Monoclonal antibody against human carbohydrate antigen CA125 and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human carbohydrate antigen CA125 monoclonal antibody and application thereof. The amino acid sequences of CDR1-3 on a light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on a heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.8-10. The monoclonal antibody provided by the invention can specifically recognize and bind to a natural carbohydrate antigen CA125 expressed by human cells or tissues, has good affinity and relatively high sensitivity of binding of the antibody and the antigen, has strong anti-interference ability to complex non-target protein components in the cells / tissues, is beneficial to significantly improving the accuracy and reliability of immunodetection of human CA125 protein, and has good application prospects. The method is suitable for high-specificity, high-sensitivity and high-reliability detection of the human CA125 protein, and has good applicability in a plurality of detection systems such as immunoblotting, immunoprecipitation, immunohistochemistry and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, in particular to a monoclonal antibody against human carbohydrate antigen CA125 and applications thereof. Background Art

[0002] Carbohydrate antigen CA125 (CA125), also known as cancer antigen 125 and mucin 16 (MUC16), is encoded by the MUC16 gene in the human chromosome 19p13.3 region and is the glycosylated cell membrane glycoprotein with the highest molecular weight in the mucus glycoprotein family. CA125 has a large and complex structure, consisting of a protein backbone of 14,507 amino acid residues. Its secondary structure is primarily composed of the following components: a) Extracellular tandem repeats: The CA125 protein core contains multiple tandem repeats rich in serine, threonine, and proline, providing abundant potential sites for O-glycosylation; b) The N-terminal domain: Contains numerous N-glycosylation sites and is the primary attachment point for sugar chains within the CA125 molecule; c) The C-terminal domain: Contains a transmembrane domain and an intracellular domain. The transmembrane region is anchored to the cell membrane, while the intracellular region contains phosphorylation sites that mediate CA125-related intracellular signaling and are involved in the shedding of CA125 from the cell membrane. CA125 has a wide molecular weight distribution range (200-1000 kDa), which is related to the complexity of its sugar chain structure and the degree of glycosylation modification. Furthermore, the rich sugar chain structure of CA125 is highly heterogeneous, which contributes to the diversity of CA125 in terms of immunogenicity and biological function. Under normal physiological conditions, human luminal epithelial cells, such as the pleura, pericardium, peritoneum, endometrium, endocervical lining, and normal lung tissue, including columnar epithelial cells and glands of the respiratory mucosa, secrete CA125, forming a protective barrier on the cell surface to prevent mechanical damage and pathogen invasion. CA125 can also interact with cell surface receptors such as mesothelin and selectins through its sugar chain structure, participating in biological processes such as intercellular adhesion and migration, signal transduction, and cell proliferation.

[0003] CA125 is a tumor marker that can be elevated in a variety of pathological conditions, including both benign and malignant conditions. It is currently the most widely used tumor marker for ovarian cancer in clinical practice. Serum CA125 levels are often significantly elevated in ovarian cancer patients, particularly in serous ovarian cancer, where the positive rate can reach over 80%. In clinical practice, CA125 immunoassays are often combined with imaging tests such as gynecological ultrasound for early screening, diagnosis, and disease monitoring of ovarian cancer. CA125 measurement is also crucial for assessing treatment response and monitoring recurrence in ovarian cancer patients. During surgery, chemotherapy, and other treatments for malignant tumors such as ovarian cancer, persistently elevated CA125 levels are often associated with progressive disease or poor treatment response, while decreasing levels indicate a favorable prognosis and effective treatment. Besides ovarian cancer, elevated CA125 levels have also been associated with other malignancies, including liver cancer, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, lymphoma, and certain gynecological tumors such as fallopian tube cancer and endometrial cancer. It has been demonstrated that CA125 can be used alone or in combination with other markers to diagnose these diseases.

