CN111434686A - An anti-human PBX1 monoclonal antibody, its preparation method and its use in the clinical diagnosis of recurrent miscarriage - Google Patents

Abstract

A monoclonal antibody, its preparation method and its use in the clinical diagnosis of recurrent miscarriage. Specifically, the present invention relates to a monoclonal antibody, which is secreted by the hybridoma cell line 2D11 and can be used in the diagnosis of recurrent miscarriage, with good specificity and high sensitivity.

Description

A kind of anti-human PBX1 monoclonal antibody, its preparation method and its purposes in the clinical diagnosis of recurrent miscarriage

technical field

The present invention relates to the field of biotechnology, in particular to a monoclonal antibody, a hybridoma cell line expressing the monoclonal antibody and the use of the monoclonal antibody in disease diagnosis.

Background technique

There are about 45 million infertility patients in my country, and the number is increasing by hundreds of thousands every year. Solving the problem of infertility is of great significance to family happiness and social harmony and stability. Recurrent miscarriage has become a common pregnancy disease that plagues women of childbearing age, seriously affecting reproductive health; about 50% of them are difficult to diagnose clinically. , clinically manifested as unexplained recurrent miscarriage, and current studies have shown that it is mainly associated with abnormal immune function of decidual NK cells (Fu B, Zhou Y et al. (2017) Immunity.; 47(6):1100-1113.e6. and Fu B, Li X et al. (2013) Proc Natl Acad Sci US A.; 110(3):E231-40). As the main immune cells at the maternal-fetal interface, NK cells undertake important physiological functions, such as participating in the induction of immune tolerance, promoting trophoblast cell invasion, participating in the remodeling process of spiral arteries, and promoting placental and fetal development (Vento-Tormo R, Efremova M, (2018) Nature.;563(7731):347-353). The transcription factor PBX1 is involved in the maintenance of the number of decidual NK cells and regulates the execution of normal functions of decidual NK cells.

Transcription factor PBX1, also known as: CAKUHED, PRL. It is encoded by the pre-B-cell leukemia homeobox gene (pre-B-cellleukemia homeobox 1, PBX1) and is one of the important members of the homeobox gene family. Transcription factor PBX1 was identified by American scholar Kamps MP et al in patients with pre-B cell leukemia (Kamps MP et al (1990) Cell.; 60(4): 547-55), and later studies suggested that it is involved in embryonic development, organogenesis and Cell differentiation plays an important role; it is associated with a variety of malignancies. The gene of human PBX1 is located at 1q23 and consists of 9 exons and 8 introns. The relative molecular weight of the encoded protein is 47kD, which is composed of 430 amino acids, and there are two main isoforms, PBX1a (430aa, 47kD) and PBX1b (347aa, 38kD), of which the 1st to 333rd amino acids in PBX1b are related to PBX1a. Completely consistent, the two homologous isomers are similar in structure, both contain the C-terminal homology domain, which plays a role in stabilizing the ternary complex structure formed by PBX1, HOX and DNA, and the N-terminal contains two PBC domains. protein-protein interaction region.

SUMMARY OF THE INVENTION

The present invention relates to a monoclonal antibody, its preparation method and its use in the clinical diagnosis of recurrent miscarriage. Specifically, the monoclonal antibody of the present invention is secreted by the hybridoma cell line 2D11 deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO.C201912, which can be applied to detect the expression of PBX1 in the NK cells of human early pregnancy decidual tissue, As one of the limited clinical diagnostic indicators for recurrent miscarriage (unexplained recurrent miscarriage) disease associated with abnormal decidual NK cells. Therefore, the monoclonal antibody of the present invention has great clinical significance for the diagnosis of recurrent miscarriage and other pregnancy-related diseases and for guiding further targeted and precise treatment.

In one aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof comprising:

The amino acid sequence is the heavy chain variable region CDR-H1 of SEQ ID No: 6, preferably, its coding sequence is SEQ ID No: 5;

The amino acid sequence is the heavy chain variable region CDR-H2 of SEQ ID No: 8, preferably, its coding sequence is SEQ ID No: 7;

The amino acid sequence is the heavy chain variable region CDR-H3 of SEQ ID No: 10, preferably, its coding sequence is SEQ ID No: 9;

The amino acid sequence is the light chain variable region CDR-L1 of SEQ ID No: 12, preferably, its coding sequence is SEQ ID No: 11;

The amino acid sequence is the light chain variable region CDR-L2 of SEQ ID No: 14, preferably, its coding sequence is SEQ ID No: 13; and

The amino acid sequence is the light chain variable region CDR-L3 of SEQ ID No: 16, preferably, its coding sequence is SEQ ID No: 15;

wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or dAb.

In one aspect, the present invention provides the monoclonal antibody or antigen-binding fragment thereof as described above, which is secreted by the hybridoma cell line deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912.

In one aspect, the present invention provides a conjugate comprising any of the monoclonal antibodies or antigen-binding fragments thereof as described above and a label bound thereto that allows its detection, wherein the label is selected from the group consisting of fluorophores, biotin, radioactive One or more of isotopes, metals and enzymes.

In one aspect, the present invention provides hybridoma cell lines that produce any of the monoclonal antibodies or antigen-binding fragments thereof described above.

In one aspect, the present invention provides a method for the preparation of any of the monoclonal antibodies or antigen-binding fragments thereof described above, or of any of the conjugates described above.

In one aspect, the present invention provides the use of a monoclonal antibody or antigen-binding fragment thereof of any of the above, or a conjugate of any of the above, in detecting the protein expression of PBX1a and/or PBX1b.

In one aspect, the present invention provides an immunoassay kit comprising any of the monoclonal antibodies or antigen-binding fragments thereof described above, or any of the conjugates described above.

