CN117603357A - Rabbit monoclonal antibody for human progestogen receptor and application thereof - Google Patents

Rabbit monoclonal antibody for human progestogen receptor and application thereof Download PDF

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CN117603357A
CN117603357A CN202311633384.6A CN202311633384A CN117603357A CN 117603357 A CN117603357 A CN 117603357A CN 202311633384 A CN202311633384 A CN 202311633384A CN 117603357 A CN117603357 A CN 117603357A
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human
monoclonal antibody
rabbit monoclonal
seq
antibody
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雷雅君
程瑶
胡沁
吴海
涂雅伦
胡文娟
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Wuhan Abclonal Inc
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Wuhan Abclonal Inc
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Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody aiming at a human progestogen receptor and application thereof. The amino acid sequences of the complementarity determining regions LCDR1-3 on the light chain variable region of the rabbit monoclonal antibody are respectively shown as SEQ ID NO. 3-5; the amino acid sequences of the complementarity determining regions HCDR1-3 on the heavy chain variable region are shown in SEQ ID NOS.8-10, respectively. The rabbit monoclonal antibody prepared by the invention can specifically identify the progestogen receptor expressed by cells and tissues, has good binding affinity with the progestogen receptor, high specificity and good anti-interference capability, can simultaneously identify the progestogen receptors of PRA and PRB subtypes, has wider applicability, and is suitable for detecting the progestogen receptor with high specificity, high accuracy and high sensitivity, in particular to detecting by adopting an immunoblotting method and an immunohistochemical method.

Description

Rabbit monoclonal antibody for human progestogen receptor and application thereof
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody aiming at a human progestogen receptor and application thereof.
Background
The progestogen receptor (progesterone receptor, PR) is one of the nuclear receptor superfamily members and its ligands comprise natural progestogens, progestogen derivatives, synthetic progestogens, and the like. PR is distributed in the human body mainly in the reproductive organs, mammary glands, central nervous system, and is used to mediate the physiological effects of progesterone, playing an important role in various reproductive events related to the establishment and maintenance of pregnancy, alveolar development and sexual behavior in the breast. PR is mainly of two subtypes: PRA and PRB, two isoforms transcribed from different sites of the same gene induced by different estrogens, PRB is the product of complete transcription of the gene and PRA lacks 164 amino acids from the amino end, when combined with ligand progestogen to transcribe and regulate downstream gene expression, PRB has stronger transcription activity, and PRA is inactive in transcription as a dominant repressor of strong ligand dependence of steroid hormone receptor transcription activity in most cell and promoter environments, and the degree of repression is related to the ratio of PRA/PRB.
The expression of the progestogen receptor is considered a biomarker for Estrogen Receptor (ER) function and prognosis of Breast Cancer (BC). Current studies indicate that PR is not only an ER-induced gene target, but also an ER-related protein that modulates its behavior. PR and ER are commonly expressed in breast cancer, and the common detection indexes of the breast cancer include ER, PR, HER, ki-67 and the like, which are used for determining the molecular type of the breast cancer and providing basis for later treatment; these indices are closely related to tumor growth infiltration, metastasis and recurrence, and have important reference value for the selection of personalized treatment schemes. PR detection is the cornerstone of treatment for patients with invasive breast cancer, the most common cancer in young women, and also the leading cause of cancer death, with Invasive Ductal Carcinoma (IDC) being the most common type of pathology. Breast cancer can be divided into different subtypes based on the expression of the hormone receptors ER, PR and human epidermal growth factor receptor 2 (HER 2): luminel a breast cancer, luminel B breast cancer, HER2 positive (HR positive) breast cancer, and triple negative breast cancer. The Luminal A breast cancer is defined as ER+, HER2-, PR > 20% and Ki67 < 14% according to the PR threshold value, which indicates that the patient has recurrence risk and endocrine treatment is effective; luminal type B breast cancer is defined as ER+, HER2-, and meets any condition that Ki67 is more than or equal to 20%, PR-or less than 20%, the type of breast cancer has high recurrence risk, and chemotherapy is combined with endocrine therapy; whereas HER2 positive breast cancer is effective for targeted therapy, a comprehensive treatment scheme of chemotherapy combined with herceptin is adopted, and the prognosis is worse than that of Luminal A type and Luminal B type; triple negative breast cancers are prone to recurrent metastasis, have the worst prognosis, are effective only for chemotherapy, and are ineffective for targeting and endocrine treatment. Therefore, PR expression level detection has important significance in breast cancer typing and prognosis evaluation, plays a decisive factor in treatment decision, and can be used as a prediction index for whether patients are suitable for endocrine treatment.