[0004] Based on the outstanding characteristics of CA125 in clinical diagnosis, the development of a detection method for CA125 for clinical diagnosis and prognosis assessment has become an urgent need. At present, the detection method of CA125 is mainly based on immunoassay technology, which is based on the specific binding reaction of antigen and antibody, and is highly dependent on the antibody's immune recognition sensitivity and binding specificity to CA125. However, there are currently few highly specific anti-CA125 monoclonal antibodies on the market, and there are only a handful of antibodies that can simultaneously meet the detection needs of scientific research samples and pathological specimens for immunoassay techniques such as immunoblotting, immunoprecipitation, and immunohistochemistry. Therefore, the development of anti-human CA125 monoclonal antibodies that can efficiently and specifically bind to the human CA125 antigen and have good detection performance is the trend of the times. Summary of the Invention

[0005] To address the existing problems of a lack of monoclonal antibodies against the human carbohydrate antigen CA125 with high immune recognition sensitivity and / or strong binding specificity for use in immunoassays, the present invention provides monoclonal antibodies against the human carbohydrate antigen CA125, their encoding genes, and recombinant vectors. Furthermore, the present invention provides the use of the monoclonal antibodies or antibody conjugates thereof in the preparation of immunoassay kits for the human carbohydrate antigen CA125, as well as related immunoassay kits. To achieve the aforementioned objectives, the present invention is specifically implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides a monoclonal antibody against the human carbohydrate antigen CA125, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NOs. 3-5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NOs. 8-10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0010] The second aspect of the present invention provides a nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal antibody against the human carbohydrate antigen CA125 as described above.

[0011] Furthermore, the recombinant vector is a pBR322 expression vector.

[0012] The third aspect of the present invention provides the use of the above-mentioned monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate in the preparation of a human carbohydrate antigen CA125 immunoassay kit, wherein the antibody conjugate comprises the monoclonal antibody and a detection label connected to the monoclonal antibody.

[0013] In a fourth aspect, the present invention provides a human carbohydrate antigen CA125 immunoassay kit, which comprises the above-mentioned monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate, wherein the antibody conjugate comprises the monoclonal antibody and a detection label connected to the monoclonal antibody.

[0014] Furthermore, the immunoassay kit is selected from an immunoblotting kit, an immunohistochemistry kit or an immunoprecipitation kit.

[0015] The advantages and positive effects of the present invention are:

[0016] The monoclonal antibody provided by the present invention, which has the above-mentioned complementary determining region sequence, can specifically recognize and bind to the natural carbohydrate antigen CA125 expressed by human cells or tissues. The antibody has good affinity and high sensitivity for binding to the antigen, and has strong anti-interference ability against complex non-target protein components in cells / tissues. It is beneficial to significantly improve the accuracy and reliability of immunoassays for human CA125 protein, effectively avoid the generation of false positive or false negative results, and improve the degree of consistency between the test results and the actual situation. It is suitable for high-specificity, high-sensitivity and high-reliability detection of human CA125 protein, has good applicability in multiple immunoassay systems such as immunoblotting, immunoprecipitation, and immunohistochemistry, and has broad application prospects and good market potential in clinical diagnosis and scientific research testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a map of the expression vectors for constructing the monoclonal antibody against the human carbohydrate antigen CA125 in Example 1 of the present invention, including, from left to right, the pRB322 vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0019] Figure 2 This is a diagram showing the results of immunoblotting using a monoclonal antibody against human carbohydrate antigen CA125 binding to human positive cells OVCAR-3 and negative cells U-937 according to Example 2 of the present invention;

[0020] Figure 3 This is a diagram showing the immunoprecipitation results of Example 2 of the present invention using a monoclonal antibody against the human carbohydrate antigen CA125 combined with human positive cells OVCAR-3;

[0021] Figure 4 This is a diagram showing the immunohistochemical detection results of Example 2 of the present invention using a monoclonal antibody against human carbohydrate antigen CA125 in combination with human tonsil tissue and human serous ovarian cancer tissue sections. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0024] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

[0025] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those skilled in the art.

[0026] The terms "include", "comprising", "containing", "having" and the like are non-limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0027] The term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is considered to include the following situations: (i) A, (ii) B, and (iii) A and B.

[0028] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and similar expressions have synonymous meanings and, unless otherwise specified, refer to antibodies that specifically bind to the human carbohydrate antigen CA125. The terms "carbohydrate antigen CA125," "CA125," "CA125 / MUC16," "MUC16 / CA125," "MUC16," "mucin 16," and "cancer antigen 125" have synonymous meanings. The modifier "rabbit" indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences.

[0029] An antibody is an immunoglobulin molecule that is capable of specifically binding to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and encompasses various antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen-binding activity. Antibody fragments can be one or more portions or fragments of a full-length antibody that retain the antibody's ability to specifically bind to the target antigen.

