CN111434686B

CN111434686B - Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion

Info

Publication number: CN111434686B
Application number: CN201910037029.XA
Authority: CN
Inventors: 魏海明; 周永刚; 徐秀秀; 傅斌清; 孙汭; 田志刚
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2019-01-15
Filing date: 2019-01-15
Publication date: 2022-09-30
Anticipated expiration: 2039-01-15
Also published as: CN111434686A

Abstract

A monoclonal antibody, a preparation method thereof and application thereof in clinical diagnosis of recurrent abortion. Specifically, the monoclonal antibody is secreted by a hybridoma cell strain 2D11, can be used for diagnosing recurrent abortion diseases, and has good specificity and high sensitivity.

Description

Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion

Technical Field

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

Background

Nearly 4500 ten thousand of infertility patients exist in China, and the number of the infertility patients increases gradually at hundreds of thousands of speeds every year. Solves the problem of infertility, and has very important significance for family happiness and social harmony and stability. Recurrent abortion has become a common pregnancy disease affecting women of childbearing age, seriously affecting reproductive health; of these, about 50% of patients have difficulty in clinical diagnosis of the cause (Qiagen (2018) J.Utility gynecology and obstetrics, 12 th 1309-131 pages) and clinical manifestations are recurrent abortion of unknown origin, and current studies show that it is mainly associated with immune dysfunction of decidua 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 U S.A.; 110 (3); E231-40). NK cells, as immune cells of the maternal-fetal interface, are responsible for important physiological functions, such as induction of immune tolerance, promotion of trophoblast invasion, participation in spiral artery remodeling processes, promotion of placental and fetal development, etc. (Vento-Tormo R, Efremova M, et al (2018) Nature.; 563(7731): 347-. The transcription factor PBX1 is involved in the maintenance of the number of decidua NK cells and regulates the execution of the normal functions of the decidua NK cells.

Transcription factor PBX1, also known as: cakured, PRL. Encoded by the pre-B-cell leukemia homeobox gene (PBX 1), is one of the important members of the homeobox gene family. The transcription factor PBX1 was identified by the American scholars Kamps MP et al in pre-B cell leukemia patients (Kamps MP et al (1990) cell.; 60(4):547-55), and later studies suggested that it plays an important role in embryonic development, organogenesis, and cell differentiation; is associated with various malignant tumors. The gene of human PBX1 maps to 1q23 and consists of 9 exons and 8 introns. The relative molecular weight of the encoded protein is 47kD, the encoded protein is composed of 430 amino acids, and two main homologous isomers PBX1a (430aa,47kD) and PBX1b (347aa,38kD) exist at the same time, wherein the 1 st to 333 rd amino acids in PBX1b are completely consistent with PBX1a, the two homologous isomers are similar in structure and respectively comprise a C-terminal homologous structural domain which plays a role in stabilizing a ternary complex structure formed by PBX1, HOX and DNA, and the N terminal comprises two PBC structural domains which are protein-protein interaction regions.

Summary of The Invention

The invention relates to a monoclonal antibody, a preparation method thereof and application thereof in clinical diagnosis of recurrent abortion. Specifically, the monoclonal antibody is secreted by a hybridoma cell strain 2D11 which is preserved in China center for type culture Collection and has the preservation number of CCTCC NO. C201912, can be applied to detecting the expression of PBX1 in human decidua prolifera NK cells, and is used as one of limited clinical diagnosis indexes of diseases related to recurrent abortion (unexplained recurrent abortion) caused by decidua NK cell abnormality. Therefore, the monoclonal antibody has great clinical significance for diagnosing and guiding pregnancy-related diseases such as recurrent abortion and the like and further treating the pregnancy-related diseases in a targeted manner.

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

heavy chain variable region CDR-H1 having amino acid sequence SEQ ID No. 6, preferably, encoding sequence SEQ ID No. 5;

the heavy chain variable region CDR-H2 having the amino acid sequence of SEQ ID No. 8, preferably, the coding sequence thereof is SEQ ID No. 7;

heavy chain variable region CDR-H3 having amino acid sequence of SEQ ID No. 10, preferably, it encodes the polypeptide having amino acid sequence of SEQ ID No. 9;

light chain variable region CDR-L1 having amino acid sequence SEQ ID No. 12, preferably, encoding sequence SEQ ID No. 11;

light chain variable region CDR-L2 having amino acid sequence SEQ ID No. 14, preferably, encoding sequence SEQ ID No. 13; and

light chain variable region CDR-L3 having amino acid sequence SEQ ID No. 16, preferably, encoding sequence 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 the antigen binding fragment thereof as described above, which is secreted by a hybridoma cell line which is deposited in the chinese typical culture collection with the collection number of CCTCC No. c 201912.

In one aspect, the present invention provides a conjugate comprising any one of the monoclonal antibodies or antigen-binding fragments thereof as described above and a label bound thereto that allows detection thereof, wherein the label is selected from one or more of a fluorophore, biotin, a radioisotope, a metal and an enzyme.

In one aspect, the invention provides a hybridoma cell line that produces any one of the monoclonal antibodies or antigen-binding fragments thereof described above.

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

In one aspect, the invention provides the use of a monoclonal antibody or antigen-binding fragment thereof as described in any one of the above or a conjugate as described in any one of the above for detecting protein expression of PBX1a and/or PBX1 b.

In one aspect, the 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 one of the monoclonal antibodies or antigen-binding fragments thereof as described above, or any one of the conjugates as described above, in the manufacture of a diagnostic agent for the auxiliary diagnosis of unexplained recurrent spontaneous abortion.