In one aspect, the present invention provides the use of any of the monoclonal antibodies or antigen-binding fragments thereof or the conjugates thereof in the preparation of a diagnostic agent for assisting in the diagnosis of unexplained recurrent miscarriage.

In one aspect, the present invention provides a kit for assisting in the diagnosis of unexplained recurrent miscarriage, comprising any of the monoclonal antibodies or antigen-binding fragments thereof described above or any of the conjugates described above.

Brief Description of Drawings

Figure 1. The construction flow chart of the prokaryotic recombinant expression vector pET21a-PBX1b and the eukaryotic recombinant expression vector pPBX1b. Figure 1A shows the flow chart of constructing prokaryotic recombinant expression vector pET21a-PBX1b based on pET21a plasmid; Figure 1B shows the flow chart of constructing eukaryotic recombinant expression vector pPBX1b based on pEGFP-N1 plasmid.

Figure 2. Human transcription factor PBX1b recombinant protein expression, purification and assay. Figure 2A shows the induced expression results of the PBX1b recombinant protein; Figure 2B shows the results before and after nickel agarose affinity chromatography of the PBX1b recombinant protein; Figure 2C shows the immunoblot identification results of the purified PBX1b recombinant protein.

Figure 3. Serum ELISA results before and after immunization of rats with PBX1b recombinant protein.

Figure 4.2D11 monoclonal antibody purity identification results.

Figure 5. 2D11 monoclonal antibody specific identification results. Figure 5A shows the identification of 2D11 monoclonal antibody specificity by immunoblotting; Figure 5B shows the identification of 2D11 monoclonal antibody specificity by flow cytometry.

Figure 6. ALEXA FLUOR ^TM 488-labeled 2D11 monoclonal antibody was used to detect the expression of NK cell transcription factor PBX1 in normal human early pregnancy decidua tissue by flow cytometry. Figure 6A shows that 2D11 mAb can bind to PBX1a and PBX1b in decidual tissue mononuclear cells and peripheral blood mononuclear cells, respectively. Figure 6B shows the expression of transcription factor PBX1 in human decidual tissue NK ( ^dNK ) cells and peripheral blood NK (pNK) cells by flow cytometry using 2D11 monoclonal antibody; and a statistical plot of the proportions in pNK cells.

Figure 7. Application of ALEXA FLUOR ^™ 488-labeled 2D11 monoclonal antibody in the diagnosis of recurrent unexplained miscarriage by flow cytometry. Figure 7A shows the expression of transcription factor PBX1 in decidua NK cells of patients with recurrent miscarriage (RSA) detected by flow cytometry, with normal human dNK cells as control; Figure 7B shows recurrent miscarriage patients and normal human PBX1 ⁺ dNK cells Statistical plot of proportion (top) and quantity (bottom). Figure 7C shows real-time PCR detection of PBX1 gene expression in decidual tissue of patients with recurrent miscarriage; Figure 7D shows the receiver operating characteristic curve of reduced PBX1 expression in unexplained recurrent miscarriage and recurrent miscarriage with fetal chromosomal abnormalities; Figure 7E shows Receiver operating characteristic curve of reduced PBX1 expression in recurrent miscarriage.

Material preservation

The following materials have been deposited in: China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China) (CCTCC):

Materials: Rat anti-human PBX1 antibody hybridoma cell line 2D11

Deposit number: C201912

Deposit date: January 8, 2019

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art. Generally, the terms used herein are well known and routinely used in the art.

The main terms used in the present invention are defined as follows:

As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody Light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In light chains and Within the heavy chain, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Variable region (VH) and heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (eg, effector cells) and the classical complement system Binding of the first component (C1q). The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs) Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. Variable for each heavy/light chain pair The regions (VH and VL) respectively form the antibody binding site. The assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J MoI. Biol. 196:901-917; definition by Chothia et al. (1989) Nature 342:878-883.

As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of a full-length antibody, typically the antigen-binding or variable region of the full-length antibody. The fragments retain the ability to bind the same antigen to which the antibody binds (eg, PBX1), competing with the intact antibody for specific binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies (diabody) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

As used herein, the term "monoclonal antibody" refers to a uniform antibody directed against only a particular epitope. In contrast to common polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" denotes a uniform characteristic of an antibody and is not to be construed as requiring that the antibody be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by a single hybridoma resulting from the fusion of spleen cells and myeloma cells.

Antibody-encoding DNA can be modified to generate "chimeric or fusion antibody polypeptides", for example by substituting human heavy and light chain constant domain (CH and CL) sequences for homologous murine sequences (US 4816567; and Morrison et al. (1984) Proc . NatlAcad. Sci. USA.; 81:6851), or by fusing an immunoglobulin coding sequence with all or part of a coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can be substituted for the constant domains of antibodies, or they can be substituted for the variable domains of one antigen-binding site of an antibody to create chimeric bivalent antibodies that contain an antigen-binding site specific for one antigen and another antigen-binding site specific for a different antigen.

"Humanized" forms of non-human (eg, rodent) antibodies refer to chimeric antibodies that minimally contain sequences derived from non-human antibodies. To a large extent, a humanized antibody refers to the use of hypervariable region residues in a human immunoglobulin (acceptor antibody) with a non-human species (donor antibody) (such as a mouse) having the desired antibody specificity, affinity and capacity , rat, rabbit, or non-human primate) immunoglobulins with the hypervariable region residues replaced. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may contain residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin. All FRs above are FRs of human immunoglobulin sequences. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. The Fc fragment contains the carboxy-terminal portions of all two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the part recognized by the Fc receptor (FcR) found on certain types of cells (Jones et al. (1986) Nature.; 321: 522-525; Riechmann et al (1988) Nature.; 332: 323-329; Presta (1992) Curr. Op. Struct. Biol.; 2: 593-596; Verhoeyen, et al (1988) Science,; 239: 1534-1536). Other methods use specific framework regions derived from consensus sequences of all human antibodies of a specific subgroup of light or heavy chains (Carter et al. (1992) Proc. Natl. Acad. Sci. USA.; 89:4285; Presta et al. ( 1993) J. Immunol.; 151:2623).