[0030] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types: kappa (κ) and lambda (λ); heavy chains can be classified into five types: μ, δ, γ, α, and ε, which define antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of heavy and heavy chains vary greatly, while the remaining amino acid sequences are relatively constant. The regions of the light and heavy chains with the most variable amino acid sequences near the N-terminus are called the variable region (V), while the regions with relatively stable amino acid sequences near the C-terminus are called the constant region (C). The heavy chain variable region (VH) and light chain variable region (VL) are generally the most variable parts of antibodies and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs). The HVRs, also known as complementarity-determining regions (CDRs), are circular structures. The heavy and light chain CDRs are closely aligned and interact with each other through the FRs, forming a surface that complements the three-dimensional structure of the target antigen or epitope. This determines the antibody's specificity and is the site of antigen recognition and binding. The FRs are the more conserved portions of the VH and VL sequences. They generally follow a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop. Each VH and VL sequence typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, a cumulative of the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define CDRs.

[0032] The light chain constant region (CL) and heavy chain constant region (CH) are not directly involved in antibody-antigen binding, but they exhibit different effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). The CL length is generally consistent across different Ig types (κ or λ), but the CH length varies across different Ig classes. For example, IgG, IgA, and IgD comprise CH1, CH2, and CH3, while IgM and IgE comprise CH1, CH2, CH3, and CH4. The amino acid sequences of the heavy and light chain constant regions of antibodies are well known in the art and can be obtained by querying the IMGT database.

[0033] A full-length antibody is the most complete antibody molecular structure and has a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "intact antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0034] Antibody fragments are one or more parts or fragments of a full-length antibody that essentially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR regions as the full-length antibody, and more preferably the same variable regions, thereby retaining complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, particularly to the same epitope. Typical examples include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antibody fragments can be obtained using conventional techniques in the art.

[0035] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of the heavy chain (variable and first constant regions). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, IgG can be degraded into two Fab fragments and an Fc fragment by papain; and into an F(ab')2 fragment and a pFc' fragment by pepsin. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab contains both the antigen-binding region and a portion of the constant region, it not only possesses the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0036] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0037] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). The three CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the intact antibody.

[0038] (iv) (Fv)2: Consists of two covalently linked Fv fragments.

[0039] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (typically consisting of 10-25 amino acids). It retains the antigen-binding specificity of the original antibody. The linker in this invention is not particularly limited, as long as it does not hinder the expression of the antibody variable regions connected to it. Compared to full-length antibodies, scFv has a smaller molecular weight, resulting in higher penetration and lower immune side effects.

[0040] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected by a linker or the like.

[0041] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include CDR regions and FR regions from rabbit immunoglobulin sequences. In other embodiments, the antibody may contain amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which part of the antibody is derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" is a chimeric antibody with a CDR region of a non-human antibody such as a rabbit antibody and a FR region from a human. In some cases, the variable region of a non-human antibody is combined with the constant region of a human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is combined with the FR region and constant region derived from a human antibody sequence, that is, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, and this framework sequence is derived from the FR sequence of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric or humanized antibodies are derived from rabbit-derived CDR regions.

[0042] The terms "monoclonal antibody" or "single antibody" and other similar terms are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a small amount of mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0043] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer-lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0044] In order to make the objects and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0045] An embodiment of the present invention provides a monoclonal antibody against the human carbohydrate antigen CA125, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each include three complementarity determining regions (CDRs), respectively named CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0046] The antibody provided by the present invention can specifically recognize and bind to the human carbohydrate antigen CA125, is suitable for a variety of detection methods in the field of immunology, and has good applicability in multiple immune detection systems, particularly immunoblotting, immunoprecipitation and immunohistochemistry detection systems. Positive cells (OVCAR-3 cells expressing human CA125 protein) and negative cells (U-937 cells that do not express human CA125 protein) were detected by immunoblotting, and the target band was detected only in the cell lysate of the positive sample and was consistent with expectations. The OVCAR-3 cell line was detected by immunoprecipitation, and obvious specific bands were presented in the positive sample, and the detection signal of the immunoprecipitation group was significantly enriched compared with the input group. The above-mentioned test results show that the antibody of the present invention has good specificity and good affinity with the antigen CA125 protein. Further, multiple tissue samples were detected by immunohistochemistry, and it was seen that the stained parts in the positive tissue were accurate and free of nonspecific staining, the background interference was low, and the detection sensitivity and specificity could reach 100%.

