CN111434686A

CN111434686A - Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion

Info

Publication number: CN111434686A
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: 2020-07-21
Anticipated expiration: 2039-01-15
Also published as: CN111434686B

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

The current research shows that about 50 percent of patients are clinically difficult to diagnose the cause (Qiagen (2018) J.C. J.J. and J.C. J.J.J.C. 131 page 12) and clinically show that the recurrent abortion is caused by unknown reasons, and the current research shows that the recurrent abortion is mainly related to the immune dysfunction of decidua NK cells (Fu B, ZHOU Y et al (2017) Immunity; 47(6) PBX 1100-1113.e6. and Fu B, L i X et al (2013) Proc Natl Acad U S.A.; 110 (633) E231-40) and the NK cells are used as the immune cells of the maternal interface main body, and bear important physiological functions, such as participation in immune cell induction, promotion of arterial wall, promotion of fetal spiral invasion, placental cell induction, placenta hominis cell recruitment and placenta induction, placenta hominis cell recruitment (20131) and placenta hom cell recruitment, and placenta hom cell recruitment and placenta recruitment (21. sub.C. 21. the like).

The transcription factor PBX1, also known as CAKUHED, PR L, encoded by the pre-B-cell leukemia homeobox gene (pre-B-celeukemia homeobox 1, PBX1), 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 patients with pre-B cell leukemia (Kamps MP et al (1990) cell.; 60(4):547-55), and later studies considered to play an important role in embryonic development, organogenesis and cellular differentiation, and in relation to various malignancies, the gene of human PBX1 was localized at 1q23, consisting of 9 exons and 8 introns, its encoded protein has a relative molecular weight of 47, consisting of 430 amino acids, while there are two major isoforms PBX1 kD a (430aa, 47) and 1B (PBX 347aa,38kD), wherein the 1 st to 333 amino acids in PBX1 PBX 2 are completely identical to those of the homologous isoforms, and the structural domains of the

PBX

3, 23, 493, 23, 3, three homologous domains are involved in the structural domains.

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 of recurrent abortion (unexplained recurrent abortion) related to 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:

the heavy chain variable region CDR-H1 having the amino acid sequence of SEQ ID No. 6, preferably, the coding sequence thereof is 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;

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 light chain variable region CDR-L1 with amino acid sequence SEQ ID No. 12, preferably, the coding sequence is SEQ ID No. 11;

the light chain variable region CDR-L2 having the amino acid sequence of SEQ ID No. 14, preferably, the coding sequence thereof is SEQ ID No. 13, and

the light chain variable region CDR-L3 having the amino acid sequence of SEQ ID No. 16, preferably, the coding sequence thereof 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 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 invention provides a kit for aiding in the 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.

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 of 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 results of the E L ISA test on serum before and after the rat immunized with the PBX1b recombinant protein.

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.A L EXA F L UOR^TMThe 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⁺Statistical plots of the proportion of NK cells in dNK and pNK cells, respectively.

FIG. 7A L EXA F L UOR^TMThe 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 fluorescent quantitative PCR detection of PBX1 gene expression in decidua tissue of patients with recurrent spontaneous 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 profile of the subjects' performance in recurrent miscarriage with decreased PBX1 expression.

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 antihuman 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 pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain, the light chains of the antibody may be classified as kappa and lambda light chains, the heavy chains may be classified as mu, gamma, α or, 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 linked 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, the heavy chains each consist of a heavy chain variable region (VH) and a heavy chain constant region (CH), the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3), the light chains each light chain consists of a light chain variable region (V L) and a light chain constant region (C L), the light chain constant regions consist of one domain C L may mediate binding of the immunoglobulin to host tissues or factors, including the constant regions of the light chains (C L, V constant regions) and light chains, the constant regions (C3626, VH, constant regions, V, CDR3, CDR 12, CDR 21, CDR3, CDR 7, CDR 27, CDR3, CDR 7, CDR3, CDR 9, CDR 27, CDR 18, CDR 27, CDR 9, CDR 35, CDR 27.