In one aspect, the present invention provides a kit for aiding in the diagnosis of recurrent abortion of unknown origin, comprising any of the monoclonal antibodies or antigen binding fragments thereof as described above or any of the conjugates as described above.

Brief description of the drawings

FIG. 1 is a flow chart of the construction of prokaryotic recombinant expression vector pET21a-PBX1b and eukaryotic recombinant expression vector pPBX1 b. FIG. 1A shows a flow chart for the construction of prokaryotic recombinant expression vector pET21A-PBX1b based on pET21A plasmid; FIG. 1B shows a scheme for the construction of the eukaryotic recombinant expression vector pPBX1B based on the pEGFP-N1 plasmid.

FIG. 2 expression, purification and assay of recombinant protein of human transcription factor PBX1 b. FIG. 2A shows the result of induced expression of PBX1b recombinant protein; FIG. 2B shows the results before and after nickel sepharose affinity chromatography of PBX1B recombinant protein; figure 2C shows the immunoblot identification of purified PBX1b recombinant protein.

FIG. 3 shows the serum ELISA detection results before and after PBX1b recombinant protein immunization of rats.

FIG. 4.2D11 monoclonal antibody purity identification results.

FIG. 5.2D11 monoclonal antibody specificity identification results. FIG. 5A shows immunoblotting to identify 2D11 monoclonal antibody specificity; figure 5B shows flow cytometry to identify 2D11 monoclonal antibody specificity.

FIG. 6.ALEXA FLUOR ^TM The 488-labeled 2D11 monoclonal antibody is applied to flow cytometry for detecting the expression of NK cell transcription factor PBX1 of the decidua tissue of the early pregnancy of a normal person. Fig. 6A shows that 2D11 monoclonal antibody can bind PBX1a and PBX1b in decidua tissue mononuclear cells and peripheral blood mononuclear cells, respectively. FIG. 6B shows the detection of transcription factor PBX1 expression in human decidua tissue NK (dNK) cells and peripheral blood NK (pNK) cells using 2D11 monoclonal antibody using flow cytometry; figure 6C shows PBX1 ⁺ NK cell proportion in dNK and pNK cellA statistical map of (2).

FIG. 7.ALEXA FLUOR ^TM The 488-labeled 2D11 monoclonal antibody is applied to flow cytometry for diagnosing recurrent abortion with unknown causes. FIG. 7A shows flow cytometry detection of transcription factor PBX1 expression in NK cells of decidua tissue of patients with Recurrent Spontaneous Abortion (RSA) against normal human dNK cells; FIG. 7B shows PBX1 of patients with recurrent abortion and normal person ⁺ Statistical plots of dNK cell ratio (top) and number (bottom). FIG. 7C shows the fluorescent quantitative PCR detection of PBX1 gene expression in decidua tissue of patients with recurrent abortion; FIG. 7D shows the working characteristics of subjects with reduced PBX1 expression in unexplained recurrent abortion and recurrent abortion with fetal chromosomal abnormalities; figure 7E shows the working characteristics of subjects with reduced PBX1 expression in recurrent miscarriage.

Material preservation

The following materials have been deposited: china center for type culture Collection (Wuhan city, Wuchang, Hubei, China, No. 299 eight channels) (CCTCC):

materials: rat anti-human PBX1 antibody hybridoma cell strain 2D11

The preservation number is as follows: c201912

Preservation day: 1 month and 8 days 2019

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In general, the terminology used herein is well known and is conventionally 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; the antibody light chains may be classified as kappa and lambda light chains; the heavy chains may be classified as mu, delta, gamma, alpha or epsilon; and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively, within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, the heavy chains further comprise a "D" region of about 3 or more amino acids; each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH 3.) each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). the light chain constant region is composed of one domain CL; the constant regions of an antibody may mediate the interaction of an immunoglobulin with host tissue or factor, including the binding of various cells of the immune system (e.g., effector cells) to the first component of the classical complement system (C1 q). The VH and VL regions may also be subdivided into regions of high denaturation (termed Complementarity Determining Regions (CDRs) interspersed with regions more conserved termed Framework Regions (FRs) each VH and VL consisting of 3 CDRs and 4 FRs arranged from amino-to carboxy-terminus FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, respectively, the variable regions (VH and VL) of each heavy/light chain pair form antibody binding sites, respectively the assignment of amino acids to each region or domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 917; Choia et al (1989) Nature 878;. 3;. 88342;. 917;. 342.

As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of a full-length antibody, typically to the antigen-binding or variable region of the full-length antibody. The fragment retains the ability to bind to the same antigen (e.g., PBX1) to which the antibody binds, competing with the intact antibody for specific binding to the antigen. Antigen-binding fragments can be generated 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 (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding capability on the polypeptide.

As used herein, the term "monoclonal antibody" refers to a homogeneous antibody directed against only a particular epitope of an antigen. 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 antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies of the invention are preferably produced by a single hybridoma, which results from the fusion of a spleen cell with a myeloma cell.

The DNA encoding the antibody may be modified to produce a "chimeric or fused antibody polypeptide", for example by replacing the homologous murine sequences with human heavy and light chain constant domain (CH and CL) sequences (US 4816567; and Morrison et al (1984) Proc. Natl Acad. Sci. USA.; 81:6851), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may replace constant domains of an antibody, or replace variable domains of one antigen binding site of an antibody with them to create a chimeric bivalent antibody comprising an antigen binding site with specificity for one antigen and another antigen binding site with specificity for a different antigen.