[0047] Combined with the results of the above-mentioned immunoblotting, immunoprecipitation and immunohistochemistry, it is confirmed that the antibody prepared by the present invention having the above-mentioned CDR sequence can specifically recognize and bind to the natural CA125 protein expressed by human cells or tissues, the antibody has good affinity and high sensitivity for binding to the antigen, and has strong anti-interference ability against complex non-target protein components in cells / tissues, which is conducive to significantly improving the accuracy and reliability of immunodetection of human CA125 protein, effectively avoiding the generation of false positive or false negative results, and improving the degree of consistency between the test results and the actual situation. It is suitable for high-specificity, high-sensitivity and high-reliability detection of human CA125 protein, and has broad application prospects and good market potential in clinical diagnosis and scientific research detection.

[0048] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), wherein the four FRs and three CDRs are arranged in a staggered order to form a variable region. The amino acid sequence of the light chain variable region (VL) of the antibody of the present invention is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 7.

[0049] Optionally, the antibodies of the present invention further comprise a light chain constant region (CL) and a heavy chain constant region (CH), wherein the CL and VL constitute a complete light chain (FL), and the CH and VH constitute a complete heavy chain (FH). Antibody constant regions are generally available through public searches, such as searching the IMGT online database (www.imgt.org) for rabbit IgG gamma C reign to obtain CH, and searching for rabbit IgG kappa C reign to obtain CL.

[0050] Specifically, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6. Correspondingly, CL is of κ type and CH is of IgG type.

[0051] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0052] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal antibody against the human carbohydrate antigen CA125 as described above.

[0053] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0054] The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0055] The original vector for constructing the recombinant vector is a conventional various vectors in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., for transferring nucleic acid molecules into a host and multiplying them in large quantities in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecules inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, which is then introduced into the host cell and cultured under specific conditions to express the antibody. This is a well-known technology in the art and will not be described in detail here.

[0056] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and light chain are expressed in different host cells, each chain can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains are mixed and incubated under appropriate conditions to form antibodies. In other embodiments, the nucleic acid molecules of the monoclonal antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence linked to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a different promoter, or the nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a single promoter so that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0057] Conventional techniques are used to transfect or transform recombinant vectors into host cells. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2. Alternatively, transfection can be accomplished by microinjection, electroporation, or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or gene gun bombardment to achieve gene introduction.

[0058] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining a host cell transfected or transformed with the recombinant vector described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain purified antibodies.

[0059] In a preferred embodiment, the above-mentioned recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0060] In a typical embodiment, a method for preparing a monoclonal antibody comprises: concatenating the heavy and light chain genes of the antibody with a signal peptide, loading the genes separately into the expression vector pBR322, co-transfecting 293F cells, culturing and collecting the cell culture supernatant, and purifying the target antibody. The choice of signal peptide is designed based on the host cell and is not particularly limited in the present invention.

[0061] Yet another embodiment of the present invention provides the use of the above-mentioned monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate in the preparation of a human carbohydrate antigen CA125 immunoassay kit, wherein the antibody conjugate comprises the above-mentioned monoclonal antibody against human carbohydrate antigen CA125 and a detection label linked to the monoclonal antibody.

[0062] The advantages of using the monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate in preparing a human carbohydrate antigen CA125 immunoassay kit are the same as the advantages of the monoclonal antibody against human carbohydrate antigen CA125 over the prior art as described above, and will not be repeated here.

[0063] It should be emphasized that the antibodies of the present invention can be used alone or linked to a detection label (covalently or non-covalently) to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to CA125 in the sample to be tested, and then qualitatively or quantitatively detect CA125 by analyzing the detection label signal linked thereto; in other embodiments, the anti-carbohydrate antigen CA125 antibody is not labeled (as a primary antibody or capture antibody), and a secondary antibody or other molecule that can bind to the primary antibody is labeled and coupled (as a detection antibody). For example, if the anti-CA125 antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, thereby qualitatively or quantitatively detecting CA125 by analyzing the changes in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention, such as the multiple detection systems established in Example 2 below.

[0064] The detection marker is used to generate a recognizable signal change to identify the antibody of the present invention or its secondary antibody according to the signal change, and then identify the expression of CA125 in the sample to be tested through the specific reaction of the antigen and antibody. The detection marker includes but is not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0065] The above-mentioned immunoassay methods include but are not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), immunoprecipitation (IP) and flow cytometry (FC).

