] The tenth aspect of the present invention provides a kit for detecting human D-dimer, the kit comprising the detection reagent described in the ninth aspect of the present invention, and instructions;

[0057] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting human D-dimer expression in a test subject.

[0058] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the nucleic acid electrophoresis diagram: a 700bp band in the first round of PCR and a 400bp band in the second round of PCR.

[0060] Figure 2 This is a plate scan: a plate containing bacterial culture solutions with 10-fold dilution differences for library capacity calculation.

[0061] Figure 3 The nucleic acid electrophoresis diagram shows 24 randomly selected monoclonal colonies used to detect the insertion rate of VHH fragments.

[0062] Figure 4 Plate scan: streaking plate for phage display biopanning.

[0063] Figure 5 The SDS-PAGE protein electrophoresis diagram of the antibody of the present invention.

[0064] Figure 6 This is a specific analysis of the antibodies of the present invention.

[0065] Figure 7 This is an analysis of the binding activity of the antibody of the present invention.

[0066] Figure 8 This is a thermal stability analysis of the antibodies of the present invention.

[0067] Fig. 9 A double antibody sandwich ELISA was constructed based on the antibody of the present invention to quantitatively detect D-dimer molecules. DETAILED DESCRIPTION

[0068] After in-depth research and a large amount of panning, the inventors successfully obtained a group of anti-D-dimer single-domain antibodies. Specifically, the present invention uses high-purity D-dimer antigens derived from human plasma to immunize camels, and obtains an immune library containing single-domain antibody gene fragments by genetic engineering. By coating the same D-dimer antigen molecule on an ELISA plate to display its epitope, the single-domain antibody immune library is further screened using phage display technology to obtain a D-dimer-specific single-domain antibody gene. Finally, this gene is transferred into an Escherichia coli expression strain to obtain a single-domain antibody of D-dimer. The experimental results show that the D-dimer single-domain antibody obtained by the present invention can effectively bind to human D-dimer. The present invention was completed on this basis.

[0069] the term

[0070] In order to better understand the present invention, the following terms are defined.

[0071] The singular terms also include the plural form, active tense and past tense of the terms unless otherwise specified.

[0072] The term "about" includes values ​​within a standard deviation of the stated value unless otherwise indicated.

[0073] Throughout the specification and claims, the words "comprises", "including", "having" and the like should be understood to have an inclusive rather than an exclusive meaning; that is, the meaning of "including but not limited to", unless otherwise indicated.

[0074] As used herein, the terms "antibody of the present invention", "single domain antibody of the present invention", "D-dimer single domain antibody", "anti-D-dimer single domain antibody" and "anti-human D-dimer single domain antibody" have the same meaning and their interchange does not change the meaning. They all refer to single domain antibodies that specifically recognize and have binding activity to D-dimer (including human D-dimer or D-dimer derived from human plasma).

[0075] As known to those skilled in the art, D-dimer in the human body is derived from fibrinogen in its natural state. The specificity of D-dimer antibodies generally refers to the ability to bind to D-dimer molecules but not to fibrinogen. Antibodies that cross-react with fibrinogen are still defined as D-dimer antibodies, but it is necessary to point out that they have the characteristic of cross-reaction with fibrinogen.

[0076] In this article, the terms "single domain antibody", "VHH antibody" and "nanoantibody" have the same meaning and can be interchanged without changing the meaning. They all refer to single domain antibodies consisting of only one heavy chain variable region, which are constructed by cloning the variable structural region of camel heavy chain antibodies. They are the smallest active antigen-binding fragment with complete functions.

[0077] Herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0078] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0079] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to D-dimer, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0080] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0081] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0082] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0083] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0084] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be:

[0085] (1) A polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code;

[0086] (2) polypeptides having a substitution group in one or more amino acid residues;

[0087] (3) a polypeptide formed by the fusion of a mature polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol);

[0088] (4) A polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag);

[0089] Based on the teachings herein, these fragments, derivatives and analogs are well within the scope known to those skilled in the art.