Immunoblotting (WB), immunohistochemistry (IHC) and other immunological methods are commonly used clinically to detect the localization and expression level of PR in cells or tissues, and the PR is easily interfered by normal components of the cells, so that a certain proportion of false positive and false negative results exist in detection results. Therefore, developing a monoclonal antibody with good sensitivity and strong specificity aiming at human Progestogen Receptor (PR) has great significance for realizing immunological detection of PR expression level.
Disclosure of Invention
Aiming at the problems that an anti-human progestogen receptor antibody is difficult to be used for immunological detection and the like in the prior art, the invention provides a rabbit monoclonal antibody aiming at a human progestogen receptor, and provides the application of the rabbit monoclonal antibody in preparing a human progestogen receptor immunoassay kit, and the antibody has the advantages of high specificity, high accuracy, high sensitivity, excellent anti-interference capability and the like when being used for immunological detection.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
in a first aspect the invention provides a rabbit monoclonal antibody directed against a human progestogen receptor comprising a light chain variable region and a heavy chain variable region wherein: the amino acid sequences of the complementarity determining regions LCDR1, LCDR2 and LCDR3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of the complementarity determining regions HCDR1, HCDR2 and HCDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.
Further, 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.
Further, a light chain constant region and a heavy chain constant region are included, the light chain constant region and the light chain variable region comprising a light chain, the heavy chain constant region and the heavy chain variable region comprising a heavy chain; the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the constant region of the light chain is a kappa chain; the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the heavy chain is of an IgG type.
Further, the rabbit monoclonal antibody is a full-length antibody or an antibody fragment having immunological activity; the antibody fragment is selected from Fab fragment, F (ab) 2 Fragments, (Fv) fragments 2 Fragments, scFv fragments and sc (Fv) 2 At least one of the fragments.
Further, the immunogen for preparing the rabbit monoclonal antibody is a human progestogen receptor 260-330 site polypeptide fragment, and the amino acid sequence of the human progestogen receptor 260-330 site polypeptide fragment is shown as SEQ ID NO. 11.
In a second aspect the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody directed against a human progestogen receptor as described above.
In a third aspect the invention provides a recombinant vector or host cell comprising a nucleic acid molecule as described above.
In a fourth aspect, the invention provides the use of a rabbit monoclonal antibody directed against a human progestogen receptor as described above, a nucleic acid molecule as described above, a recombinant vector or a host cell as described above for the preparation of a human progestogen receptor immunoassay kit.
In a fifth aspect the invention provides a human progestogen receptor immunoassay kit comprising a rabbit monoclonal antibody directed against a human progestogen receptor as described above or an immunoconjugate of said rabbit monoclonal antibody conjugated to a detectable label.
Further, the kit is one of an enzyme immunoassay kit, an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.
Further, the immunodetection sample includes cell lysates and naturally expressed progestogen receptors in tissue samples; wherein the cells comprise human breast ductal carcinoma cells, human breast cancer cells or human triple negative breast cancer cells, and the tissue comprises human breast invasive ductal carcinoma tissue, human cervical tissue or human triple negative breast cancer tissue.
The invention has the advantages and positive effects that:
the invention takes the polypeptide fragment of human progestogen receptor 260-330 amino acids as immunogen to prepare the rabbit monoclonal antibody for recognizing and combining human progestogen receptor, the obtained antibody can specifically recognize the progestogen receptor expressed by cells and tissues, has good binding affinity with the progestogen receptor, high specificity and good anti-interference capability, can simultaneously recognize the progestogen receptors of PRA and PRB subtype, has wider applicability, and is suitable for detecting the progestogen receptor with high specificity, high accuracy and high sensitivity, in particular for detecting by adopting an immunoblotting method and an immunohistochemical method.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a SDS-PAGE gel of purified immunogens expressed by the prokaryotic expression system of example 1 of the present invention;
FIG. 2 is a diagram of a mammalian vector pBR322 used for constructing a rabbit monoclonal antibody expression vector for a human progestogen receptor according to example 1 of the present invention, in which pRB322 vector maps carrying light chain constant regions and heavy chain constant regions in advance are shown from left to right, respectively;
FIG. 3 is a graph showing the results of immunoblotting detection of binding of a rabbit monoclonal antibody against a human progestogen receptor to breast ductal carcinoma cells expressing a progestogen receptor and breast carcinoma cells not expressing a progestogen receptor in example 2 of the present invention;
fig. 4 is a graph showing the results of immunohistochemical detection of a rabbit monoclonal antibody against a human progestin receptor and a tissue section sample according to example 2 of the present invention, wherein human breast invasive ductal carcinoma tissue, human triple negative breast carcinoma tissue and human cervical tissue are sequentially arranged from left to right.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" will be considered to encompass the following: (i) A, (ii) B, and (iii) A and B.