As used herein, the term "sequence identity" refers to the percentage of identical residues in an amino acid sequence variant after aligning the sequences and introducing gaps where necessary to achieve maximum percent sequence identity. Methods and computer programs for alignment are well known in the art.

As used herein, the term "specifically binds" or "specifically binds" to a specific molecular target or antigen of interest or to an epitope on a specific molecular target or antigen of interest for which it is "specific" to mean a measurable difference binding to nonspecific interactions. Specific binding can be measured, for example, by assaying the binding of a molecule and comparing the binding to a control molecule, typically a molecule that is structurally similar but has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, eg, an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. Typically, an antibody binds an epitope with an affinity (KD) of less than about ^10-6 M, eg, less than about ^10-7 M, ^10-8 M, ^10-9 M, or ^10-10 M or less.

"Binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigen more rapidly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

As used herein, the term "specificity of a monoclonal antibody" refers to the property of a monoclonal antibody to recognize and bind to a particular epitope or antigenic determinant on an antigen.

As used herein, the term "reactivity of a monoclonal antibody" refers to the ability of a monoclonal antibody to bind to an antigen under appropriate reaction conditions.

As used herein, the term "hybridoma" includes not only hybridoma parent cells but also subclones and progeny cells of hybridomas.

As used herein, the term "cell line" refers to a single cell culture obtained from a primary culture or cell line by screening or limiting dilution methods.

As used herein, the term "kit" can include a variety of forms of presentation suitable for use, eg, can be in boxes, bottles, bags, and the like.

As used herein, the term "PBX1" refers herein to PBX1a and PBX1b.

In one aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof comprising:

The amino acid sequence is the heavy chain variable region CDR-H1 of SEQ ID No: 6, preferably, its coding sequence is SEQ ID No: 5;

The amino acid sequence is the heavy chain variable region CDR-H2 of SEQ ID No: 8, preferably, its coding sequence is SEQ ID No: 7;

The amino acid sequence is the heavy chain variable region CDR-H3 of SEQ ID No: 10, preferably, its coding sequence is SEQ ID No: 9;

The amino acid sequence is the light chain variable region CDR-L1 of SEQ ID No: 12, preferably, its coding sequence is SEQ ID No: 11;

The amino acid sequence is the light chain variable region CDR-L2 of SEQ ID No: 14, preferably, its coding sequence is SEQ ID No: 13; and

The amino acid sequence is the light chain variable region CDR-L3 of SEQ ID No: 16, preferably, its coding sequence is SEQ ID No: 15;

wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or dAb.

In one aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof comprising:

The amino acid sequence is the heavy chain variable region of SEQ ID No: 18, preferably, its coding sequence is SEQ ID No: 17; and

The amino acid sequence is the light chain variable region of SEQ ID No:20, preferably, its coding sequence is SEQ ID No:19;

wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or dAb.

In one embodiment, any of the monoclonal antibodies or antigen-binding fragments thereof described above are capable of specifically binding to the PBX1a and/or PBX1b protein. In one embodiment, the subclass of Ig of any one of the monoclonal antibodies or antigen-binding fragments thereof as described above is _IgG2a ,κ. In one embodiment, any one of the monoclonal antibodies or antigen-binding fragments thereof described above is a humanized antibody, a chimeric antibody, a single-chain antibody, or a diabody.

In one embodiment, any one of the monoclonal antibodies or antigen-binding fragments thereof described above is secreted by the hybridoma cell line 2D11 deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912.

In another aspect, the present invention provides a conjugate comprising any one of the monoclonal antibodies or antigen-binding fragments thereof as described above and bound thereto a label allowing its detection, wherein the label is selected from the group consisting of a fluorophore, biotin, One or more of radioisotopes, metals and enzymes.

In one aspect, the present invention provides a hybridoma cell line, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912. In one embodiment, the hybridoma cell line produces a monoclonal antibody or antigen-binding fragment thereof against the human transcription factor PBX1 protein. In one embodiment, the hybridoma cell line is capable of stably secreting a monoclonal antibody against the human transcription factor PBX1 protein or an antigen-binding fragment thereof.

In another aspect, the present invention provides the use of any one of the monoclonal antibodies or antigen-binding sheets thereof, or any one of the conjugates described above, in detecting the protein expression of PBX1a and/or PBX1b. In one embodiment, the detected sample is a plasma sample. In one embodiment, the detected sample is of human origin. In one embodiment, the detected samples are NK cells in the decidua tissue of early pregnancy patients with recurrent miscarriage, preferably patients with unexplained recurrent miscarriage. In one embodiment, the method of detection is to use any one of the monoclonal antibodies or antigen-binding fragments thereof described above or any one of the conjugates described above as the primary antibody. In one embodiment, the method of detection is the detection of human cellular transcription factor PBX1 by immunoblotting. In one embodiment, the fluorophore in the conjugate is the labeled fluorescein ALEXAFLUORTM488. In one embodiment, the method of detection is the detection of human cellular transcription factor PBX1 by flow cytometry.

In another aspect, the present invention provides an immunoassay kit comprising any one of the monoclonal antibodies or antigen-binding fragments thereof described above or any one of the conjugates described above.