[0066] The test samples include serum, cell or tissue samples, wherein cells include but are not limited to: human OVCAR-3 cell line; tissues include but are not limited to: human tonsil tissue, human serous ovarian cancer tissue samples, etc.

[0067] Based on the same inventive concept, an embodiment of the present invention further provides a human carbohydrate antigen CA125 immunoassay kit, comprising the above-mentioned monoclonal antibody against human carbohydrate antigen CA125 or an antibody conjugate thereof.

[0068] Optionally, the immunoassay kit is an enzyme-linked immunosorbent assay kit, an ELISpot kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, an immunoprecipitation kit, or a flow cytometry kit. Preferably, the immunoassay kit is an immunoblotting kit, an immunohistochemistry kit, or an immunoprecipitation kit.

[0069] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0070] Example 1 Preparation of rabbit monoclonal antibodies against human carbohydrate antigen CA125

[0071] 1.1. Antigen Preparation: In this example, rabbits were immunized with the recombinant carbohydrate antigen CA125 (Human MUC-16 / CA125, purchased from Abclonal, Catalog No. RP01417). Monoclonal antibodies were prepared based on single B lymphocyte labeling and sorting technology to obtain the heavy and light chain sequences of anti-CA125 protein rabbit monoclonal antibodies.

[0072] 1.2. Animal immunization: Three Japanese white rabbits were immunized with 300 μg of antigen per rabbit. Before the first immunization, the antigen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to form an emulsion, which was injected subcutaneously at multiple points on the rabbit's abdomen and back. After the first immunization, 150 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to form an emulsion, which was injected subcutaneously at multiple points on the rabbit's abdomen and back every three weeks for two booster immunizations. After three immunizations, rabbit serum samples were collected and their titers against human CA125 were determined by enzyme-linked immunosorbent assay (ELISA). The serum after the last immunization was purified into antibodies, and the binding specificity of the antibodies to the naturally expressed CA125 protein on the cells was detected by immunoblotting (WB). The rabbits with high serum titers and the best WB results were selected and boosted once with 400 μg of the immunogen by multiple subcutaneous injections. The animals were sacrificed three days later and the spleens were removed.

[0073] 1.3. Isolation of B lymphocytes from the spleen and sorting of antigen-specific B lymphocytes: For related methods, please refer to the published patents "Method for Efficient Isolation of Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and "A B Lymphocyte In Vitro Culture System and Application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0074] 1.4 Cloning of the rabbit monoclonal antibody gene: The cultured B lymphocyte supernatant was used to identify antigen-specific B lymphocytes using an ELISA coated with human MUC-16 / CA125 protein. The screening criteria was an OD value greater than 0.7. Positive clones of single B lymphocytes with an OD value greater than 0.7 were collected and lysed, and then analyzed using Quick-RNA TM RNA was extracted using a MicroPrep kit (ZYMO, Cat. No. R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR and sequenced. The PCR system consisted of 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2× Gloria HiFi (ABclonal, Cat. No. RK20717), and 6.5 μL H₂O. The PCR protocol consisted of 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min. The reaction mixture was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent forward and reverse primers, respectively.

[0075] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO. 11);

[0076] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO. 12);

[0077] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO. 13);

[0078] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO. 14).

[0079] The amplified DNA product was sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7. The IMGT online database (www.imgt.org) was then queried to obtain the sequence of the constant region, and an antibody with a complete light chain (FL) shown in SEQ ID NO.1 and a complete heavy chain (FH) shown in SEQ ID NO.6 was obtained, and named 7E11.

[0080] Antibody sequencing was completed by Jinkairui Biotechnology Co., Ltd. The antibody amino acid (AA) sequence is shown in Table 1 , where LCDR1-3 represent the complementarity determining regions CDR1-3 of the light chain variable region, and HCDR1-3 represent the complementarity determining regions CDR1-3 of the heavy chain variable region, respectively. The variable region numbering system is the Kabat numbering system.

[0081] Table 1 Sequence information of rabbit monoclonal antibody 7E11 in this example

[0082]

[0083]

[0084] 1.5. Expression and large-scale production of antibody 7E11: Using genetic engineering recombinant expression technology, the amplified VL and VH genes were inserted into an expression vector in series with the light chain constant region (CL) and the heavy chain constant region (CH). The monoclonal antibody 7E11 was mass-produced by recombinant expression of the antibody genes. In this example, the CL and CH genes were pre-inserted into the mammalian expression vector pBR322. The expression pattern is shown in FIG. Figure 4In the figure, the pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, the CMV promoter is a transcription promoter, the SV40 PA terminator is a tailing signal, the light chain constant is the nucleic acid sequence of CL (left), and the heavy chain constant is the nucleic acid sequence of CH (right). The VL and VH genes were then ligated via homologous recombination with the expression vector pBR322 carrying the CL and CH genes, which had been linearized with the restriction enzymes XbaI (955 bp) and NheI (949 bp), respectively. The VL and VH genes were inserted upstream of the CL and CH genes of the pBR322 vector, respectively, to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors. Sequencing verified the successful construction of the vectors.