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 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 an antibody that is homogeneous 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 "chimeric or fused antibody polypeptides" for example by replacing homologous murine sequences with human heavy and light chain constant domain (CH and C L) sequences (US 4816567; and Morrison et al (1984) Proc. Natl Acad. Sci. USA.; 81:6851), or by fusing immunoglobulin coding sequences to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). the non-immunoglobulin polypeptide sequences may replace the constant domains of the antibody, or replace the variable domains of one antigen binding site of the antibody with them to create a chimeric bivalent antibody comprising an antigen binding site specific for one antigen and another antigen binding site specific 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 nonhuman 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 moleculeSimilar to the target, e.g., excess 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^-6M, e.g. less than about 10^-7M、10^-8M、10^-9M or 10^-10M or less binds to an epitope of the antigen with an affinity (KD).

"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 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_2aAnd κ. In one embodiment, any of the aboveA monoclonal antibody or an antigen-binding fragment thereof 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 as described above is secreted by hybridoma cell line 2D11 which is deposited in the chinese type culture collection with the collection number CCTCC No. c 201912.

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 line produces a monoclonal antibody or antigen-binding fragment thereof against the 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 any of the conjugates as described above for detecting protein expression of PBX1a and/or PBX1b in one embodiment, the sample being tested is a plasma sample in one embodiment, the sample being tested is of human origin in one embodiment, the sample being tested is decidua prematurely pregnant tissue NK cells of recurrent abortion patients, preferably patients with unexplained recurrent abortion in one embodiment, the method of testing is with any of the monoclonal antibodies or antigen-binding fragments thereof as described above or any of the conjugates as described above as an antibody in one embodiment, the method of testing is with immunoblotting to detect human cell transcription factor PBX1 in one embodiment, the fluorophore in the conjugate is labeled fluorescein a L EXAF L uortm488 in one embodiment, the method of testing is with flow cytometry to detect human cell 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 preparation of a diagnostic agent for the aided diagnosis of unexplained recurrent abortion, in one embodiment, the sample for aided diagnosis is decidua tissue NK cells of the early pregnancy of an unexplained recurrent abortion patient in one embodiment, the fluorophore in the conjugate is labeled fluorescein a L EXA F L uortm488 in one embodiment, the method of diagnosis is to detect the transcription factor PBX1 of the decidua tissue NK cells of the early pregnancy of an unexplained recurrent abortion patient by flow cytometry, and further to diagnose whether the cause of the disease is low expression of the transcription factor PBX1, NK cell abnormality.

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

The general biosystems (Anhui) Limited company is entrusted with artificially synthesizing a full-length human transcription factor PBX1b gene (NCBIReference Sequence: NM-001204961.1, encoding amino acids 1 to 347), 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 end, the C end is directly cloned to a recombinant expression vector pET21A (Shanghai Bionical Co., Ltd.) through NdeI and XhoI enzyme cutting sites, the obtained recombinant expression vector is transferred into DH5 α competent cells, and prokaryotic clones are screened for DNA sequencing identification, and the sequencing result shows that the construction of the recombinant expression vector pET21A-PBX1b is successful, and the construction flow of the recombinant expression vector pET 21A-1 b is shown in a figure 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, Excellent medical equipment, Inc., Hunan-Ke-Ai), the product number is VT 1B, the obtained recombinant expression vector is transferred into a DH5 α competent cell, positive clones are screened for DNA sequencing identification, and the sequencing result shows that the recombinant expression vector pPBX1B is successfully constructed, and the construction process of the eukaryotic recombinant expression vector pPBX1B is shown in figure 1B.

Example two: 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 (Beijing Pan-jin Biotechnology Co., Ltd., product No. CD801), heat-shocked at 42 ℃ for 90 seconds, allowed to stand on ice for 2 minutes, spread on L B solid plates containing ampicillin at a final concentration of 50. mu.g/m L and chloramphenicol at 34. mu.g/m L, and cultured overnight at 37 ℃.