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human antibodies. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise 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, the humanized antibody will comprise substantially all of at least one, and typically 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 FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The Fc fragment contains the carboxy-terminal portions of all two heavy chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also a part recognized by Fc receptors (FcRs) 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 the consensus sequence of all human antibodies of a particular 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 amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the 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 particular molecular target or antigen of interest or to which an epitope on a particular molecular target or antigen of interest is "specific" means that binding is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of the molecule and comparing it to the binding of a control molecule, which is typically a molecule of similar structure but lacking binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. Typically, the antibody is present in an amount less than about 10 ^-6 M, e.g. less than about 10 ^-7 M、10 ^-8 M、10 ^-9 M or 10 ^-10 M or less binds to an epitope.

"binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., 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 readily, while high affinity antibodies generally bind antigen more rapidly and tend to remain bound for a longer period of time. A variety of methods for measuring binding affinity are known in the art, any of which may 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 suitable reaction conditions.

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

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" may include a variety of forms of presentation suitable for use, such as may be in the form of a box, bottle, pouch, or the like.

As used herein, the term "PBX 1" refers herein to PBX1a and PBX1 b.

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

the heavy chain variable region CDR-H3 having the amino acid sequence of SEQ ID No. 10, preferably, the coding sequence thereof is SEQ ID No. 9;

the heavy chain variable region having the amino acid sequence of SEQ ID No. 18, preferably, the coding sequence thereof is SEQ ID No. 17; and

the light chain variable region having the amino acid sequence of SEQ ID No. 20, preferably, the coding sequence thereof is SEQ ID No. 19;

In one embodiment, any of the monoclonal antibodies or antigen binding fragments thereof described above is capable of specifically binding to PBX1a and/or PBX1b proteins. In one embodiment, the subclass of Ig of any one of the monoclonal antibodies or antigen binding fragments thereof described above is IgG _2a And κ. In one embodiment, any 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 hybridoma cell strain 2D11 with the collection number of CCTCC No. c201912, which is collected in the chinese center for type culture collection.

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 a label bound thereto that allows detection thereof, wherein the label is selected from one or more of a fluorophore, biotin, a radioisotope, a metal and an enzyme.

In one aspect, the invention provides a hybridoma cell strain, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO. C201912. In one embodiment, the hybridoma cell strain produces a monoclonal antibody or antigen-binding fragment thereof against human transcription factor PBX1 protein. In one embodiment, the hybridoma cell strain can stably secrete the monoclonal antibody or the antigen-binding fragment thereof for resisting the human transcription factor PBX1 protein.

In another aspect, the invention provides the use of any of the monoclonal antibodies or antigen-binding fragments thereof as described above, or of any of the conjugates as described above, for detecting protein expression by PBX1a and/or PBX1 b. In one embodiment, the sample tested is a plasma sample. In one embodiment, the sample tested is of human origin. In one embodiment, the sample to be tested is decidua early pregnancy tissue NK cells of a patient with recurrent abortion, preferably a patient with unexplained recurrent abortion. In one embodiment, the method of detection is by using as a primary antibody any of the monoclonal antibodies or antigen-binding fragments thereof described above or any of the conjugates described above. In one embodiment, the method of detection is detection of the human cell transcription factor PBX1 using immunoblotting. In one embodiment, the fluorophore in the conjugate is the labeled fluorescein alexafluor 488. In one embodiment, the method of detection is flow cytometry detection of the human cellular transcription factor PBX 1.

In another aspect, the 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 another aspect, the present invention provides the use of any one of the monoclonal antibodies or antigen-binding fragments thereof as described above or any one of the conjugates as described above for the manufacture of a diagnostic agent for aiding in the diagnosis of unexplained recurrent abortion disease, in one embodiment the sample for aiding in the diagnosis is decidua early pregnancy tissue NK cells of an unexplained recurrent abortion patient. In one embodiment, the fluorophore in the conjugate is the labeled fluorescein ALEXA fluor 488. In one embodiment, the diagnosis method is to detect the transcription factor PBX1 of decidua early pregnancy tissue NK cells of an unexplained recurrent abortion patient by using flow cytometry, and further diagnose whether the pathogenesis of the recurrent abortion patient is low expression of the transcription factor PBX1 and abnormal NK cells.

In another aspect, the present invention provides a kit for the auxiliary diagnosis of recurrent spontaneous abortion of unknown origin, comprising any of the monoclonal antibodies or antigen-binding fragments thereof as described above or any of the conjugates as described above.

Examples

The following examples will assist one of ordinary skill in the art in further understanding the invention, but are not intended to limit the invention in any way.

The experimental procedures in the examples, unless otherwise specified, were carried out by techniques conventional in the art, and the experimental reagents were commercially available.

The first embodiment is as follows: construction of prokaryotic recombinant expression vector pET21a-PBX1b and eukaryotic recombinant expression vector pPBX1b

Entrusted with general biosystems (Anhui) Limited company to artificially synthesize a full-length gene of a human transcription factor PBX1b (NCBI Reference Sequence: NM-001204961.1, encoding amino acids 1 to 347), wherein the gene Sequence and the encoded protein Sequence are respectively sequences shown in

SEQ ID NO

1 and 2, 6His-Tag and a stop codon are introduced into the C-terminal, the gene Sequence and the encoded protein Sequence are directly cloned to a recombinant expression vector pET21a (Shanghai Bionical corporation) through NdeI and XhoI enzyme cutting sites, the obtained recombinant expression vector is transferred into DH5 alpha competent cells, positive clones are screened for DNA sequencing identification, and the sequencing result shows that the construction of the recombinant expression vector pET21a-PBX1b is successful. The construction process of prokaryotic recombinant expression vector pET21A-PBX1b is shown in FIG. 1A.