[0090] The antibody of the present invention refers to a polypeptide having D-dimer binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0091] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0092] The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0093] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0094] Table 1

[0095] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0096] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0097] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0098] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0099] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to:

[0100] (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C;

[0101] (2) Denaturing agents are added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.;

[0102] (3) Hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0103] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0104] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0105] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0106] The sequences of the present invention are shown in Table 2:

[0107] Table 2

[0108]

[0109]

[0110] Note: The underlined part is the linker in the bivalent

[0111] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0112] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0113] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0114] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0115] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0116] The antibodies of the present invention may be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes).

[0117] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0118] Detection Methods

[0119] The present invention also relates to a method for detecting D-dimer. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of D-dimer in the dissolved sample.

[0120] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

[0121] Reagent test kit

[0122] The present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0123] The present invention also provides a detection kit for detecting D-dimer levels, which includes an antibody that recognizes D-dimer, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0124] application

[0125] As described above, the single domain antibody of the present invention has a wide range of biological and clinical application values, and its application involves diagnosis and treatment of diseases related to D-dimer, basic medical research, biological research, etc. A preferred application is for clinical diagnosis of D-dimer.

[0126] The main advantages of the present invention include:

[0127] (1) The single domain antibody of the present invention can effectively bind to D-dimer.

[0128] (2) The single domain antibody of the present invention has high purity, good specificity, better binding activity than commercially available monoclonal antibodies, and stronger thermal stability, providing a new option for the detection of D-dimer and the diagnosis of diseases with abnormal D-dimer levels.

[0129] (3) The single domain antibody of the present invention is suitable for constructing a variety of qualitative and quantitative detection methods for human D-dimer based on the principle of antigen-antibody recognition, including immunoturbidimetry, colloidal gold, ELISA, lateral flow chromatography, etc.

[0130] (4) The single domain antibody of the present invention has no cross-reaction to fibrinogen.

[0131] The present invention will be further described below in conjunction with specific examples. It should be understood that these specific examples are only further detailed descriptions of the present invention and are not intended to limit the scope of the present invention in any form. The experimental methods in the following examples without specifying specific conditions are usually based on conventional parameters, such as the conditions described in (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-ALaboratory Manual) (3rd edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Percentages and parts are calculated by weight unless otherwise indicated.

[0132] Example 1: Construction and screening of D-dimer single domain antibody library

[0133] Construction and validation of VHH antibody library:

[0134] (1) High-purity D-dimer purchased from human plasma (Medix Biochemica) was mixed with an equal volume of Freund's adjuvant, and the mixture of D-dimer antigen and Freund's adjuvant was injected subcutaneously at multiple points per week into a Bactrian camel.

[0135] (2) After 7 immunizations, peripheral blood was collected from camels, and the mononuclear cells (PBMCs) were isolated and total RNA was extracted from them, which was then reverse transcribed into cDNA.

[0136] (3) Obtaining the gene encoding the single-domain antibody VHH fragment by two rounds of PCR amplification, such as Figure 1 shown.

[0137] (4) The VHH fragment was double-digested with restriction endonucleases Pst I and Not I and then recombined and ligated.

[0138] (5) The library was constructed by electroporation into TG1 Escherichia coli cells.

[0139] (6) The quality of the constructed library was evaluated by testing the library capacity and the accuracy of VHH fragment insertion. Figure 2 As shown, the library capacity is 1.96×10 9 CFU (colony forming units). 24 monoclonal colonies were randomly selected for colony PCR detection, such as Figure 3 As shown, the library insertion accuracy was 100%.

[0140] Enrichment and screening of D-dimer positive clones:

[0141] (1) Use 100 mM NaHCO3, pH 8.4 to dilute D-dimer antigen to 50 ug / mL (and dilute human fibrinogen to 50 ug / mL as a control), coat the diluted D-dimer and fibrinogen on NUNC high-binding ELISA plates (coating volume 100 uL / well), and place in a refrigerator at 2-8°C for overnight coating.