1.2 selection of optimal Induction conditions in Small-scale cultures

Selecting single colony of recombinant protein expression strain Transetta (DE3) PBX1B in 10m L L B culture medium (containing 50 ug/m L ampicillin and 34 ug/m L chloramphenicol) at 37 deg.C and 220 rpm, shaking overnight, inoculating overnight cultured bacterial liquid in 10m L L B (containing 50 ug/m L ampicillin and 34 ug/m L chloramphenicol) culture medium at 1:100 ratio, shaking at 37 deg.C and 220 rpm, and culturing while OD is not changed_600nmWhen 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. Centrifugally collecting thallus, ultrasonic breaking and cracking thallus, centrifugally collecting supernatant and precipitate, and depositingDissolving the mixture by using 500 mu L inclusion body dissolving solution (8Murea, 50mM Tris-HCl, 300mM NaCl, pH8.0), respectively taking 40 mu L samples to carry out 12% SDS-PAGE electrophoresis detection, wherein the loading amount is 10 mu L, after the gel electrophoresis is finished, staining the samples by Coomassie brilliant blue for 20 minutes, and imaging after decoloration, as shown in figure 2A, compared with other control conditions, the supernatant of IPTG induced bacteria lysate has a remarkable band at 38kDa, and the 20 ℃ induction overnight condition is better than the 37 ℃ induction for 4 hours.

1.3 Mass inducible expression of fusion proteins

According to the results of the 1.2 small test, it was finally confirmed that 20 ℃ induction overnight was selected, and the uninduced monoclonal inoculum used in the 1.2 small test was inoculated in L B resistant medium 4L at a ratio of 1:100 for mass induction expression.

2. Purification of fusion proteins

The lysate precipitate induced in the 1.3 step was collected by centrifugation, the lysate precipitate was sonicated in ice bath, the lysate supernatant was collected after centrifugation and purified by nickel agarose affinity chromatography, 5M L Ni-IDA was taken, the equilibrated column was washed with 10 column volumes of equilibration solution (1M Tris, 150mM NaCl, pH8.0) at a flow rate of 5M L/min, the lysate supernatant obtained in the two steps 1.3 step of the above example was applied at a flow rate of 2M L/min, the permeate was collected as a control, the column was washed with 10 column volumes of equilibration solution at a flow rate of 5M L/min, the eluate was washed with 20/50mM Imidazole (1M Tris, 150mM NaCl, 20/50mM Imidazole, pH8.0) at a flow rate of 5M L/min, the eluate was collected as a control, the eluate was finally dialyzed with 500mM Imidazole solution (1M, 150mM NaCl, 500mM pH8.0), the eluate was collected as a control, the eluate was concentrated with a flow rate of 150mM Imidazole, the target protein was analyzed by centrifugation at a flow rate of 150mM Tris, the target protein, the Tris was collected as a comparative example Tris buffer, the target protein was collected as a dialysis buffer, the supernatant was concentrated by centrifugation at a flow rate of 500mM Tris buffer (150 mM Tris, the concentration of 150mM Tris buffer, the target protein was collected as shown in 150mM Tris buffer, the concentration of 500mM Tris buffer, the concentration of 150mM Tris buffer, the target protein was obtained in 150mM Tris buffer, the concentration of 150mM Tris buffer, the target protein was obtained in 150mM Tris buffer, the concentration of 150 mM.

3. Identification of recombinant PBX1b protein

The concentration and purity of the target protein purified in the second step 2 of example were measured using a non-interfering protein concentration measurement kit (Shanghai Biotech Co., Ltd., cat # C503071) to obtain a protein having a purity of about 90% and a concentration of 0.5mg/m L, and the target protein was subjected to immunoblotting, in which rabbit anti-His-tag antibody (CST Co., cat #2365)1:1000 was diluted as a primary antibody, horseradish peroxidase-labeled goat anti-rabbit IgG (Boster Co., cat # BA1054)1:5000 was diluted as a secondary antibody, and subjected to chemiluminescence (Thermo scientific, cat # 34080) detection, as shown in FIG. 2C, indicating that the protein induced to be expressed and purified in the second step 2 of example was recombinant PBX1b protein.