In the context of the present invention, the terms "rabbit monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to the Human (Human) Progestogen Receptor (PR). The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different 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. Where "antibody fragment" refers to one or more portions or fragments of a full-length antibody, in typical examples, the antibody fragment comprises: fab, fab', F (ab) 2 、F(ab’) 2 、Fv、(Fv) 2 、scFv、sc(Fv) 2 。
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 and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate to form a surface complementary to the three-dimensional structure of the antigen or epitope of interest, which determines the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs may be identified according to Kabat definitions, chothia definitions, a combination of both Kabat and Chothia definitions, abM definitions, contact definitions, IMGT unique numbering definitions and/or conformational definitions, or any CDR determination method known in the art. As used herein, CDRs are defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.
Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.
The term "Antigen binding fragment (Fab)" is the region of an antibody molecule that binds Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain (variable and one constant region fragment), whereby the full-length antibody is proteolytically cleaved to give Fab, F (ab') 2 Fragments such as Fab'. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab') under the action of pepsin 2 Fragments and a pFC' fragment. F (ab') 2 The fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has antibody-antigen affinity like a single chain antibody (scFv), excellent tissue penetrating power and the like, but also has a more stable structure, and is widely applied to clinical diagnosis and treatment.
The term "variable fragment (Fv)" is located at the N-terminus of an antibody Fab fragment, contains only the variable region, and consists of one variable region of one light chain and one heavy chain, is a dimer of one VH and one VL that are non-covalently bound (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than an intact antibody.
The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker, typically consisting of 10 to 25 amino acids), which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.
The full length sequences of the antibodies or antibody fragments of the invention may be from a single species, such as rabbit, or may be chimeric or humanized antibodies to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" antibody refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases, the variable regions of a non-human antibody bind to the constant regions of a human antibody, e.g., a human rabbit chimeric antibody; in other cases, the CDR regions of a non-human antibody bind to FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of a non-human antibody are grafted onto human antibody Framework (FR) sequences derived from single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.
The term "monoclonal antibody" refers to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the structure, source, or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a rabbit monoclonal antibody aiming at a human progestogen receptor, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs), and the amino acid sequences of a light chain complementarity determining region 1 (LCDR 1), a light chain complementarity determining region 2 (LCDR 2) and a light chain complementarity determining region 3 (LCDR 3) on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), and heavy chain complementarity determining region 3 (HCDR 3) on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.
The invention takes polypeptide fragments of human progestogen receptor 260-330 amino acids as immunogens, and then obtains rabbit monoclonal antibodies for recognizing and binding human progestogen receptor based on monoclonal antibody development technology of single B lymphocyte screening and culturing. The obtained antibody is used for immunodetection methods such as immunoblotting and immunohistochemistry, only has specific binding in breast duct cancer cells expressing a progestogen receptor, human breast invasive duct cancer tissues and human cervical tissue samples, and the antibody is accurately positioned, but does not bind in breast cancer cells or triple negative breast cancer cells and tissues which do not express the progestogen receptor, so that the obtained antibody can specifically identify the progestogen receptor expressed by the cells and the tissues, has good binding affinity with the progestogen receptor, has high specificity and good anti-interference capability, is beneficial to improving the accuracy of an immunodetection result, effectively avoids false positive and false negative results, can simultaneously identify PRA and PRB subtype progestogen receptors, has wider applicability, and therefore, the rabbit monoclonal antibody aiming at the human progestogen receptor, which is prepared by the invention, is suitable for detecting the progestogen receptor with high specificity, high accuracy and high sensitivity, and particularly adopts immunoblotting and immunohistochemistry for detection.
Optionally, each of the light chain variable region and the heavy chain variable region further comprises 4 Framework Regions (FR), 4 FR and 3 CDRs are sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 7.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region (CH) and a heavy chain constant region (VH), CL and VL comprising the complete light chain, CH and VH comprising the complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: through IMGT online database (www.imgt.org), rabbit source IgG gamma C reign is searched for CH and rabbit source IgG Kappa C reign is searched for CL.
Alternatively, the amino acid sequence of the light chain comprising CL is shown in SEQ ID No.1, correspondingly the light chain constant region is a kappa chain; the amino acid sequence of the heavy chain comprising CH is shown in SEQ ID NO.6, correspondingly the heavy chain is of the IgG type.
The rabbit monoclonal antibody of the invention may be a full length antibody or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full length form of the rabbit monoclonal antibody, in particular, an antibody fragment comprising the CDR regions (SEQ ID No.3-5 and SEQ ID No. 8-10) as described above, more preferably having the variable regions (SEQ ID No.2 and SEQ ID No. 7) as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, in particular to the same epitope, as the full length antibody. Such antibody fragments include, but are not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) F (ab) 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) 2 Fragments consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) sc (Fv) 2 A fragment is a fragment in which two heavy chain variable regions and two light chain variable regions are linked by a linker or the like. These antibody fragments can be obtained by conventional techniques known in the art.