[0142] (2) The next day, human fibrinogen was added to the phage to be screened, mixed and allowed to stand for 1 h to block antibodies displayed on the phage coat protein that cross-reacted with fibrinogen.

[0143] (3) Wash the coated ELISA plate overnight with sterile 1xPBST, then add filter-sterilized 0.1% BSA and block at room temperature for 2 h.

[0144] (4) After washing again to remove excess blocking solution, the negatively screened phage library was added to the wells of the D-dimer and fibrinogen ELISA plates and allowed to bind at room temperature for 1 h.

[0145] (5) Wash the D-dimer and fibrinogen control wells with 1x PBST to remove nonspecific phages.

[0146] (6) Add triethylamine solution to the D-dimer and control wells and let stand to dissociate specific phages, then add an equal volume of Tris-HCl for neutralization.

[0147] (7) Finally, the neutralized phages were infected with TG1 cells that had been cultured to the logarithmic growth phase for amplification and enrichment and used for the next round of screening.

[0148] (8) Figure 4 As shown, four rounds of screening were performed, and a total of 380 monoclonal colonies were picked from the screening plates of the last two screening rounds.

[0149] (9) 380 monoclonal colonies were inoculated into TB medium (containing ampicillin) in a sterile deep-well plate, cultured in a shaking incubator at 37°C until the logarithmic growth phase, and IPTG was added to induce expression.

[0150] (10) E. coli cells were lysed by osmotic shock to obtain periplasmic proteins, and positive clones were identified by periplasmic extract ELISA (PE-ELISA). When the OD value of the positive well was more than 3 times that of the negative well (Ratio + / - > 3), it was defined as a positive clone.

[0151] (11) The positive clones were inoculated into LB medium (containing ampicillin) for amplification and sent for sequencing.

[0152] (12) After obtaining the differential sequences, the differentially positive clones were inoculated into small shakes, amplified overnight, and stored in glycerol stocks. The plasmids were then extracted for subsequent transformation.

[0153] Example 2: Expression of D-dimer single domain antibody in E. coli WK6 cells:

[0154] (1) The pMECS-Nb recombinant plasmid extracted from the positive clone was added into Escherichia coli WK6 cells, and the exogenous plasmid was introduced into the WK6 cells by electroporation.

[0155] (2) After culturing at 37°C in a shaking incubator for 1 hour, the culture was spread on an LB plate containing ampicillin and glucose and cultured at 37°C overnight.

[0156] (3) On the second day, a single clone was picked and inoculated into LB medium (containing ampicillin) for activation and cultured at 37°C overnight.

[0157] (4) On the second day, the activated bacteria were cultured in shake flasks and induced to express by IPTG.

[0158] (5) The supernatant containing the D-dimer single domain antibody was obtained by swelling the WK6 E. coli cells with high concentration sucrose, osmotic pressure lysis with low concentration double volume sucrose, and high-speed centrifugation.

[0159] (6) D-dimer single domain antibody was obtained after Ni column affinity purification, imidazole gradient elution and ultrafiltration concentration.

[0160] (7) Detect the molecular weight and purity of the D-dimer single domain antibody by SDS-PAGE protein electrophoresis. Figure 5 As shown, gel electrophoresis of the D-dimer single domain antibody Nb10 showed a molecular weight of approximately 15 kDa and good purity.

[0161] Example 3: Transient expression of bivalent D-dimer single domain antibody in HEK293F cells

[0162] (1) The constructed bivalent D-dimer single domain antibody Nb10 (the corresponding amino acid sequence is shown in SEQ ID NO: 10) recombinant plasmid was transferred into Escherichia coli DH5α by chemical transformation and plated.

[0163] (2) On the second day, a single clone was picked from the ampicillin-resistant LB plate and inoculated into LB medium (containing ampicillin) for activation and cultured at 37°C overnight.