In another aspect, the present invention provides any one of the above monoclonal antibodies or antigen-binding fragments thereof or any one of the above conjugates in the preparation of a diagnostic agent for auxiliary diagnosis of unexplained recurrent miscarriage disease Uses, in one embodiment, the sample for auxiliary diagnosis is NK cells in the decidua tissue of early pregnancy of patients with unexplained recurrent miscarriage. In one embodiment, the fluorophore in the conjugate is the labeled fluorescein ALEXA FLUORTM488. In one embodiment, the method of diagnosis is to detect the transcription factor PBX1 of NK cells in the decidual tissue of patients with unexplained recurrent miscarriage in early pregnancy by flow cytometry, and then diagnose whether the cause of the disease is the low expression of transcription factor PBX1, NK cells abnormal.

In another aspect, the present invention provides a kit for assisting diagnosis of unexplained recurrent miscarriage, which contains any one of the monoclonal antibodies or antigen-binding fragments thereof or any one of the conjugates described above.

Example

The following examples will help those of ordinary skill in the art to further understand the present invention, but do not limit the present invention in any form.

The experimental methods in the examples, unless otherwise specified, use conventional techniques in the art, and the experimental reagents are all commercially available products.

Example 1: Construction of prokaryotic recombinant expression vector pET21a-PBX1b and eukaryotic recombinant expression vector pPBX1b

General Biosystems (Anhui) Co., Ltd. was entrusted to artificially synthesize the full-length gene of human transcription factor PBX1b (NCBI Reference Sequence: NM_001204961.1, encoding amino acids 1 to 347), and its gene sequence and encoded protein sequence are SEQ ID NO: The sequences shown in 1 and 2, and 6His-Tag and a stop codon were introduced at the C-terminus, and were directly cloned into the recombinant expression vector pET21a (Shanghai Sangon Biotechnology Co., Ltd.) through the NdeI and XhoI restriction sites, and the resulting recombinant expression vector The cells were transformed into DH5α competent cells, and positive clones were screened for DNA sequencing identification. The sequencing results showed that the recombinant expression vector pET21a-PBX1b was successfully constructed. The construction flow of the prokaryotic recombinant expression vector pET21a-PBX1b is shown in Figure 1A.

The full-length gene of the synthetic human transcription factor PBX1b was directly cloned into the recombinant expression vector pEGFP-N1 (Hunan Keai Medical Equipment Co., Ltd. (Youbao Bio), Cat. No.: VT1110) through EcoRI and BamHI restriction sites, named pPBX1b, The obtained recombinant expression vector was transferred into DH5α competent cells, and positive clones were screened for DNA sequencing identification. The sequencing results showed that the recombinant expression vector pPBX1b was successfully constructed. The construction flow of the eukaryotic recombinant expression vector pPBX1b is shown in Figure 1B.

Example 2: Expression, purification and determination of recombinant protein of human transcription factor PBX1b

1. Induce the expression of recombinant protein

1.1 Transformation

The recombinant expression vector pET21a-PBX1b obtained in Example 1 was transformed into Transetta (DE3) competent cells (Beijing Quanshijin Biotechnology Co., Ltd., product number CD801), and placed on ice after heat shock at 42°C for 90 seconds. After 2 minutes, it was spread on LB solid plates containing a final concentration of 50 μg/mL ampicillin and 34 μg/mL chloramphenicol, and cultured at 37°C overnight.

1.2 Select the best induction conditions for small test culture

Pick a single colony of the PBX1b recombinant protein expression strain Transetta (DE3) in 10 mL of LB medium (containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol) at 37°C, 220 rpm, and shake overnight for culture. The overnight cultured bacterial solution was inoculated into 10 mL LB (containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol) medium at a ratio of 1:100, and cultured with shaking at 37°C and 220 rpm. When OD _600nm = 0.6, IPTG with a final concentration of 0.5 mM was added at 220 rpm to induce expression by shaking culture, overnight at 20°C and 4 hours at 37°C. No IPTG inducer was added as a negative control. The cells were collected by centrifugation, the cells were lysed by ultrasonication, the supernatant and the precipitate were collected by centrifugation, and the precipitate was dissolved with 500 μL inclusion body lysis solution (8MUrea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), and 40 μL samples were taken for 12% SDS-PAGE electrophoresis detection, the loading volume is 10 μL, after gel electrophoresis, Coomassie brilliant blue staining for 20 minutes, and imaging after destaining. As shown in Figure 2A, compared with other control conditions, the supernatant of IPTG-induced bacterial lysate had an obvious band at 38kDa, and the overnight induction at 20°C was better than the induction at 37°C for 4 hours.

1.3 Induction and expression of fusion proteins in large quantities

According to the results of the small test in 1.2, it is finally determined: select the condition of overnight induction at 20 °C, and inoculate the uninduced monoclonal culture bacteria solution used in the small and medium-sized test in the above 1.2 at a ratio of 1:100 in 4L of LB resistance medium for large-scale induction. Express.