The full-length gene of the human transcription factor PBX1b is synthesized artificially, the full-length gene is directly cloned to a recombinant expression vector pEGFP-N1 (Excellent biological products, Inc.) through EcoRI and BamHI enzyme cutting sites and is named as pPBX1b, the obtained recombinant expression vector is transferred into DH5 alpha competent cells, positive clones are screened for DNA sequencing identification, and the sequencing result shows that the recombinant expression vector pPBX1b is successfully constructed. The construction process of the eukaryotic recombinant expression vector pPBX1B is shown in FIG. 1B.

The second embodiment: expression, purification and determination of human transcription factor PBX1b recombinant protein

1. Inducible expression of recombinant proteins

1.1 transformation

The recombinant expression vector pET21a-PBX1b obtained in example 1 was transformed into Transetta (DE3) competent cells (Kyoto Kogyo Biotech Co., Ltd., product No. CD801), heat-shocked at 42 ℃ for 90 seconds, allowed to stand on ice for 2 minutes, spread on LB solid plates containing ampicillin at a final concentration of 50. mu.g/mL and chloramphenicol at 34. mu.g/mL, and cultured overnight at 37 ℃.

1.2 selection of optimal Induction conditions in Small-scale cultures

A single colony of the recombinant protein expression strain Transetta (DE3) from PBX1b was picked up in 10mL of LB medium (containing 50. mu.g/mL ampicillin and 34. mu.g/mL chloramphenicol) at 37 ℃ and 220 rpm and cultured overnight with shaking. The overnight-cultured bacterial suspension was inoculated into 10mL of LB medium (containing 50. mu.g/mL ampicillin and 34. mu.g/mL chloramphenicol) at a ratio of 1:100, respectively, and cultured at 37 ℃ under shaking at 220 rpm. When OD is reached _600nm When the concentration was 0.6, IPTG was added to the medium at a final concentration of 0.5mM at 220 rpm, and expression was induced by shaking culture, and the resultant was induced overnight at 20 ℃ and 4 hours at 37 ℃ respectively, and negative control was obtained without IPTG inducer. The thalli is collected by centrifugation, the thalli is cracked by ultrasonic disruption, the supernatant and the precipitate are respectively collected by centrifugation, the precipitate is dissolved by 500 mu L of inclusion body dissolving solution (8M Urea, 50mM Tris-HCl, 300mM NaCl, PH8.0), 40 mu L of samples are respectively taken to carry out 12 percent SDS-PAGE electrophoresis detection, the sample loading amount is 10 mu L, and after the gel electrophoresis is finished, Coomassie brilliant blue is used for staining for 20 minutes, and imaging is carried out after decoloration. As shown in FIG. 2A, the supernatant of IPTG-induced lysate showed a distinct band at 38kDa, and the overnight induction at 20 ℃ was better than the induction at 37 ℃ for 4 hours, compared to the other control conditions.

1.3 Mass inducible expression of fusion proteins

Final determination according to the lab results of 1.2: the overnight induction condition at 20 ℃ was selected, and the uninduced monoclonal culture broth used in the above 1.2 pilot plant was inoculated into 4L of LB resistant medium at a ratio of 1:100 for mass induction expression.

2. Purification of fusion proteins

And (3) centrifugally collecting a large amount of induced bacterial liquid sediment in the step 1.3, ultrasonically crushing the cracked bacterial liquid sediment in ice bath, centrifugally collecting cracked supernatant, and purifying by adopting a nickel-agarose affinity chromatography method. 5mL of Ni-IDA was taken and the equilibrated column was washed with 10 bed volumes of equilibration solution (1M Tris, 150mM NaCl, pH8.0) at a flow rate of 5 mL/min. The column was filled with the lysed supernatant obtained in step 1.3 of the above example 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. Further, the control was eluted with 20/50mM Imidazole eluent (1M Tris, 150mM NaCl, 20/50mM Imidazole, pH8.0) at a flow rate of 5 mL/min to remove the contaminating proteins. Finally, the target protein was eluted using a 500mM Imidazole solution (1M Tris, 150mM NaCl, 500mM Imidazole, pH8.0) at a flow rate of 2 mL/min, and the target protein eluate was collected. A sample of the eluate of the target protein was collected and subjected to 12% SDS-PAGE, as shown in FIG. 2B, and a band of the target protein was evident at 38kDa in the eluate of 500mM imidazole, compared to the control group. According to the identification result, the target protein eluted from 500mM imidazole solution was dialyzed against dialysate 1(500mM L-Arginine, 25mM Tris, 150mM NaCl, pH7.4) at 4 ℃ for 16 hours, then dialyzed against dialysate 2(250mM L-Arginine, 25mM Tris, 150mM NaCl, pH7.4) for 6 hours, concentrated, centrifuged, dispensed, and lyophilized, and stored at-80 ℃.

3. Identification of recombinant PBX1b protein

The concentration and purity of the target protein purified in step 2 of example were measured using a non-interfering protein concentration measuring kit (Shanghai Biotech, cat # C503071) to obtain a protein with a purity of about 90% and a concentration of 0.5 mg/mL. Immunoblot detection of the target protein was performed by diluting rabbit anti-His-tag antibody (CST, cat #2365)1:1000 as a primary antibody, and horse radish peroxidase (enzyme) -labeled goat anti-rabbit IgG (Boster, cat # BA1054)1:5000 as a secondary antibody, and performing chemiluminescence detection (Thermo scientific, cat # 34080), as shown in fig. 2C, indicating that the protein obtained by purification in example 2 and induced expression was recombinant PBX1b protein.