Example three: preparation of monoclonal antibodies

1. Immunized rats, cell fusion and monoclonal screening

1.1 preparation of rat immune and splenocytes

The purified human PBX1b recombinant protein with the final concentration of 100 mug/ml obtained in the example II and an equal volume of complete Freund's adjuvant are fully mixed into a water-in-oil state, primary immunization is carried out on 5 female SD rats (respectively marked as 1#, 2#, 3#, 4#, and 5#) with the age of 8 weeks through subcutaneous multipoint injection at the back, 100 mug of PBX1b recombinant protein is injected into each rat, then, 50 mug of human PBX1b recombinant protein is mixed with incomplete Freund's adjuvant every 15 days, subcutaneous immunization is carried out once, repeated immunization is carried out for three times, total 45 days, after 15 days, 50 mug of human PBX1b recombinant protein and incomplete Freund's adjuvant are injected into the abdominal cavity, 10 days after the last immunization, tail vein blood is taken, the serum titer is indirectly measured through E L, and as shown in figure 3, 2 persons with the highest antiserum are selected, and 50 mug of human PBX1b protein ISA is injected into the abdominal cavity for strengthening immunization on 15 days after vein blood taking.

The method for detecting antibody titer by E L ISA comprises diluting the purified protein obtained in example two with coating solution (0.1M carbonate buffer, pH 9.6) to a final concentration of 10. mu.g/ml, coating a 96-well microplate with 100. mu.l/well, standing overnight at 4 deg.C, washing 3 times with TBST (50mM Tris-HCl, 150mM NaCl, 0.05% Tween20, pH 7.5), blocking with 1% BSA, incubating at 37 deg.CAnd raising 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, and reading absorbance (OD 405 nm) of light₄₀₅)。

1.2 cell fusion

3 days after the boosting, splenocytes from the boosted mice obtained in example three were aseptically taken and fused with myeloma cells SP2/0(ATCC No. CR L-1581) in logarithmic growth phase by the conventional PEG method, and the prepared 10 cells were washed with incomplete RPMI-1640 medium⁸Spleen cells and 2.5 × 10⁷Myeloma cells SP2/0, centrifuged to remove supernatant, gently flicked, added 0.7ml of 40 ℃ PEG (molecular weight: 1,000-6,000) solution at a final concentration of 50% (W/V) PEG, 60 seconds later, initially added incomplete RPMI-1640 medium (5 minutes later), 1ml first, 4ml after 1 minute later, 20ml after 2 minutes later, 300g at 4 ℃ for 10 minutes to collect cells, gently suspended in 37 ℃ incomplete RPMI-1640 medium containing 2 × HAT (containing hypoxanthine, aminopterin and thymidine) to give a final concentration of 2 × 10⁶And/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 fused cells (i.e., hybridoma cells) were spread across the area of the well bottom 1/10. Enzyme label coated by human PBX1b recombinant proteinPlates were screened for positive clones by the indirect E L ISA 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 preparation of the D11 monoclonal antibody

The 2D11 monoclonal antibody is prepared by adopting a nude mouse abdominal cavity inoculation method, firstly, the nude mouse of 8 weeks old is injected with 500 mul sterile liquid paraffin in the abdominal cavity, and after one week, the nude mouse is injected with 1 × 10 in the abdominal cavity⁶And (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. The potency of the 2D11 monoclonal antibody measured by indirect E L ISA is about 10^-9。