2. Purification of the fusion protein

Centrifuge to collect a large amount of the bacterial liquid precipitate induced in the above 1.3, ultrasonically break the lysed bacterial liquid precipitate in an ice bath, collect the lysed supernatant after centrifugation, and purify by nickel agarose affinity chromatography. Take 5 mL of Ni-IDA and wash the equilibrated column with 10 bed volumes of equilibration solution (1M Tris, 150 mM NaCl, pH 8.0) at a flow rate of 5 mL/min. The lysis supernatant obtained in step 1.3 of Example 2 above was applied to the column at a flow rate of 2 mL/min, and the permeate was collected as a control. The column was washed with 10 bed volumes of equilibration solution at a flow rate of 5 mL/min. Then use 20/50 mM imidazole eluate (1M Tris, 150 mM NaCl, 20/50 mM Imidazole, pH 8.0) to wash off the impurity protein, the flow rate is 5 mL/min, and the eluate is collected as a control. Finally, 500 mM imidazole solution (1 M Tris, 150 mM NaCl, 500 mM Imidazole, pH 8.0) was used to elute the target protein at a flow rate of 2 mL/min, and the target protein eluate was collected. The target protein eluate samples were collected for 12% SDS-PAGE detection. As shown in Figure 2B, compared with the control group, there was an obvious target protein band at 38kDa in the 500mM imidazole eluate. According to the identification results, the target protein eluted with 500mM imidazole solution was dialyzed into dialysate 1 (500mM L-Arginine, 25mM Tris, 150mMNaCl, pH 7.4), and after dialysis at 4°C for 16h, it was changed to dialysate 2 (250mM L- Arginine, 25mM Tris, 150mM NaCl, PH7.4) dialyzed for 6h, concentrated and centrifuged, aliquoted and lyophilized, and stored at -80°C.

3. Identification of recombinant PBX1b protein

The concentration and purity of the target protein purified in step 2 of Example 2 were determined by using a non-interfering protein concentration assay kit (Shanghai Sangon Biotechnology Co., Ltd., product number: C503071), and the final protein purity was about 90%, and the concentration was 0.5 mg /mL. The target protein was detected by western blotting, that is, a rabbit anti-His-tag antibody (CST company, catalog #2365) was diluted 1:1000 as the primary antibody, and horseradish peroxidase (enzyme)-labeled goat anti-rabbit IgG (Boster Company, product number BA1054) 1:5000 dilution as a secondary antibody, chemiluminescence color (Thermo scientific company, product number 34080) detection, as shown in Figure 2C, indicating that the purified protein obtained in Example 2 2 was a recombinant PBX1b protein.

Example 3: Preparation of Monoclonal Antibodies

1. Immune rats, cell fusion and monoclonal screening

1.1 Rat immunization and preparation of splenocytes

The purified human PBX1b recombinant protein with a final concentration of 100 μg/ml obtained in Example 2 was thoroughly mixed with an equal volume of complete Freund's adjuvant to a water-in-oil state, and then placed in five 8-week-old female SD rats (respectively marked as 1#, 2#, 3#, 4#, 5#) were subcutaneously injected at multiple points on the back for primary immunization, and each rat was injected with 100 μg of PBX1b recombinant protein. Then every 15 days, after mixing 50 μg of human PBX1b recombinant protein with incomplete Freund's adjuvant, subcutaneous immunization was injected once, and the immunization injection was repeated three times for a total of 45 days. After another 15 days, 50 μg of recombinant human PBX1b protein mixed with incomplete Freund's adjuvant was injected intraperitoneally. 10 days after the last immunization, blood was collected from the tail vein, and the serum titer was determined by indirect ELISA, as shown in Figure 3. Two animals with the highest antiserum titer were selected for booster immunization by intraperitoneal injection of 50 μg recombinant human PBX1b on the 15th day after blood was drawn from the tail vein.

ELISA method for detecting antibody titer: Dilute the purified protein obtained in Example 2 with coating solution (0.1M carbonate buffer, pH 9.6), the final concentration is 10 μg/ml, and 100 μl per well is coated with 96-well enzyme label Plates, overnight at 4°C, washed 3 times with TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5), blocked with 1% BSA, and incubated at 37°C for 2 hours. Wash 3 times with TBST, add the multi-fold dilution to be tested (1/1000, 1/2000, 1/4000, 1/8000, 1/16000, 1/32000, 1/64000, 1/128000, 1/256000, 1/ 512000, 1/1024000) of immunized rat serum (experimental group), tail vein serum of unimmunized rats as negative control, 100 μl/well, incubated at 37°C for 1 hour. After washing with TBS three times, 100 μl of alkaline phosphatase (AP)-labeled rabbit anti-rat secondary antibody (Boster Company, product number BA1012) diluted 1:10000 was added to each well, and incubated at 37° C. for 1 hour. After washing 3 times with TBST, add substrate color development solution containing 1 mg/ml p-nitrophenyl phosphate (PNPP, Thermoscientific company, product number 34045), 100 μl/well, dark for 10-15 minutes, add stop solution (1 M HCl) 100 μl/well, detected with a microplate reader immediately after termination, and read the absorbance value (OD ₄₀₅ ) at a wavelength of 405 nm.

1.2 Cell fusion

Three days after booster immunization, the booster immunized rat splenocytes obtained in Example 3 were aseptically taken, and fused with myeloma cells SP2/0 (ATCC number CRL-1581) in logarithmic growth phase using conventional PEG method. Wash the prepared 10 ⁸ splenocytes and 2.5×10 ⁷ myeloma cells SP2/0 with incomplete RPMI-1640 medium, discard the supernatant after centrifugation, gently flick the cells, and add 0.7 ml of PEG at 40°C (Molecular weight 1,000-6,000) solution, the final concentration of PEG is 50% (W/V), after 60 seconds, start adding incomplete RPMI-1640 medium preheated at 40°C (5 minutes), first add 1ml, 1 After 2 minutes add 4ml and after 2 minutes add 20ml. Cells were collected by centrifugation at 300g at 4°C for 10 minutes, and the cells were gently resuspended in incomplete RPMI-1640 medium containing 2×HAT (containing hypoxanthine, aminopterin, and thymidine) at 37°C to make the final cell concentration 2× 10 ⁶ /ml. Add 100 μl of the resuspended cells to each well of the 96-well plate, and continue to culture. HAT incomplete RPMI-1640 medium was supplemented the day after the cells were confluent. After one week of cell fusion, the medium was changed with RPMI-1640 complete medium, supplemented with HAT-containing RPMI-1640 complete medium, and the HAT selective medium was maintained for two weeks. -1640 complete medium. The production of specific antibodies was detected when fused cells (ie, hybridoma cells) covered 1/10 of the well bottom area. The ELISA plate was coated with human PBX1b recombinant protein, and positive clones were screened by indirect ELISA.