Example three: preparation of monoclonal antibodies

1. Immunized rats, cell fusion and monoclonal screening

1.1 rat immunization and preparation of splenocytes

The purified human PBX1b recombinant protein obtained in example two, at a final concentration of 100 μ g/ml, was mixed well with an equal volume of complete freund's adjuvant in a water-in-oil form, and primary immunization was performed by subcutaneous multi-point injection into the back of 5 female SD rats (labeled 1#, 2#, 3#, 4#, and 5#, respectively) aged 8 weeks, each rat being injected with 100 μ g of PBX1b recombinant protein. Then, every 15 days, 50 μ g of the human PBX1b recombinant protein was mixed with incomplete freund's adjuvant, and the subcutaneous immune injection was performed once, and the immune injection was repeated three times for 45 days. After another 15 days, 50 μ g of human PBX1b recombinant protein was intraperitoneally injected with incomplete freund's adjuvant. 10 days after the last immunization, blood was taken via the tail vein and the serum titer was determined by indirect ELISA, as shown in FIG. 3.2 subjects with the highest antiserum titers were selected and boosted by intraperitoneal injection of 50 μ g of human PBX1b recombinant protein on day 15 after tail vein bleeds.

The ELISA method for detecting the antibody titer comprises the following steps: the purified protein from example two was diluted with coating solution (0.1M carbonate buffer, pH 9.6) to a final concentration of 10. mu.g/ml, coated with 100. mu.l/well of a 96-well microplate, overnight at 4 ℃, washed 3 times with TBST (50mM Tris-HCl, 150mM NaCl, 0.05% Tween20, pH 7.5), blocked with 1% BSA, and incubated at 37 ℃ for 2 hours. TBST was washed 3 times, and serum (experimental group) of immunized rats diluted to be tested in multiple ratios (1/1000, 1/2000, 1/4000, 1/8000, 1/16000, 1/32000, 1/64000, 1/128000, 1/256000, 1/512000 and 1/1024000) was added, and the serum of tail vein of non-immunized rats was used as negative control, 100. mu.l/well and incubated at 37 ℃ for 1 hour. TBS was washed 3 times, 100. mu.l of Alkaline Phosphatase (AP) -labeled rabbit-anti-rat secondary antibody (Boster Co., cat. No. BA1012) diluted 1:10000 was added to each well, and incubated at 37 ℃ for 1 hour. Washing TBST for 3 times, adding substrate developing solution containing 1mg/ml p-nitrophenyl phosphate (PNPP, Thermoscientific, Inc., Cat. 34045), developing for 10-15 min in dark, adding 100 μ l stop solution (1M HCl), detecting with microplate reader immediately after stopping, readingAbsorbance (OD) at wavelength of 405nm ₄₀₅ )。

1.2 cell fusion

3 days after the boosting, the spleen cells of the boosted mice obtained in example three were aseptically taken and fused with myeloma cells SP2/0(ATCC accession number CRL-1581) in the logarithmic growth phase using the conventional PEG method. Washing of prepared 10 with incomplete RPMI-1640 Medium ⁸ Spleen cells and 2.5X 10 ⁷ Myeloma cells SP2/0 were centrifuged to discard the supernatant, the cells were gently flicked, 0.7ml of 40 ℃ PEG (molecular weight: 1,000-6,000) solution was added to the solution, the final concentration of PEG was 50% (W/V), and after 60 seconds, the incomplete RPMI-1640 medium preheated at 40 ℃ was started to be added (after 5 minutes), 1ml was added first, 4ml was added after 1 minute, and 20ml was added after 2 minutes. The cells were collected by centrifugation at 300g for 10 minutes at 4 ℃ and suspended gently in incomplete RPMI-1640 medium containing 2 XHAT (containing hypoxanthine, aminopterin and thymidine) at 37 ℃ to give a final cell concentration of 2X 10 ⁶ And/ml. The resuspended cells were added to a 96-well plate at 100. mu.l per well and the culture was continued. HAT incomplete RPMI-1640 medium was supplemented the next day after cell fusion. Replacing the culture medium with RPMI-1640 once after cell fusion for one week, supplementing the complete culture medium with RPMI-1640 containing HAT, culturing with HT culture medium after HAT selective culture medium is maintained for two weeks, and replacing with RPMI-1640 complete culture medium after two weeks. The production of specific antibodies was detected when the fused cells (i.e., hybridoma cells) were spread across the area of the bottom 1/10 of the well. The ELISA plate is coated with human PBX1b recombinant protein, and positive clones are screened by an indirect ELISA method.

1.3 monoclonal screening

The positive hybridomas screened in the three steps 1.2 of example were cloned by limiting dilution method into positive well hybridoma cells, and feeder cells were prepared from normal mouse spleen cells. And obtaining the hybridoma cell strain after multiple screening. After the hybridoma cell strain is continuously cultured in vitro for more than 2 months or is frozen for 6 months, the cell strain can still stably secrete a large amount of anti-human PBX1 antibody, the antibody is named as a 2D11 monoclonal antibody, the hybridoma cell strain is named as 2D11 and is preserved in China center for type culture Collection, and the preservation number is CCTCC NO. C201912.