[0085] To facilitate antibody purification, secretory expression of the antibody is achieved by adding signal peptides upstream of the VL and VH genes. Signal peptides commonly used in the art can be used for antibody expression. For example, the patents "Anti-human interferon α2 rabbit monoclonal antibodies and their applications (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and "High-affinity human IL-5 rabbit monoclonal antibodies and their applications (Publication No.: CN115819578A, Publication Date: 2023-03-21)" contain the signal peptide "MDTRAPTQLLGLLLLWLPDARC" upstream of the VL gene, and the signal peptide "METGLRWLLLVAVLKGVQC" upstream of the VH gene. Of course, those skilled in the art can also replace other signal peptides for antibody expression after obtaining the antibody sequences of the present invention. Therefore, the signal peptide sequences are not shown in the antibody sequences in Table 1 of this example.

[0086] The constructed FL and FH expression vectors were co-transfected into 293F cells and cultured for 72-96 hours. The culture supernatant contained recombinant rabbit monoclonal antibodies that recognized human CA125. The target antibody was purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). The antibody concentration was 0.95 mg / mL and the purity was greater than 90%. The purified antibody was aliquoted and stored at -20°C until needed.

[0087] Example 2 Establishment and Effect Evaluation of Immunoassay Method Based on Anti-Human CA125 Antibody 7E11

[0088] In this example, immunoblotting, immunoprecipitation, and immunohistochemistry analysis systems were established for the rabbit monoclonal antibody 7E11 to evaluate the performance of the antibody for immunoassays.

[0089] 2.1 Establishment of Western blot (WB) detection system

[0090] The positive cell sample in this example is human ovarian adenocarcinoma cell line OVCAR-3 that highly expresses CA125, and the negative (NC) cell sample is human histiocytic lymphoma cell line U-937 that does not express CA125. The WB experimental analysis operation is as follows: (1) Electrophoresis: Lyse the cells to obtain protein lysate, and perform 6% polyacrylamide gel electrophoresis (SDS-PAGE); (2) Transfer: Transfer the gel protein band to a PVDF membrane in an electrotransfer system according to the conventional method; (3) Blocking: Place the PVDF membrane in a TBST blocking solution containing 3% skim milk powder and block it at room temperature for 1 hour; (4) Primary antibody incubation: Add the antibody 7E11 prepared in Example 1 (primary antibody working concentration is 0.95 μg / mL) and incubate at 4°C overnight; (5) Secondary antibody incubation: Wash the membrane with TBST, add goat anti-rabbit IgG secondary antibody (purchased from Abclonal, product number AS014, secondary antibody dilution ratio 1:5000), and incubate at room temperature for 1 hour; (6) Color development: Wash the membrane with TBST, add ECL ultrasensitive color development solution, and develop.

[0091] WB results are shown in Figure 2 CA125 is a high molecular weight glycoprotein. According to the uniprot database, CA125 protein has 102 glycosylation modification sites. Glycosylation increases the molecular weight of the protein. High molecular weight glycosylated proteins take longer to migrate in the gel, thus forming larger bands. Moreover, since the protein contains more glycosylation modification sites, the degree of glycosylation at different sites during the post-translational modification of the protein is different, which will lead to the production of CA125 with different molecular weights. Therefore, the molecular weight distribution of CA125 protein is relatively wide, so the target band with diffuse distribution will be observed in the WB gel image. Figure 2 As can be seen, the target band was not detected in the negative cell sample, indicating that no specific antigen-antibody binding reaction occurred with the non-target protein, resulting in a negative signal. In contrast, a diffuse band matching the expected molecular weight of CA125 was observed in the positive cells, indicating a positive signal. This demonstrates that antibody 7E11 can sensitively recognize CA125 protein in cell lysates, demonstrates high specificity, good affinity for CA125 protein, and effectively resists interference from cellular components.