1.3 Monoclonal Screening

The positive hybridomas screened in step 1.2 of Example 3 were immediately cloned by the limiting dilution method to clone the hybridoma cells in the positive wells, and feeder cells were prepared from normal mouse spleen cells. Hybridoma cell lines were obtained after multiple screening. Hybridoma cell lines were continuously cultured in vitro for more than 2 months or after cryopreservation for 6 months, and the cell lines were still able to stably and massively secrete anti-human PBX1 antibody, the antibody was named 2D11 monoclonal antibody, and the hybridoma cell line was named It is 2D11 and is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912.

2.2 Mass production of D11 monoclonal antibody

The 2D11 monoclonal antibody was prepared by intraperitoneal inoculation of nude mice. First, 8-week-old nude mice were intraperitoneally injected with 500 μl of sterile liquid paraffin. One week later, 1×10 ⁶ hybridoma cells were intraperitoneally injected. The ascites was collected in about 7-10 days, and the supernatant was collected by high-speed centrifugation. For the antibody obtained by the above method, the 2D11 antibody was purified by Protein A affinity chromatography, and the purity of the antibody was identified by SDS-PAGE. As shown in Figure 4, the purity of the purified 2D11 monoclonal antibody was higher than 95%, and the heavy chain of the antibody was all It is about 45kDa and the light chain is about 25kDa.

Example 4: Identification of 2D11 monoclonal antibody

1. The titer of 2D11 monoclonal antibody measured by indirect ELISA was about 10 ^-9 .

2. The affinity constant of 2D11 monoclonal antibody determined by competitive ELISA was 6.37×10 ⁸ L/M.

The purified PBX1 monoclonal antibody obtained in Example 3 was diluted with coating solution (0.1M carbonate buffer, pH 9.6), the final concentration was 2 μg/ml, 100 μl per well was coated on a 96-well microtiter plate, and incubated overnight at 4°C . Purified PBX1b was diluted to 2μg/ml, 1μg/ml, 0.5μg/ml, 0.25μg/ml, 0.125μg/ml 0.0625μg/ml, 0.03125μg/ml, 0μg/ml, 50μl of each, and 50μl of monoclonal antibody The culture supernatant of the hybridoma cell line 2D11 was incubated at 4°C overnight to prepare a mixture of 2D11 monoclonal antibodies for antigen competition. 100 μl of the incubation solution of each concentration of antigen and the supernatant of hybridoma 2D11 was added to each well, and incubated at 37° C. for 1 hour. After washing three times with PBST, 100 μl of horseradish peroxidase (HRP)-labeled goat anti-rat secondary antibody (1:10000 dilution, Abcam, product number: ab97057) was added to each well, and incubated at 37°C for 1 hour. After washing 3 times with PBST, add TMB (eBioscience, product number 00-4201-56) substrate, 100 μl/well, dark for 10-15 minutes, add stop solution (1M HCl) 100 μl/well, immediately after termination, use enzyme labeling OD ₄₅₀ and OD ₆₃₀ measurements were performed on the instrument. The antibody affinity constant calculation formula is A0/(A0-A)=1+Kd/a0, where A0 is the OD value when the competing antigen is 0, A is the OD value of each antigen concentration, a0 is the total amount of antigen, and Kd is dissociation constant. K is obtained according to K=1/Kd, where K is an affinity constant, and the unit is L/M, and the calculated affinity constant is 6.37×10 ⁸ L/M.

3.2 Determination of Ig subclass of D11 monoclonal antibody, which is IgG _2a , κ

The rat antibody subtype rapid detection card provided by Antaiji (Beijing) Biotechnology Co., Ltd. was used for the determination.

4.2 Specificity identification of D11 monoclonal antibody

The specificity of the 2D11 monoclonal antibody was identified by immunoblotting and flow cytometry.

Human embryonic kidney 293T cells (ATCC number CRL-3216) express the transcription factor PBX1, and the siRNA (synthesized by Shanghai Gema Company) targeting the PBX1b gene, the sequences are siRNA-1 (SEQ ID NO: 3) and siRNA-2 ( SEQ ID NO: 4) PBX1b knockdown (PBX1-K.D.) 293T cell line was established, and PBX1b expression was restored by transfecting 1 μg and 5 μg of pPBX1b overexpression plasmid into PBX1-K.D.293T cell line. The specificity of the 2D11 monoclonal antibody was identified by immunoblotting (Figure 5A) to detect the expression of PBX1b in 293T cells, PBX1b-K.D.293T cells, and PBX1b-K.D.293T cells that restored PBX1b expression. Extract the nuclear protein from the above grouped cells of equal amount, take 20 μl for 4-20% SDS-PAGE electrophoresis, then transfer the target protein to 0.22 μm PVDF membrane by wet transfer method, block with 5% skim milk at room temperature for 1 hour, and then use 5% Dilute 2D11 monoclonal antibody to 2μg/ml in skim milk, incubate overnight at 4°C, after washing off non-specifically bound antibody with TBST, incubate with secondary antibody (HRP-labeled goat anti-rat antibody, 1:5000 dilution) for 1 hour at room temperature, TBST Wash 3 times. Chemiluminescence color rendering. As shown in Figure 5A, it was identified by immunoblotting that there was a specific band at the relative molecular mass of 38 kDa in 293T cells, and the band was significantly weakened in PBX1b-K.D.293T cells, and the band was restored in PBX1b-K.D.293T cells expressing PBX1b Significant recovery.