2.2 Mass production of the D11 monoclonal antibody

The 2D11 monoclonal antibody is prepared by a nude mouse abdominal cavity inoculation method. The nude mice of 8 weeks old were first intraperitoneally injected with 500. mu.l of sterile liquid paraffin, and one week later were intraperitoneally injected with 1X 10 ⁶ And (3) collecting ascites after 7-10 days of hybridoma cells, and performing high-speed centrifugation to collect supernatant. The antibody obtained by the above method was purified by Protein A affinity chromatography to obtain 2D11 antibody, and the purity of the antibody was confirmed by SDS-PAGE, as shown in FIG. 4, the purity of the purified 2D11 monoclonal antibody was higher than 95%, the heavy chains of the antibody were all about 45kDa, and the light chain was about 25 kDa.

Example four: identification of 2D11 monoclonal antibody

1. 2D11 mAb titers were determined to be about 10 by indirect ELISA ^-9 。

2. The affinity constant of the 2D11 monoclonal antibody was 6.37X 10 in a competitive ELISA assay ⁸ L/M。

The purified PBX1 monoclonal antibody obtained in example three was diluted with a coating solution (0.1M carbonate buffer, pH 9.6) to a final concentration of 2. mu.g/ml, and 100. mu.l per well of a 96-well microplate and incubated overnight at 4 ℃. Purified PBX1b was diluted 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.03125. mu.g/ml, 0. mu.g/ml, 50. mu.l each, and 50. mu.l of the culture supernatant of monoclonal antibody hybridoma cell line 2D11 was incubated overnight at 4 ℃ to prepare an antigen-competitive 2D11 monoclonal antibody mixture. Mu.l of the incubation solution of each concentration of antigen and hybridoma 2D11 supernatant was added to each well and incubated at 37 ℃ for 1 hour. PBST was washed 3 times, and 100. mu.l of a horseradish peroxidase (HRP) -labeled goat-anti-rat secondary antibody (1:10000 dilution, Abcam Co., cat # ab97057) was added to each well, followed by incubation at 37 ℃ for 1 hour. PBST was washed 3 times, then TMB (eBioscience, cat. 00-4201-56) substrate was added thereto, 100. mu.l/well was subjected to color development in the dark for 10-15 minutes, 100. mu.l/well of stop buffer (1M HCl) was added thereto, and OD was performed immediately after completion using a microplate reader ₄₅₀ And OD ₆₃₀ And (6) detecting. The antibody affinity constant is calculated as a0/(a0-a) ═ 1+ Kd/a0, where a0 is the OD value at which the competing antigen is 0, a is the OD value at each antigen concentration, a0 is the total amount of antigen, and Kd is the dissociation constant. The affinity calculated from K1/Kd, K being the affinity constant in L/MConstant of 6.37X 10 ⁸ L/M。

Determination of Ig subclass of the monoclonal antibody 2D11, which is IgG _2a ，κ

The determination is carried out by adopting a rat antibody subtype rapid detection card provided by Antaiji (Beijing) biotechnology limited.

4.2 specificity identification of the D11 monoclonal antibody

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

Human embryonic kidney 293T cells (ATCC accession number CRL-3216) express the transcription factor PBX1, a PBX1b knockdown (PBX1-K.D.)293T cell line was established by siRNA (synthesized by Shanghai Jima) targeting PBX1b gene, with the sequences siRNA-1(SEQ ID NO:3) and siRNA-2(SEQ ID NO:4), respectively, and PBX1b was restored by transfecting 1. mu.g and 5. mu.g of pPBX1b over-expression plasmid into PBX1-K.D.293T cell line. The specificity of the 2D11 monoclonal antibody was identified by detecting the expression level of PBX1b in 293T cells, PBX1b-K.D.293T cells and PBX1b-K.D.293T cells expressing PBX1b by immunoblotting (FIG. 5A). The nucleoprotein of the same amount of cells was extracted from the above groups, 20. mu.l of the nucleoprotein was subjected to 4-20% SDS-PAGE, the target protein was transferred to a PVDF membrane of 0.22. mu.m by wet transfer, 5% skim milk was blocked at room temperature for 1 hour, then 2D11 monoclonal antibody was diluted to 2. mu.g/ml with 5% skim milk, incubated overnight at 4 ℃ and, after washing off the non-specifically bound antibody by TBST, a secondary antibody (HRP-labeled goat anti-rat antibody, 1:5000 dilution) was incubated at room temperature for 1 hour and washed 3 times by TBST. Chemiluminescence and color development. As shown in FIG. 5A, immunoblotting identified a specific band at 38kDa relative molecular mass in 293T cells, which was significantly attenuated in PBX1b-K.D.293T cells and significantly restored in PBX1b-K.D.293T cells expressing PBX1 b.

The specificity of the 2D11 monoclonal antibody in the live cell assay was identified by detecting the expression level of PBX1B in 293T cells, PBX1B-K.D.293T cells and PBX1B-K.D.293T cells expressing PBX1B by the flow cytometry method (FIG. 5B). Each of 10 above ⁶ After the cells were fixed and permeabilized using the eBioscience transcription factor fixed-membrane kit (cat. No. 00-5523-00), the 2D11 monoclonal antibody was diluted with the kit washing solution20 μ g/ml, incubated at 4 ℃ for 30 minutes, and after washing away the non-specifically bound antibody with PBS, a 1:500 dilution of Alexa Fluor 647-labeled secondary goat anti-rat antibody (AF647) (BioLegend, cat. No. 405416) was incubated at 4 ℃ for 30 minutes, and after washing with PBS, it was detected using an LSR II flow cytometer from BD.