2. The 2D11 monoclonal antibody has an affinity constant of 6.37 × 10 determined by competitive E L ISA⁸L/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 of each well was coated with 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. The antigen was mixed with the supernatant of hybridoma 2D11 at each concentrationMu.l of the incubation solution 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 (1MHCl) was added thereto, and OD was performed immediately after the termination with a microplate reader₄₅₀And OD₆₃₀The antibody affinity constant calculation formula is A0/(A0-A) ═ 1+ Kd/a0, wherein A0 is the OD value when the competitive antigen is 0, A is the OD value of each antigen concentration, a0 is the total amount of antigen, Kd is the dissociation constant, K is found from K ═ 1/Kd, K is the affinity constant, unit L/M, the calculated affinity constant is 6.37 × 10⁸L/M。

Determination of Ig subclass of the 2D11 monoclonal antibody, 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

Identification of the specificity of the 2D11 monoclonal antibody immunoblotting and flow cytometry were used.

Human embryonic kidney 293T cells (ATCC accession number CR L-3216) express the transcription factor PBX1, a PBX1b knockdown (PBX1-K.D.)293T cell line was established by siRNA-1(SEQ ID NO:3) and siRNA-2(SEQ ID NO:4) targeting the PBX1b gene (synthesized by Shanghai Gilma), the expression of PBX1b was restored by transfecting 1. mu.g and 5. mu.g of pPBX1b overexpressing plasmid into PBX1-K.D.293T cell line, the expression of PBX1b was restored by immunoblotting (FIG. 5A), the nuclear protein of 293T cells, PBX1 b-K.293T cells, PBX1b-K.D.293T cells expressing PBX1b were restored, the specificity of 2D11 monoclonal antibody was identified, the nuclear protein of above grouped cells extracted was washed with 20. mu.l of PBX 4-20%, the protein was recovered by blotting with a wet SDS-5000. mu.D.293T 3, the antibody was recovered by washing with a reagent after the supernatant of the supernatant, the supernatant of the bovine fetal kidney 293T cells was recovered by blotting antibody, the supernatant was recovered by blotting antibody, the supernatant after the supernatant was diluted with a supernatant of the bovine serum albumin, the supernatant was recovered by the supernatant, the antibody was recovered by the supernatant after the supernatant, the antibody was diluted with a supernatant, the antibody was recovered by the antibody was diluted with a supernatant, the antibody was recovered by the antibody, the antibody was recovered by the antibody was diluted with a supernatant after the antibody, the antibody was recovered by the antibody was diluted with a supernatant after the antibody, the antibody.

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). Above groups are each 10⁶After the cells were fixed and permeabilized with the use of the transcription factor fixed-permeabilization kit (cat No. 00-5523-00) from eBioscience, the 2D11 monoclonal antibody was diluted to 20 μ g/ml with a kit washing solution, incubated at 4 ℃ for 30 minutes, and after the unspecifically bound antibodies were washed away with PBS, 1:500 diluted Alexa Fluor 647-labeled goat-anti-rat secondary antibody (AF647) (Bio L egend, cat No. 405416) was incubated at 4 ℃ for 30 minutes, and then washed with PBS and detected with the use of the flow cytometer L SR II 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 of 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^TM488 fluorescein labeled 2D11 monoclonal antibody

Alexa Fluor, Invitrogen corporation was used^TM488 antibody labeling kit (cat # A10235) labels 500. mu.l of 2D11 monoclonal antibody at a concentration of 2mg/ml the labeled eluate, and the antibody concentration M and labeling efficiency C were calculated by measuring the absorbance of solutions at 280nm and 494nm (A280 and A494), M ═ A280-A494 × 0.11.11)]The concentration of the antibody after being labeled is 1mg/ml, the labeling efficiency is 5.8, and 5.8 fluorescein labels are marked on each antibody on average.

Example five: application of 2D11 monoclonal antibody

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

Collecting decidua tissue from early pregnancy of a person who wishes to terminate pregnancy, removing residual blood clot in PBS, cutting the tissue in culture medium, digesting the tissue with collagenase IV (Sigma) with a final concentration of 5mg/ml to chyle, filtering the cell suspension with 200 mesh sieve to 50ml centrifuge tube, centrifuging, collecting precipitate, separating blood (Solebao, Cat. No. P8610) by human lymphocyte, performing density gradient centrifugation, collecting leukocyte of decidua tissue, performing immunoblotting with monocyte of peripheral blood of a person as control, and collecting 5 × 10⁶Extracting 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.