The expression of PBX1b in 293T cells, PBX1b-KD293T cells, and PBX1b-KD293T cells restored to express PBX1b was detected by flow cytometry (Fig. 5B), and the specificity of 2D11 monoclonal antibody in live cell detection was identified. 10 ⁶ cells in each of the above groups, use eBioscience company transcription factor immobilization transmembrane kit (Cat. No. 00-5523-00), after immobilization and transmembrane, the kit wash solution diluted 2D11 monoclonal antibody to 20μg/ml, 4 ℃ Incubate for 30 minutes. After washing off non-specifically bound antibodies with PBS, incubate with Alexa Fluor 647-labeled goat anti-rat secondary antibody (AF647) (BioLegend, Cat. No. 405416) at a dilution of 1:500 at 4°C for 30 minutes. After washing with PBS, use BD The company LSR II flow cytometer detection.

The above detection results indicate that the 2D11 monoclonal antibody secreted by the hybridoma cell line 2D11 specifically binds to PBX1b, and the 2D11 monoclonal antibody can be used for western blot detection and flow cytometry detection.

5. Sequencing the purified 2D11 monoclonal antibody

The 2D11 monoclonal antibody was entrusted to be sequenced and purified by Jinweizhi Company. Based on the nucleic acid sequence obtained by Sanger sequencing, the corresponding amino acid sequence was obtained by comparing with the International Immunogenetics Database (IMGT). The sequencing results are: see SEQ ID NOs: 5-20 for details.

6. Alexa Fluor ^™ 488 Fluorescein-labeled 2D11 Monoclonal Antibody

Use Invitrogen Alexa Fluor ^™ 488 Antibody Labeling Kit (Cat. No. A10235) to label 500 μl of 2D11 monoclonal antibody at a concentration of 2 mg/ml. After labeling, the antibody concentration M and labeling efficiency C were calculated by measuring the absorbance of the solutions (A280 and A494) at 280 nm and 494 nm, M=[(A280-A494×0.11)]/203,000, C=A494×dilution Multiples/71,000×M. By calculation, the concentration of our labeled antibody is M=1 mg/ml, the labeling efficiency is C=5.8, and each antibody is labeled with 5.8 fluorescein on average.

Example 5: Application of 2D11 monoclonal antibody

1. Flow cytometry detection of human decidual NK cell transcription factor PBX1

Take the decidual tissue of voluntary termination of pregnancy, normal human early pregnancy decidua, remove residual blood clots in PBS, cut the tissue into pieces in the culture medium, and digest the tissue to chyle with a final concentration of 5 mg/ml collagenase IV (Sigma), 200 mesh Filter the cell suspension into a 50ml centrifuge tube with a sieve, collect the pellet by centrifugation, and perform density gradient centrifugation through human lymphocyte separation blood (Solebold, Product No. P8610) to collect decidua albuginea mononuclear cells. Take normal human peripheral blood mononuclear cells as control, carry out western blot detection, take 5×10 ⁶ decidual tissue mononuclear cells and peripheral blood mononuclear cells respectively to extract nuclear protein, take 20μl for 4-20% SDS-PAGE electrophoresis , the target protein was transferred to 0.22 μm PVDF membrane by wet transfer method, and the immunoblotting experiment operation was consistent with the above-mentioned 293T cell detection method. As shown in Figure 6A, identified by immunoblotting, decidual tissue mononuclear cells and peripheral blood mononuclear cells have specific bands at relative molecular weights of 47kDa (PBX1a) and 38kDa (PBX1b), and the dominant band is 38kDa , the 2D11 monoclonal antibody can specifically bind to PBX1a and PBX1b, that is, in the present disclosure, the monoclonal antibody can specifically bind to PBX1.

The expression of NK cell PBX1 in mononuclear cells was further detected by flow cytometry. 1×10 ⁶ mononuclear cells were suspended in 100 μl PBS, and the surface molecules of human NK cells, including CD3, CD45, CD56, etc., were labeled and identified, and incubated at 4°C for 30 minutes. Fixing transmembrane kit (Cat. No. 00-5523-00), after fixing and transmembrane, dilute the Alexa Fluor ^TM 488-labeled 2D11 monoclonal antibody obtained in Example 4 to 20 μg/ml in the kit wash, and incubate at 4°C After 30 minutes, non-specifically bound antibodies were washed away with PBS and detected by flow cytometry. As shown in Figure 6B/C, CD3 ^- CD45 ⁺ CD56 ⁺ human decidual tissue NK cells highly express the transcription factor PBX1, and the statistical results show that the proportion of PBX1 ⁺ decidual NK (dNK) cells (96.7) is significantly higher than that of peripheral The proportion of blood NK (pNK) cells (1.96).

2. Application of flow cytometry in the diagnosis of unexplained recurrent miscarriage

Taking normal human decidual tissue NK cells as control, the decidual tissue of early pregnancy patients with unexplained recurrent miscarriage was taken as a control, and 2D11 monoclonal antibody was used to detect the expression of transcription factor PBX1 by flow cytometry. The experimental operation is consistent with the detection method of normal human dNK cells. The results are shown in Figure 7A/B. Combining the statistics, it can be seen that the expression of PBX1 in the NK cells of the decidual tissue of the first trimester of patients with unexplained recurrent miscarriage is significantly reduced ^. significantly reduced.