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

5. Sequencing the purified 2D11 monoclonal antibody

The purified 2D11 monoclonal antibody was sequenced by Kingchi corporation and the corresponding amino acid sequences were obtained by aligning the International immunogenetics database (IMGT) with the Sanger sequencing to obtain the nucleic acid sequences. The sequencing result is as follows: see SEQ ID NO 5-20 for details.

6.Alexa Fluor ^TM 488 fluorescein labeled 2D11 monoclonal antibody

Alexa Fluor, Invitrogen corporation was used ^TM The 488 antibody labeling kit (cat # A10235) labeled 500. mu.l of 2D11 monoclonal antibody at a concentration of 2 mg/ml. The labeled eluate was subjected to absorbance measurement at 280nm and 494nm (A280 and A494) to calculate the antibody concentration M and the labeling efficiency C, M ═ A280-A494X 0.11]203,000, C is A494 × dilution factor/71,000 × M. By calculation, the concentration of the antibody after labeling was 1mg/ml, the labeling efficiency C was 5.8, and 5.8 fluorescein was labeled on average per antibody.

Example five: application of 2D11 monoclonal antibody

1. Flow cytometry detection applied to human decidua NK cell transcription factor PBX1

The decidua tissue of the early pregnancy of the volunteer, the normal person was taken, the residual blood clot and the like were removed in PBS, the tissue was minced in a culture medium, the tissue was digested to chyle form with collagenase IV (Sigma) having a final concentration of 5mg/ml, the cell suspension was filtered through a 200-mesh sieve to a 50ml centrifuge tube, the precipitate was collected by centrifugation, blood was separated by human lymphocytes (Solebao, Cat. No. P8610) and mononuclear cells of the decidua tissue of the tunica albuginea were collected by density gradient centrifugation. The normal human peripheral blood mononuclear cells are used as a control to carry out immunizationDetecting epidemic blotting, and respectively taking 5 × 10 ⁶ Extracting nucleoprotein from decidua tissue mononuclear cells and peripheral blood mononuclear cells, performing 4-20% SDS-PAGE electrophoresis on 20 mu l of nucleoprotein, transferring the target protein to a PVDF membrane with the thickness of 0.22 mu m by a wet transfer method, wherein the operation of the immunoblotting experiment is consistent with that of the 293T cell detection method. As shown in fig. 6A, immunoblotting identified that there were specific bands at the relative molecular masses of 47kDa (PBX1a) and 38kDa (PBX1b) in decidua tissue mononuclear cells and peripheral blood mononuclear cells, the dominant band was 38kDa, and the 2D11 monoclonal antibody could specifically bind to PBX1a and PBX1b, i.e., in the present disclosure, the monoclonal antibody could specifically bind to PBX 1.

NK cell PBX1 expression was further detected in monocytes by flow cytometry. 1 x 10 ⁶ Each mononuclear cell was suspended in 100. mu.l of PBS, labeled to identify surface molecules of human NK cells, including CD3, CD45, CD56 and the like, incubated at 4 ℃ for 30 minutes, washed away with non-specifically bound antibodies by PBS, and fixed-membrane-penetrating kit (cat 00-5523-00) using a transcription factor of eBioscience, after which the Alexa Fluor obtained in example four was diluted with a washing solution of the kit ^TM The 488-labeled 2D11 monoclonal antibody was raised to 20. mu.g/ml, incubated at 4 ℃ for 30 minutes, and after washing away the non-specifically bound antibody with PBS, it was detected using a flow cytometer. As shown in FIG. 6B/C, CD3 ^- CD45 ⁺ CD56 ⁺ Human decidua tissue NK cell high expression transcription factor PBX1, and statistical results show that PBX1 ⁺ The decidua NK (dNK) cell proportion (96.7) is significantly higher than the peripheral blood NK (pNK) cell proportion (1.96).

2. Application to flow cytometry for diagnosing recurrent abortion with unknown cause

Taking normal human decidua tissue NK cells as a control, taking early pregnancy decidua tissues of patients with unexplained recurrent abortion, and performing flow cytometry on 12 cases in total by using a 2D11 monoclonal antibody to detect the expression condition of a transcription factor PBX 1. The experimental operation is consistent with the detection method of normal human dNK cells. The results are shown in FIG. 7A/B, and it can be seen from the combination of statistics that PBX1 has significantly reduced expression in NK cells of decidua prolifera tissue of unknown cause recurrent abortion patients, and that dNK cells (PBX 1) expressing PBX1 ⁺ dNK cells) ratio and number were significantly reduced in patients。