[0092] 2.2. Establishment of Immunoprecipitation (IP) Detection System

[0093] The positive cell sample in this example is OVCAR-3 cells. The IP experiment analysis operation is as follows: (1) Cell lysis: take 1×10 7Cells were added with 1 mL of IP lysis buffer for IP (without inhibitors), from Abclonal, RM00022, containing protease inhibitor cocktail (from Abclonal, RM02916), and mixed. The mixture was ultrasonicated at 30W for 1 min, 5 s for 5 s, and 10 s intervals. Avoid foaming during the ultrasonication. If there is flocculent matter, ultrasonicate for 2 min. (2) Protein concentration determination: centrifuge at 4°C and 12000 rpm for 10 min, carefully aspirate the supernatant, and determine the protein concentration in the supernatant using a BCA kit. (3) Dilute 500 μg protein to 300 μL, take 40 μL of the diluted supernatant and mark it as input, add 10 μL of 5× loading Buffer was mixed, heated at 95℃ for 10 min, and frozen at -20℃ for later use; (4) 500μg protein was diluted to 300μL, 300μL of the diluted supernatant was placed in two EP tubes, 0.5μg of antibody 7E11 was added to one tube, and 0.5μg of negative IgG antibody (Control IgG) was added to the other tube, and the mixture was placed on a flip mixer at 4℃ for 2h; (5) The above antibody-antigen binding complex was mixed with pre-blocked Protein A / G magnetic beads, and the reaction was carried out at 4℃ for 2h to allow the antibody to bind to Protein A / G, thereby collecting the magnetic bead-antibody-antigen complex by magnetic separation; (6) 1mL IP lysis buffer was added, and the magnetic beads were separated after instant centrifugation. The above operation was repeated 3 times to wash the magnetic beads; (7) 35μL 1× loading The buffer was mixed evenly, heated at 95°C for 10 min, and the supernatant was collected to obtain IgG antibody reaction samples and antibody 7E11 reaction samples respectively; (8) the input sample, IgG antibody reaction sample, and antibody 7E11 reaction sample were subjected to SDS-PAGE detection with the same sample loading amount.

[0094] The magnetic bead blocking operation is as follows: mix the Protein A / G magnetic beads by inverting or vortexing, and turn the bottle over to confirm that there is no black precipitate at the bottom; take 10-15μL Protein A / G magnetic beads and add 1mL IP lysis buffer to mix, centrifuge briefly, collect the magnetic beads, discard the supernatant, and repeat twice; then add 1mL 3% BSA and block at 4℃ for 1h; then add IP lysis buffer to mix, centrifuge briefly, collect the magnetic beads, discard the supernatant, repeat twice for later use.

[0095] IP results see Figure 3As can be seen from the figure, the input control sample showed a weak target protein band, and the band position was consistent with the expected size of the CA125 protein. The anti-CA125 antibody 7E11 reaction sample not only detected the target band that was consistent with the expected size, but also the signal was significantly stronger than the input control. No band was detected in the IgG antibody reaction sample, indicating that the antibody 7E11 can specifically recognize and bind to the CA125 protein and has high affinity and good binding sensitivity to the CA125 protein.