Taking 20 normal human decidual tissues as controls, the early pregnancy decidual tissues of 50 patients with recurrent miscarriage (including 27 cases of unexplained recurrent miscarriage and 23 cases of embryo chromosomal abnormality recurrent miscarriage) were detected by fluorescence quantitative PCR. Changes in the expression of the transcription factor PBX1 gene. The specific steps are as follows: the tissue is ground and broken with liquid nitrogen, the ground tissue is dissolved with 1 ml of Trizol (Invitrogen, product number: 15596026), the total RNA is extracted with 200 μl of chloroform, the supernatant is collected by high-speed centrifugation, and mixed with an equal volume of isopropanol. RNA was precipitated on ice for 30 minutes, and the RNA precipitate was collected by high-speed centrifugation, washed with 75% ethanol solution, and finally dissolved in DEPC-treated water. RNA was reverse transcribed into cDNA using M-MLV kit (Invitrogen, Cat. No. 28025-013), which was used as a template for subsequent fluorescence quantitative PCR detection. Use the dye method fluorescence quantitative kit (TaKaRa company, product number: RR820A) to carry out fluorescence quantitative PCR, using decidual tissue cDNA as the template, the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 are the forward direction of fluorescence quantitative PCR, respectively and reverse primer to prepare a 20 μl reaction system. The reaction conditions were pre-denaturation at 94°C for 30 seconds; denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, and 40 cycles as reaction conditions. Using the 2- ^ΔΔCt analysis method and the unpaired t-test statistical method to process the data, the results are shown in Figure 7C. Combined with statistics, it can be seen that the PBX1 gene is in all patients with unexplained recurrent miscarriage (p<0.0001) and some embryos with recurrent chromosomal abnormalities. The expression in the first trimester decidua tissue of miscarriage patients (p=0.0001) was significantly decreased. Receiver operating characteristic curve (ROC curve) was used to analyze the relative expression of PBX1 gene in decidual tissue of patients with unexplained recurrent miscarriage, patients with recurrent miscarriage with embryonic chromosomal abnormalities and two parts of patients with recurrent miscarriage as a whole. The correlation of disease occurrence, the results are shown in Figure 7D-E, the reduction of PBX1 expression can be very effective for clinical diagnosis of unexplained recurrent miscarriage (in the unexplained recurrent miscarriage group, AUC = 0.941 (95% confidence interval = 0.8601 to 1.021, p<0.0001), higher than that in the recurrent miscarriage group with chromosomal abnormalities in embryos, AUC=0.762 (95% confidence interval=0.6167 to 0.9072, p=0.003361)), effectively predicting the occurrence of recurrent miscarriage disease (overall recurrent miscarriage) , AUC=0.859 (95% confidence interval=0.7731 to 0.9439, p<0.0001)). In conclusion, the decreased expression of PBX1 in decidual tissue can be effectively used for the diagnosis of recurrent miscarriage, especially in cases of unexplained recurrent miscarriage; and 2D11 monoclonal antibody can further analyze the dominant immune cell NK in decidual tissue by flow cytometry. The expression of cellular transcription factor PBX1 provides accurate diagnosis basis for recurrent miscarriage patients, especially unexplained recurrent miscarriage with multiple pregnancy failures, and provides the possibility for subsequent targeted treatment.

Claims (15)

1. A monoclonal antibody or antigen-binding fragment thereof comprising: The amino acid sequence is the heavy chain variable region CDR-H1 of SEQ ID No:6, preferably, its coding sequence is SEQ ID No:5; The amino acid sequence is the heavy chain variable region CDR-H2 of SEQ ID No:8, preferably, its coding sequence is SEQ ID No:7; The amino acid sequence is the heavy chain variable region CDR-H3 of SEQ ID No:10, preferably, its coding sequence is SEQ ID No:9; The amino acid sequence is the light chain variable region CDR-L1 of SEQ ID No:12, preferably, its coding sequence is SEQ ID No:11; The amino acid sequence is the light chain variable region CDR-L2 of SEQ ID No: 14, preferably, its coding sequence is SEQ ID No: 13; And The amino acid sequence is the light chain variable region CDR-L3 of SEQ ID No:16, preferably, its coding sequence is SEQ ID No:15; wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or dAb. 2. A monoclonal antibody or antigen-binding fragment thereof comprising: The amino acid sequence is the heavy chain variable region of SEQ ID No: 18, preferably, its coding sequence is SEQ ID No: 17; and The amino acid sequence is the light chain variable region of SEQ ID No:20, preferably, its coding sequence is SEQ ID No:19; wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or dAb. 3. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2, which is capable of specifically binding to PBX1a and/or PBX1b protein. 4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the Ig subclass is _IgG2a ,κ. 5. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a humanized antibody, chimeric antibody, single-chain antibody, or diabody. 6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is secreted by the hybridoma cell line deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912. 7. A conjugate comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 and a label bound to it that allows its detection, wherein the label is selected from the group consisting of fluorophores, biotin , one or more of radioisotopes, metals and enzymes. 8. A hybridoma cell line, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C201912. 9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6, or the conjugate according to claim 7, in detecting the protein expression of PBX1a and/or PBX1b. 10 . An immunodetection kit comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6 or the conjugate of claim 7 . 11 . 11. The immunoassay kit of claim 10, wherein the detected sample is a plasma sample. 12. The immunodetection kit of claim 10 or 11, wherein the detected sample is of human origin. 13. The immunoassay kit according to any one of claims 11-11, wherein the detected samples are NK cells of early pregnancy decidual tissue of patients with recurrent miscarriage, preferably patients with unexplained recurrent miscarriage. 14. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the conjugate according to claim 7 in the preparation of a diagnostic agent for recurrent miscarriage, preferably the The recurrent miscarriage is defined as recurrent miscarriage of unknown cause or recurrent miscarriage caused by abnormal embryo. 15. A kit for assisting diagnosis of unexplained recurrent miscarriage, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the conjugate according to claim 7.

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