Further detecting NK cell PBX1 expression in mononuclear cells by flow cytometry 1 × 10⁶Each 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^TMThe 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⁺Decidua NK (dNK) cellsThe ratio (96.7) is significantly higher than the peripheral blood nk (pnk) cell ratio (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.

Using 20 normal human decidua tissues as a control, further detecting the change of the expression level of the transcription factor PBX1 gene in the decidua tissue of 50 recurrent abortion patients (including 27 patients with recurrent abortion due to unknown reasons and 23 patients with recurrent abortion due to abnormal embryo chromosome) by fluorescence quantitative PCR, the concrete steps are grinding and crushing the tissue by liquid nitrogen, dissolving the ground tissue by 1ml Trizol (Invitrogen, Cat. No.: 15596026), extracting the total RNA therein by 200. mu.l chloroform, centrifuging at high speed, mixing the supernatant with equal volume of isopropanol, precipitating the RNA on ice for 30 minutes, centrifuging at high speed, washing the RNA precipitate with 75% ethanol solution, finally dissolving in DEPC-treated water, reverse transcribing the RNA into cDNA by using M-M L V kit (Invitrogen, Cat. No.: 28025-013), preparing the subsequent fluorescence quantitative PCR detection template by fluorescence quantitative PCR using dye method fluorescence quantitative kit (TaRa, Cat. No.: 96 0), performing fluorescence quantitative PCR using SEQ ID PCR as a template, performing real-time PCR by PCR and denaturation by using SEQ ID primer system of 20 ℃ 20. mu.C. 20. mu.C., annealing by 20. mu.C. 20. PCR, and 20. PCR using fluorescence quantitative PCR^-ΔΔCtThe results of the analysis and non-paired t-test statistical methods 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 the group of recurrent abortion with abnormal embryo chromosome, AUC 0.762 (95% confidence interval 0.6167-0.9072, p 0.003361)), and is effective for predicting recurrent abortion (whole recurrent abortion, AUC 0.859 (95% confidence interval 0.7731-0.9439, p)<0.0001)). In conclusion, the reduced expression of decidua tissue PBX1 can be effectively used for diagnosing recurrent abortion, especially the cases of recurrent abortion of unknown cause; the 2D11 monoclonal antibody can further specifically analyze the expression quantity of PBX1 of the dominant immune cell NK cell transcription factor in decidua tissue through flow cytometry, provides accurate diagnosis basis for patients with recurrent abortion, particularly patients with recurrent abortion caused by unknown reasons due to multiple pregnancy failures, and provides possibility for subsequent targeted treatment.

Claims

1. 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;

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

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

3. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1-2, which is capable of specifically binding to PBX1a and/or PBX1b proteins.

4. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3, having the Ig subclass of IgG_2a，κ。

5. 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.

6. 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 having a collection number of CCTCC NO. C201912, which is collected in China center for type culture collection.

7.A conjugate comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 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.

8. The hybridoma cell strain is preserved in China center for type culture Collection with the preservation number of 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 for detecting protein expression of PBX1a and/or PBX1 b.

10. An immunoassay kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the conjugate according to claim 7.

11. The immunoassay kit of claim 10, wherein the sample detected is a plasma sample.

12. The immunoassay kit of claim 10 or 11, wherein the sample detected is of human origin.

13. The immunoassay kit of any one of claims 11-11, wherein the sample tested is decidua prolifera NK cells of recurrent abortion patients, preferably patients with unexplained recurrent abortion.

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 for the preparation of a diagnostic agent for recurrent abortion, preferably the recurrent abortion is one caused by an unexplained recurrent abortion or an embryonic abnormality.

15. 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 1-6 or the conjugate of claim 7.