[0096] 2.3 Construction of Immunohistochemistry (IHC) Detection System

[0097] CA125 is an important marker for ovarian serous carcinoma and is valuable for distinguishing between serous and mucinous carcinomas. This antigen is also present in the ovaries under certain normal physiological conditions, such as pregnancy and menstruation. Furthermore, CA125 can also be detected in adenomas of the cervix, endometrium, breast, gastrointestinal tract, and thyroid. The samples used in this example were tissue biopsies of human tonsil epithelial, human breast cancer, human breast, and human ovarian serous carcinoma. The IHC detection steps are as follows: (1) Slice baking: Immerse the paraffin tissue slices baked at 56°C for 30 minutes in dewaxing solution 1. After 5 minutes, take out the slices and immerse them in dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3 in the order of immersion. Place them in dewaxing solution for 5 minutes and in anhydrous ethanol for 3 minutes. Then, wash the slices with running water for 3 minutes. Dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; (2) Antigen repair: 0.01M Tris-EDTA repair solution (pH 9.0) High-pressure heat repair; (3) Inactivation of endogenous peroxidase: Immerse the slides in PBS buffer for 3 times, 3 minutes each time, remove the buffer on the slides; then immerse the slides in 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes; (4) Blocking: Immerse the slides in PBS buffer for 3 times, 3 minutes each time, then remove the buffer, circle the area to be examined on the slide, add PBS blocking solution in the area to be examined, and incubate at room temperature for 30 minutes; (5) Primary antibody incubation: Remove the blocking solution, add antibody 7E11 dilution solution (primary antibody working concentration is 4.75μg / mL), incubate at room temperature for 60 minutes; remove the antibody working solution, rinse quickly with PBS buffer once, and soak and wash three times, 3 minutes each time; (6) Secondary antibody incubation: Add fluorescent secondary antibody (from ABclonal Ready-to-use Immunohistochemistry Mouse and Rabbit Universal Secondary Antibody Kit, Product No. RK50015), incubate at room temperature for 25 minutes; remove the secondary antibody working solution, rinse quickly with PBS buffer once, Soak and wash 3 times, 3 minutes each time; (7) Color development: add color development solution (from ABclonal ready-to-use immunohistochemistry mouse and rabbit universal secondary antibody kit, product number RK50015), closely observe the color change under a microscope, and after obtaining the appropriate staining intensity, immerse the slices in a large amount of distilled water to stop the color development, and then rinse in running water for 10 minutes; (8) Counterstaining: Immerse the slightly drained tissue slices in Mayer's hematoxylin for counterstaining for 1 minute, and rinse in running water for 3 minutes. n; (9) Re-blueing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, and then rinse with running water for 3 minutes; (10) Dehydration: Immerse the slices in anhydrous ethanol twice, lifting them up and down several times during the soaking period, and take them out after 10 seconds; Dry the slices at high temperature (54-58℃); (11) Sealing: Add an appropriate amount of neutral gum to the center of the slice and cover it with a coverslip. The amount of glue added should be appropriate. After sealing the coverslip, the tissue should be completely covered and no glue should overflow. Finally, scan the slices.

[0098] Immunohistochemical staining results are classified as positive or negative. Positive results are those with brown staining at specific antigen sites in the corresponding cells and tissues and low or no background. Negative results refer to the absence of brown staining in specific tissues and cells. The cell nucleus appears blue after counterstaining with hematoxylin, which can reveal the background outline of cells specifically immunostained and is used to determine the subcellular localization of the target protein. The antibody of the present invention showed strong positivity in the detection of human breast cancer, human mammary gland, and human serous ovarian cancer tissues. The positive signal was localized in the cytoplasm and cell membrane, as expected. Human tonsil tissue can be used as both a positive and a negative control. Its epithelial cells express CA125, while the remaining cells are negative. Figure 4 From left to right, examples of immunohistochemical staining of human tonsil and human ovarian serous cancer tissue samples are shown. 1:200 represents the primary antibody dilution ratio. The results demonstrate that antibody 7E11 is accurately localized to the cytoplasm and cell membrane of positive cell populations, with staining consistent with the actual CA125 expression in the tissue samples. The staining is clear and the background is clean, demonstrating the antibody's high specificity and ability to effectively recognize naturally expressed CA125 protein in tissue samples. Binding to the target protein is unaffected by the complexity of the tissue cells, resulting in high sensitivity and ensuring the accuracy and reliability of the test results.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made to the present invention within the scope of implementation of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody against human carbohydrate antigen CA125, characterized in that: It includes a light chain variable region and a heavy chain variable region, the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The monoclonal antibody against human carbohydrate antigen CA125 according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against human carbohydrate antigen CA125 according to claim 2, characterized in that The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against human carbohydrate antigen CA125 according to claim 1, characterized in that The monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against human carbohydrate antigen CA125 according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 5.

7. The recombinant vector according to claim 6, characterized in that The recombinant vector is a pBR322 expression vector.

8. Use of the monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate according to any one of claims 1 to 4 in the preparation of a human carbohydrate antigen CA125 immunoassay kit, characterized in that: The antibody conjugate comprises the monoclonal antibody and a detection label linked to the monoclonal antibody.

9. A human carbohydrate antigen CA125 immunoassay kit, characterized in that: The invention comprises the monoclonal antibody against human carbohydrate antigen CA125 or its antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate comprises the monoclonal antibody and a detection label connected to the monoclonal antibody.

10. The human carbohydrate antigen CA125 immunoassay kit according to claim 9, characterized in that: The immunoassay kit is selected from an immunoblotting kit, an immunohistochemistry kit or an immunoprecipitation kit.

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