Yet another embodiment of the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody directed against a human progestogen receptor as described above.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody.
The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.
Another embodiment of the invention provides a recombinant vector or host cell comprising a nucleic acid molecule as described above.
Typical vectors for constructing the recombinant vector include plasmids, viral vectors, phages, cosmids and minichromosomes, as long as they can harbor the nucleic acid molecule. Plasmids are the most common form of vector, and thus, in the context of the present invention, vectors are used interchangeably with plasmids. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The cloning vector may contain a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements (e.g., promoters, enhancers) for expression in the specified host cell. The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is well known in the art and will not be described in detail herein.
Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, nucleic acid molecules encoding the heavy and light chains of a rabbit monoclonal antibody of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or alternatively, the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.
Transformation of host cells with recombinant vectors can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 By a method or MgCl 2 And (5) processing. Microinjection, electroporation, or liposome encapsulation may also be used if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as insect cells of yeast, drosophila S2 or Sf9, CHO, COS7, 293 series cells, etc. After obtaining a host cell transformed with the expression vector as described above, the cell is cultured under appropriate conditions to express the monoclonal antibody, and then isolated to obtain a purified antibody.
In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322 and the host cell is a human kidney epithelial cell (293F cell).
In a further embodiment the invention provides the use of a rabbit monoclonal antibody of a progestogen receptor as described above against a human progestogen receptor, a nucleic acid molecule as described above or a recombinant vector or host cell as described above for the preparation of a kit for the immunoassay of a human progestogen receptor.
The advantages of the rabbit monoclonal antibody, nucleic acid molecule, recombinant vector or host cell of the progestogen receptor against the human progestogen receptor in preparing the human progestogen receptor immunoassay kit are the same as those of the rabbit monoclonal antibody of the progestogen receptor against the human progestogen receptor as described above in comparison with the prior art, and are not described herein.
Based on the same inventive concept as described above, the embodiment of the present invention also provides a human progestogen receptor immunoassay kit comprising the rabbit monoclonal antibody directed against a human progestogen receptor or the immunoconjugate of the rabbit monoclonal antibody conjugated with a detectable label as described above.
In the case of immunoassay, the sample to be examined is contacted with an anti-human progestogen receptor monoclonal antibody, after which the monoclonal antibody is detected. In some embodiments, a detectable label may be conjugated to an anti-human progestogen receptor monoclonal antibody, and qualitative or quantitative detection of the progestogen receptor may be achieved by analyzing the change in the identifiable signal produced by the detectable label. In other embodiments, the anti-human progestogen receptor monoclonal antibody (primary antibody or capture antibody) is not labeled, and the detectable label is conjugated to a secondary antibody (detection antibody) or other molecule that can bind to the monoclonal antibody, e.g., if the anti-human progestogen receptor monoclonal antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, whereby the secondary antibody conjugated with the detectable label specifically binds to the primary antibody to produce a change in the recognizable signal.
Such detectable labels for producing identifiable signal changes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.
Alternatively, immunodetection methods include, but are not limited to: enzyme immunoassay (Enzyme immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunosorbent assay (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), flow Cytometry (FCM), etc., and thus the kit may be an Enzyme immunoassay kit, an Enzyme-linked immunosorbent kit, an immunohistochemical kit, an Immunofluorescence kit, an immunoblotting kit, or a Flow cell kit. In a preferred embodiment, the kit is an immunoblotting kit or an immunohistochemical kit.
Alternatively, the test sample includes plasma, serum, cell lysate, cell culture fluid, and naturally expressed progestogen receptor in a tissue sample. Among them, cells include, but are not limited to: human ductal breast cancer cells, human triple negative breast cancer cells, tissues including but not limited to: human breast invasive ductal carcinoma tissue, human cervical tissue, human triple negative breast carcinoma tissue.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
Example 1 preparation of Rabbit monoclonal antibodies directed against human Progestogen Receptor (PR)
1. Antigen preparation
In this embodiment, the human PR protein 260 th to 330 th amino acid polypeptide fragments with biological activity obtained by in vitro recombinant expression by a prokaryotic expression system are taken as immunogens, and the prepared anti-human PR rabbit monoclonal antibody can simultaneously recognize PRA and PRB subtype according to the amino acid region of the immunogens, wherein the full length of the human PR protein is shown in Uniprot number P06401-1, and the amino acid sequences of the 260 th to 330 th amino acids are shown as follows:
AAAGGVALVPKEDSRFSAPRVALVEQDAPMAPGRSPLATTVMDFIHVPILPLNHALLAA RTRQLLEDESYD (see SEQ ID NO. 11).