[0164] (3) Take the activated bacteria and culture them in a shake flask at 37°C overnight.

[0165] (4) Use a transfection-grade plasmid extraction kit to extract the plasmid, perform agarose gel electrophoresis on the extracted plasmid to verify the band size and perform sequencing.

[0166] (5) HEK293 cells were passaged to 2E6, and the cell viability was greater than 90%.

[0167] (6) Add plasmid DNA to F17 medium, mix well, and filter to sterilize.

[0168] (7) Add PEI to F17 medium, mix well, and then mix it with the filter-sterilized plasmid. After standing, slowly add the mixture to HEK293F cells.

[0169] (8) On the second day after transfection, transfection enhancer was added, and on the fifth day after transfection, cell culture supernatant was collected for purification.

[0170] (9) The molecular weight and purity of the bivalent D-dimer single domain antibody Nb10 were detected by SDS-PAGE protein electrophoresis.

[0171] like Figure 5 As shown, gel electrophoresis of the bivalent D-dimer single domain antibody Nb10 showed a molecular weight of approximately 30 kDa and good purity.

[0172] Example 4: Specificity, binding activity and thermal stability of the antibodies of the present invention

[0173] As known to those skilled in the art, the concentration of fibrinogen in human plasma is more than 1,000 times higher than that of D-dimer. The general characteristics of D-dimer-specific antibodies are that they bind to D-dimer, not to fibrinogen, and have different binding properties to fibrinogen degradation fragments. In addition, D-dimer antibodies that cross-react with fibrinogen can still be used as detection antibodies.

[0174] Specificity of D-dimer single domain antibodies:

[0175] (1) Coat 2ug / mL D-dimer and Fibrinogen diluted in 100mM NaHCO3 onto NUNC high-binding ELISA plates and place in a 2-8℃ refrigerator overnight.

[0176] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0177] (3) After washing, add the serially diluted D-dimer single domain antibody Nb10, with the dilution as the zero concentration, and place in a 37°C incubator for 1 h.

[0178] (4) After washing, add mouse anti-HA antibody and place in a 37°C incubator for 1 h.

[0179] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0180] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0181] like Figure 6 As shown, the antibody of the present invention can specifically recognize D-dimer molecules.

[0182] Binding activity of bivalent D-dimer single domain antibodies:

[0183] (1) Coat 2ug / mL D-dimer diluted in 100mM NaHCO3 onto NUNC high-binding ELISA plates and place in a 2-8℃ refrigerator overnight.

[0184] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0185] (3) After washing, add serially diluted bivalent D-dimer single domain antibody Nb10 and control mouse monoclonal antibody (Medix Biochemica: Anti-h D-Dimer 1401SPTN-5), with the dilution solution as the zero concentration, and place in a 37°C incubator for 1 hour.

[0186] (4) After washing, the antibody of the present invention is added to the mouse anti-HA antibody (the monoclonal antibody is added to an equal amount of diluent) and placed in a 37° C. incubator for 1 hour.

[0187] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0188] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0189] like Figure 7 As shown, compared with the commercially available D-dimer monoclonal antibody, the antibody of the present invention has better binding activity.

[0190] Thermal stability of bivalent D-dimer single domain antibodies:

[0191] (1) Coat 2ug / mL D-dimer diluted in 100mM NaHCO3 onto NUNC high-binding ELISA plates and place in a 2-8℃ refrigerator overnight.

[0192] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0193] (3) After washing, 2 ug / mL of bivalent D-dimer single domain antibody Nb10 and 2 ug / mL of control mouse monoclonal antibody that had been heated in a water bath at different temperatures for 5 min and in a water bath at the same temperature for different times were added and placed in a 37°C incubator for 1 h.

[0194] (4) After washing, the antibody of the present invention is added to the mouse anti-HA antibody (the monoclonal antibody is added to an equal amount of diluent) and placed in a 37° C. incubator for 1 hour.