The expression level of the transcription factor PBX1 gene in decidua prolifera tissue of 50 patients with recurrent abortion (including 27 patients with unexplained recurrent abortion and 23 patients with recurrent abortion due to embryonic chromosome abnormality) was further determined by fluorescence quantitative PCR using 20 normal decidua tissues as controls. Specifically, the tissue was crushed by liquid nitrogen grinding, the ground tissue was dissolved with 1ml of Trizol (Invitrogen, cat # 15596026), total RNA was extracted with 200. mu.l of chloroform, the supernatant was collected by high-speed centrifugation, mixed with an equal volume of isopropanol, RNA was precipitated on ice for 30 minutes, RNA precipitate was collected by high-speed centrifugation, the precipitate was washed with 75% ethanol solution, and finally dissolved in DEPC-treated water. The RNA was reverse-transcribed into cDNA using M-MLV kit (Invitrogen, cat # 28025-013) as a template for subsequent quantitative fluorescent PCR detection. A20. mu.l reaction system was prepared by performing fluorescent quantitative PCR using a dye-based fluorescent quantitative kit (TaKaRa Co., Ltd., product No.: RR820A) using the ecdysone tissue cDNA as a template and the sequences shown in SEQ ID NO:21 and SEQ ID NO:22 as the forward and reverse primers of the fluorescent quantitative PCR, respectively. Pre-denaturation at 94 ℃ for 30 seconds; denaturation at 94 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, and 40 cycles as reaction conditions, and detection was performed using a LightCycler96 real-time fluorescent quantitative PCR instrument from Roche. Use 2 ^-ΔΔCt The results of the analysis and non-paired t-test statistical method on the data are shown in FIG. 7C, and the combination of statistics shows that PBX1 gene is present in all patients with recurrent abortion of unknown origin (p)<0.0001) and partial embryo chromosome abnormality recurrent abortion (p ═ 0.0001) were both significantly reduced in the decidua of early pregnancy. The correlation between the relative expression level of PBX1 gene and the occurrence of diseases in decidua tissue of patients with undetermined recurrent abortion, patients with abnormal fetal chromosomes recurrent abortion and two parts of patients with recurrent abortion was analyzed by the receiver operating characteristic curve (ROC curve for short), and the results are shown in FIGS. 7D-E, the reduction of PBX1 expression can be very effectively used for clinical diagnosis of undetermined recurrent abortion (in the undetermined recurrent abortion group, AUC is 0.941 (95% confidence interval is 0.8601-1.021, p is 95% confidence interval)<0.0001), higher than that in the group of recurrent abortion of an embryo chromosomal abnormality, AUC ═0.762 (95% confidence interval 0.6167-0.9072, p-0.003361)), effectively predicting the occurrence of recurrent abortion (overall recurrent abortion, AUC-0.859 (95% confidence interval 0.7731-0.9439, p)<0.0001)). In conclusion, decreased expression of decidua tissue PBX1 may be useful in diagnosing recurrent abortion, especially in cases of unexplained recurrent abortion; the 2D11 monoclonal antibody can further specifically analyze the expression level of a dominant immune cell NK cell transcription factor PBX1 in decidua tissue through flow cytometry, provides accurate diagnosis basis for patients with recurrent abortion, particularly patients with unexplained recurrent abortion due to multiple pregnancy failures, and provides possibility for subsequent targeted treatment.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof comprising:

the heavy chain variable region CDR-H1 with amino acid sequence SEQ ID No. 6;

the heavy chain variable region CDR-H2 with the amino acid sequence of SEQ ID No. 8;

heavy chain variable region CDR-H3 with amino acid sequence SEQ ID No. 10;

light chain variable region CDR-L1 with amino acid sequence SEQ ID No. 12;

light chain variable region CDR-L2 with amino acid sequence SEQ ID No. 14; and

light chain variable region CDR-L3 with amino acid sequence SEQ ID No. 16;

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region CDR-H1 encoding sequence is SEQ ID No. 5;

the coding sequence of the heavy chain variable region CDR-H2 is SEQ ID No. 7;

the coding sequence of the heavy chain variable region CDR-H3 is SEQ ID No. 9;

the coding sequence of the light chain variable region CDR-L1 is SEQ ID No. 11;

the coding sequence of the light chain variable region CDR-L2 is SEQ ID No. 13; and

the coding sequence of light chain variable region CDR-L3 is SEQ ID No. 15.

3. A monoclonal antibody or antigen-binding fragment thereof comprising:

a heavy chain variable region with an amino acid sequence of SEQ ID No. 18; and

the variable region of the light chain with the amino acid sequence of SEQ ID No. 20;

4. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein

The coding sequence of the heavy chain variable region is SEQ ID No. 17; and

the coding sequence of the light chain variable region is SEQ ID No. 19.

5. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1-4, which is capable of specifically binding to PBX1a and/or PBX1b protein.

6. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, having an Ig subclass of IgG _2a ，κ。

7. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a humanized antibody, a chimeric antibody, a single chain antibody, or a diabody.

8. The monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, which is secreted by a hybridoma cell line deposited in China center for type culture Collection with the collection number of CCTCC number C201912.

9. A conjugate comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-8 and a label bound thereto that allows detection thereof, wherein the label is selected from one or more of a fluorophore, biotin, a radioisotope, a metal, and an enzyme.

10. The hybridoma cell strain is preserved in China center for type culture Collection with the preservation number of CCTCC number C201912.

11. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the conjugate according to claim 9 in the preparation of a kit or medicament for detecting protein expression of PBX1a and/or PBX1 b.

12. An immunoassay kit comprising a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-8 or a conjugate according to claim 9.

13. The immunoassay kit of claim 12, wherein the sample to be tested is a plasma sample.

14. The immunoassay kit of claim 12 or 13, wherein the sample to be tested is of human origin.

15. The immunoassay kit of any one of claims 12-14, wherein the sample tested is decidua prematurely tissue NK cells from a recurrent abortion patient.

16. The immunoassay kit of claim 15 wherein the sample tested is a patient with recurrent abortion of unknown cause.

17. Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-8 or a conjugate according to claim 9 in the preparation of a diagnostic agent for recurrent abortion.

18. The use of claim 17, wherein the recurrent abortion is a recurrent abortion caused by an unexplained recurrent abortion or an embryonic abnormality.

19. A kit for aiding in the diagnosis of unexplained recurrent spontaneous abortion comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8 or the conjugate of claim 9.