The expression and purification method of the polypeptide fragment of the recombinant human PR protein 260-330 amino acids comprises the following steps:
1) Protein expression plasmid construction: the 260 th to 330 th fragments of human PR proteins were amplified with a pair of specific primers using a plasmid containing human PR sequences (from Wohan Botek Biotechnology Co., ltd.) as a template. Wherein, the PCR reaction system is as follows: 1. Mu.L of template, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 25. Mu.L of 2 XGloria Hi-Fi PCR Master Mix with GC Buffer (from Ebolthack Biotechnology Co., ltd., product No. RK 20717) and 22. Mu.L of pure water (N.F H) 2 O); PCR amplification procedure: the reaction solution is subjected to pre-denaturation at 98 ℃ for 30s, then 30 times of circulation are carried out according to the conditions of 10s at 98 ℃, 30s at 64 ℃ and 30s at 72 ℃, and finally the reaction solution is kept at 72 ℃ for 5min, and the obtained reaction solution is kept at 4 ℃. The nucleic acid sequences (5 '-3') of the specific primer pairs for amplifying human PR proteins are as follows:
PR-F: ccgcgtggatccccggaattcgcggcagcaggaggc (see SEQ ID NO. 12);
PR-R: ttagtggtggtgatggtgatgatgatgcggccgctcgaggtcgtaactttcgtc (see SEQ ID NO. 13).
After the amplified PCR product is purified, the amplified PCR product is loaded on an expression vector in a homologous recombination mode, the prokaryotic expression vector is pGEX-4T-1 (the commercial vector is modified, an 8X his tag is introduced at the C end), and the plasmid is preserved after the insertion sites BamHI and Xho are verified by sequencing.
2) Protein expression: pGEX-4T-1 plasmid containing PR protein 260-330aa sequence with 8X his at C-terminal was transformed into E.coli Rosetta strain and incubated overnight at 37℃on LB agar plates (containing 100. Mu.g/mL ampicillin) to obtain single colony transformant.
Single colony transformants were inoculated into 10mL polypropylene tubes containing 2mL LB medium (containing 100. Mu.g/mL ampicillin) and incubated at 220rpm for 3-4h at 37℃to OD 600nm About 0.4 to about 0.6; thereafter, 2mL of the culture of each strain was transferred to a 1L flask containing 400mL of LB expression medium, and further cultured at 220rpm at 37℃for 3-4 hours; when OD is 600nm When the bacterial strain reaches about 0.45-0.55, adding 0.8mM IPTG, inducing at 37deg.C for 3-4 hr, transferring the induced bacterial strain to a dry 500mL centrifuge bottle, balancing with electronic scale, and balancing with qualityAdding pure water if the amount is not equal, centrifuging at 4000rpm for 10min, discarding supernatant, standing the centrifuge bottle, and collecting thallus for storage at-20deg.C.
First time breaking of thallus: taking 30mL of bacteria breaking liquid (50mM Tris+300mM NaCl) to suspend the bacteria, transferring the bacteria liquid after suspension to a 50mL round bottom centrifuge tube, putting the centrifuge tube into an ice box, and fixing the centrifuge tube with ice; selecting an amplitude transformer, placing the amplitude transformer into a bacteria breaking cabin, wherein the power is 350W, the bacteria breaking is performed for 3s at intervals of 3s, counting down for 5min, then placing the amplitude transformer into an ice-water mixture for cooling for 5min, and repeating the steps for 5min; after the completion of the sterilization, the mixture was centrifuged at 9000rpm for 10min to obtain a precipitate.
And (3) crushing the thalli for the second time: 30mL of bacteria-destroying liquid (2M urea+PBS buffer solution) is measured and poured into the sediment, the sediment is uniformly blown, the mixture is transferred into a 50mL round bottom centrifuge tube, the power is 350W, the bacteria are destroyed for 3s at intervals of 3s, the bacteria are broken for 0-5min (the breaking time is determined according to the sediment amount and the texture, the bacteria liquid is placed in ice cubes), and the mixture is centrifuged at 9000rpm for 10min to obtain inclusion bodies.
3) Protein purification: PR proteins were purified by extraction from inclusion bodies by affinity chromatography using Ni affinity chromatography resin (available from Wuhan Hui Biotechnology Co., ltd., cat# HQ 060328500M) with the purification method being referred to in the description course to remove the impurity proteins. The obtained purified inclusion body is respectively diluted by 2 times and 10 times by Loading Buffer; the resulting samples of 2-fold diluted inclusion bodies (abbreviated as ". Times.2"), 10-fold diluted inclusion bodies (abbreviated as ". Times.10") were subjected to SDS-PAGE gel electrophoresis, and the results are shown in FIG. 1, wherein lanes 0.4 and 0.2 represent 0.4mg/mL and 0.2mg/mL of BSA protein, lanes 2 and 10 represent 2-fold and 10-fold diluted inclusion bodies, and lane MK represents a molecular weight Marker.