[0195] (5) After washing, add HRP-labeled rabbit anti-mouse IgG and place in a 37°C incubator for 1 h.

[0196] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0197] like Figure 8 As shown, compared with the commercially available D-dimer monoclonal antibody, the antibody of the present invention has better thermal stability.

[0198] Example 5: Construction of a double antibody sandwich method based on a bivalent D-dimer single domain antibody

[0199] (1) 2ug / mL of the divalent D-dimer single domain antibody Nb10 diluted in 100mM NaHCO3 was coated on the NUNC high binding ELISA plate and placed in a refrigerator at 2-8°C for overnight coating.

[0200] (2) After washing 4 times with 1xPBST, 1% skim milk powder was added for blocking at room temperature for 2 h.

[0201] (3) After washing, D-dimer and Fibrinogen were diluted 2-fold from 2000 to 31.25 ng / mL, respectively. The dilutions were used as the zero concentration, and the two antigens diluted to the target concentrations were added to the corresponding wells. The wells were placed in a 37°C incubator for 1 h.

[0202] (4) After washing, 100 uL of 2 ug / mL biotinylated D-dimer single domain antibody Nb1 was added to each well and placed in a 37°C incubator for 1 h.

[0203] (5) After washing, HRP-labeled streptavidin was added at a volume ratio of 1:5000 and placed in a 37°C incubator for 1 h.

[0204] (6) After washing and patting dry, add 100uL TMB substrate to each well and react at room temperature in the dark for 5-10 min or until the color is fully developed. Then add 50uL 2M H2SO4 to stop the color development.

[0205] like Fig. 9 As shown, the double antibody sandwich ELISA constructed based on the antibody of the present invention can quantitatively detect D-dimer molecules and has no cross reaction to fibrinogen.

[0206] Finally, it should be noted that the technical solution of the present invention is described in detail above in conjunction with the preferred embodiments, but it is not intended to limit the present invention. It should be understood by those skilled in the art that the present invention may be modified, deformed, improved, etc. without departing from the spirit and scope of the present invention, and any modification, deformation, improvement, etc. are within the protection scope of the present invention. The protection scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. An anti-human D-dimer single domain antibody, characterized in that: The complementary determining regions CDRs of the VHH chain in the single domain antibody are as follows: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO:

3.

2. The anti-human D-dimer single domain antibody according to claim 1, characterized in that: The VHH chain of the single-domain antibody also includes a framework region FR, and the framework region FR is as follows: FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:5, FR3 shown in SEQ ID NO:6, and FR4 shown in SEQ ID NO:

7.

3. An anti-human D-dimer antibody, characterized in that: The antibody comprises one or more anti-human D-dimer single domain antibodies as claimed in claim 1.

4. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the group consisting of the anti-human D-dimer single domain antibody of claim 1 and the anti-human D-dimer antibody of claim 3.

5. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector of claim 5, or the polynucleotide of claim 4 is integrated into its genome.

7. A method for producing an anti-human D-dimer single domain antibody, characterized in that: The method comprises the following steps: (1) culturing the host cell of claim 6 under conditions suitable for producing single-domain antibodies, thereby obtaining a culture containing the anti-human D-dimer single-domain antibody; (2) isolating or recovering the anti-human D-dimer single-domain antibody from the culture; and (3) optionally, purifying and / or modifying the anti-human D-dimer single-domain antibody obtained in (2).

8. A method for detecting D-dimer in a sample, characterized in that: The method comprises the steps of: (1) contacting a sample with the anti-human D-dimer single domain antibody of claim 1 and the anti-D-dimer antibody of claim 3; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of D-dimer protein in the sample.

9. A D-dimer protein detection reagent, characterized in that: The detection reagent comprises: (1) the anti-human D-dimer single domain antibody according to claim 1, the anti-D-dimer antibody according to claim 3; and a carrier acceptable in detection.

10. A kit for detecting D-dimer protein, characterized in that: The kit contains the detection reagent according to claim 9 and instructions.

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