According to the selected 260 th to 330 th fragments of human PR proteins and the vector, the size of the recombinant protein is predicted to be 35kDa, the band size of the target protein in figure 1 accords with expectations, but proteins which do not accord with expectations appear at about 27kDa and 20kDa, the purity of the PR recombinant protein is about 85% according to the signal intensity of the protein band, and the concentration of the PR recombinant protein is 1.5mg/mL compared with a BSA protein standard.
2. Immunization of animals
Taking the polypeptide sequence obtained by the purification as an immunogen to immunize 4 New Zealand white rabbits; each big ear white rabbit is immunized by 200 mug, the immunogen is mixed with the same amount of complete Freund's adjuvant to prepare an emulsifier before the first immunization, and the emulsifier is subcutaneously injected into the abdomen and the back of the rabbit at multiple points. 100 mug of immunogen is mixed with an equal amount of incomplete Freund's adjuvant every 3 weeks after the primary immunization to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting. Serum samples of rabbits were collected after three immunizations, titers against human Progestogen Receptors (PR) were determined by enzyme-linked immunosorbent assay (ELISA), rabbits with high serum titers were taken, boosted once by subcutaneous multipoint injection with 200. Mu.g of immunogen, and spleens were taken three days later.
3. Isolation and culture of B lymphocytes in spleen
Spleen cells were isolated: taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by rolling the tail end of the pressed part. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company, product number 64010-00-100), gently blowing off cell clusters by using a pipette, counting for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipette, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of B cell complete medium, gently blowing off cell clusters by using a pipette, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
Sorting and culturing of B lymphocytes: see patent "method for efficiently isolating individual antigen-specific B lymphocytes from spleen cells (publication No. CN110016462A, publication No. 2019-07-16)" and patent "an in vitro B lymphocyte culture system and use (publication No. CN111518765A, publication No. 2020-08-11)".
4. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B cell supernatants were subjected to antigen-coated ELISA to identify B lymphocyte positive clones capable of recognizing and binding to human PR. With Quick-RNA TM MicroPrep kit (available from ZYMO company, cat.R 1100-250) extracts RNA and reverse transcribes it into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of the naturally paired rabbit monoclonal antibodies from the cDNA of the corresponding positive clone, and the sequence is determined by sequencing, and the sequencing work is completed by Jin Kairui biotechnology Co. Wherein, the PCR reaction system comprises: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2 XGloria HiFi (from Wuhan Aibolag Biotechnology Co., ltd., cat., product No. RK 20717) and 6.5. Mu. L H 2 O. The PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃. Wherein the nucleic acid sequences of the primer pairs for amplifying the VL and VH genes are shown below, F represents the forward primer, and R represents the reverse primer:
VL-F：5'-tgaattcgagctcggtacccatggacacgagggcccccac-3'(SEQ ID NO.14)；
VL-R：5'-cacacacacgatggtgactgttccagttgccacctgatcag-3'(SEQ ID NO.15)；
VH-F：5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3'(SEQ ID NO.16)；
VH-R：5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'(SEQ ID NO.17)。
5. Production and purification of Rabbit monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies recognizing human progestogen receptors, the selected rabbit monoclonal antibody VL and VH genes were loaded onto a mammalian expression vector pBR322 carrying in advance a light chain constant region (CL) and a heavy chain constant region gene (CH), the expression patterns of which are shown in FIG. 2, in which pRB322origin and f1origin are replication promoters in E.coli (E.Coli), ampcillin is a plasmid resistance gene, CMV immearly promotor is a promoter in eukaryote, SV40 PA terminator is a tailing signal, light chain constant is a nucleic acid sequence of the light chain constant region (left panel), heavy chain constant is a nucleic acid sequence of the heavy chain constant region (right panel), and the constant region sequences are obtained by querying an IMGT online database (www.imgt.org).
The construction process of the rabbit monoclonal antibody expression vector carrying the light chain and heavy chain genes is as follows: mammalian cell expression vectors pBR322 containing rabbit monoclonal antibodies CH and CL are subjected to conventional linearization treatment by NheI and XbaI restriction enzymes respectively, and VL genes and VH genes with signal peptide coding genes at the upstream are respectively constructed at NheI (949 bp) and XbaI (955 bp) in the expression vectors pBR322 by adopting a homologous recombination mode; after sequencing verification, the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies are transfected into 293F cells together; culturing for 72-96h after transfection, and culturing to obtain supernatant containing recombinant rabbit monoclonal antibody recognizing human PR protein. The rabbit monoclonal antibodies recognizing human PR were then purified from the culture supernatant using protein A affinity gel resin (available from Tiandi and, cat. SA 023100), and the purification protocol was performed according to the protein A affinity gel resin instruction, and will not be described in detail herein. The concentration of the purified antibody is 1mg/mL, and the antibody is split charging after being verified to be qualified and is preserved at a low temperature of-20 ℃ for standby.
The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as a rabbit monoclonal antibody against human interferon alpha 2 and its use (publication No. CN116063487A, publication No. 2023-05-05), with a signal peptide "MDTRAPTQLLGLLLLWLPGATF" upstream of the light chain variable region or a signal peptide "METGLRWLLLVAVLKGVQC" upstream of the heavy chain variable region.
The amino acid sequences of the rabbit monoclonal antibodies against human progestogen receptors obtained in this example are shown in table 1, and for convenience of description, light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, and heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively.
TABLE 1 summary of amino acid sequence information of Rabbit monoclonal antibodies of the invention
EXAMPLE 2 reaction specificity and application Studies of Rabbit monoclonal antibodies
1. Immunoblot analysis of Rabbit monoclonal antibody (Western blot, WB)
Taking a human breast duct cancer cell (T-47D) sample and a breast cancer cell (MDA-MB-231 or SK-BR-3) sample for respective cleavage to obtain a protein extract, performing 7% or 8% polyacrylamide gel electrophoresis, and transferring gel protein bands to an NC membrane in an electrotransfer system according to a conventional method; placing the membrane in TBST sealing solution containing 3% skimmed milk powder, incubating for 1h at room temperature, adding rabbit monoclonal antibody of antiprogestin receptor (primary antibody dilution ratio 1:1000, primary antibody working concentration after dilution 1 μg/mL), and incubating at 4deg.C overnight; then, washing the membrane by TBST, adding goat anti-rabbit secondary antibody (purchased from Jackson ImmunoResearch, product number 111-035-003, secondary antibody dilution ratio 1:10000), and incubating for 1h at room temperature; and (3) washing the membrane again by TBST, adding ECL hypersensitive chromogenic liquid, developing, and analyzing the development conclusion to determine the specificity of the PR rabbit monoclonal antibody.
Human breast ductal carcinoma cells T-47D are T-47 differentiated epithelial sub-strains, are isolated from female patients with 54-year-old invasive duct cancer, breast cancer cells MDA-MB-231 are triple-negative breast cancer cells, ER positive, PR positive, HER2 negative or ER, PR and HER2 are positive in invasive breast cancer, three indexes of ER, PR, HER in triple-negative breast cancer are negative, breast cancer cells SK-BR-3 are isolated from pleural effusion of a 43-year-old female breast cancer patient, SK-BR-3 cells overexpress HER2/c-erb-2 gene products and do not express PR genes, so that in the embodiment, the human breast ductal carcinoma cells T-47D are taken as positive samples of anti-human PR rabbit monoclonal antibodies, and the specificity of the breast cancer cells MDA-MB-231 or the breast cancer cells SK-BR-3 are taken as negative samples of anti-human PR monoclonal antibodies is verified. The WB detection results are shown in fig. 3, and it can be seen from the figure that the prepared anti-human PR rabbit monoclonal antibody can synchronously detect two subtypes of progestogen receptors of PRA (90 kDa) and PRB (118 kDa) in human breast cancer cells T-47D, in this test, the human breast cancer cells are derived from cell lysates cultured in two batches, and slight protein degradation occurs in one batch, which is responsible for the occurrence of non-conforming target bands, so that the performance is slightly different, and in general, no non-specific bands exist in human breast cancer cells MDA-MB-231 and SK-BR-3, which proves that the specificity of the antibody is good, the progestogen receptor can be uniquely identified by avoiding the interference of cell components in the cell sample lysate, the affinity for binding with the progestogen receptor is good, and the false positive results can be avoided. And the antibody can simultaneously recognize PR proteins in two different forms of PRA and PRB, and can meet different use requirements.
2. Immunohistochemical analysis of Rabbit monoclonal antibodies (IHC)
The immunohistochemical section samples contained: a human breast invasive ductal carcinoma tissue section sample, a human triple negative breast carcinoma tissue section sample, and a human cervical tissue section sample. The IHC staining and analysis operations are referred to the pathological chip instruction book, and specifically comprise the following steps: (1) sample preparation and baking: placing paraffin slices on a slice rack in the same direction, and placing the paraffin slices into a constant temperature box at 56 ℃ to bake the slices for 30min; simultaneously placing the dewaxing liquid 1 cylinder together into a constant temperature box at 56 ℃, placing paraffin slices and a slice frame together into the dewaxing liquid 1 cylinder, taking out the paraffin slices from the constant temperature box together and placing the paraffin slices at normal temperature, taking out the paraffin slices after 5min, immersing the paraffin slices into a normal temperature dewaxing liquid 2 cylinder, and sequentially placing the paraffin slices into the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 1, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3 according to the sequence of the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3, wherein each dewaxing liquid reagent cylinder is 5min; washing the slices with running water for 3min; the dewaxing liquid 1-3 is purchased from the original industry and industry limited company of the Wuxi city river; (2) antigen retrieval: high pressure thermal remediation of 0.01M sodium citrate remediation solution (pH 6.0); (3) endogenous peroxidase inactivation: immersing and washing for 3 times by using PBS buffer solution for 1min each time, and removing the buffer solution on the slice; immersing the slices into 3% hydrogen peroxide solution completely, and incubating for 10min at room temperature; (4) closing: immersing and washing for 3 times by using PBS buffer solution for 3min each time, and removing the buffer solution on the slice; an immunohistochemical water pen is used for delineating a tissue region to be detected on a slide, and PBS blocking solution is dripped into the delineating region; horizontally placing the slices in an incubation wet box with water at the bottom, incubating for 30min at normal temperature, and starting timing from dripping the sealing liquid; (5) primary antibody incubation: removing the blocking solution, dripping rabbit monoclonal antibody diluted by antibody diluent-PBS working solution (primary antibody dilution ratio is 1:100, primary antibody working concentration after dilution is 10 mug/mL) on a tissue slice, horizontally placing in an incubation wet box, and incubating for 60min at normal temperature; removing antibody working solution, quickly rinsing with PBS buffer solution for 1 time, soaking and washing with the buffer solution PBS for 3 times, and 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing; (6) secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (purchased from DAKO, product number K5007) on a tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 25min at normal temperature; removing the secondary antibody working solution on the slice, quickly rinsing the slice for 1 time by using PBS buffer solution, soaking and washing the slice for 3 times by using the PBS buffer solution for 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing; (7) color development: dropwise adding a color development liquid working solution on the slice, closely observing the color change condition under a microscope to obtain proper dyeing intensity, immersing the slice in a large amount of distilled water to terminate color development, and washing the slice in running water for 10min after the color development is terminated; (8) counterstaining: immersing the slightly drained tissue slice into Mayer's hematoxylin for counterstaining for 1min, and then washing with running water for 3min; (9) blue returning: immersing the slightly drained slice into a saturated aqueous solution of lithium carbonate to blu for 3s, and cleaning the slice with running water for 3min; (10) dehydration: soaking the cleaned slice in absolute ethyl alcohol for 1 time, lifting up and down for several times during the soaking period, and taking out after timing for 10 seconds; completely drying at high temperature (54-58 deg.C) in constant temperature blast drying oven; (11) Dripping a proper amount of neutral gum at the center of the slice, covering a cover glass, wherein the glue adding amount is proper, and the cover glass is required to cover tissues completely and cannot overflow the glue; (12) slice scanning.
IHC staining results were divided into: positive and negative. Positive expression must appear brown-stained at specific antigen sites of cells and tissues to be positive, and no brown staining to be negative. Based on the expression of the hormone receptors ER, PR and HER2, breast cancer molecular typing is mainly: invasive breast cancer, triple negative breast cancer, HER2 positive (HR positive) breast cancer. Invasive breast cancer refers to ER positive, PR positive, HER2 negative, or breast cancer positive for ER, PR and HER 2; HER2 (HR positive) breast cancer refers to breast cancer with both ER and PR negative, HER2 positive; the three indexes of the triple negative breast cancer ER, PR, HER2 are all negative. And RP subcellular localization to the nucleus, the cytoplasm and cell membrane are negative. The IHC staining results of the human mammary invasive duct cancer (human breast invasive ductal cancer) tissue section sample, the human triple negative breast cancer (human triple negative breast cancer) tissue section sample and the human cervical (human cervix) tissue section sample are shown from left to right in the figure, and the IHC staining results of the human PR rabbit monoclonal antibody prepared by the invention are accurate in the human mammary invasive duct cancer staining location, clear in staining and free of non-specific staining, exhibit positive signals, and free of specific staining in the triple negative breast cancer, exhibit negative signals, and exhibit positive signals in the normal uterine smooth muscle cells of human cervical tissues, so that the theory positioning accords with expectations.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (10)

1. A rabbit monoclonal antibody directed against a human progestogen receptor comprising a light chain variable region and a heavy chain variable region, wherein:
the amino acid sequences of the complementarity determining regions LCDR1, LCDR2 and LCDR3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5;
the amino acid sequences of the complementarity determining regions HCDR1, HCDR2 and HCDR3 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 rabbit monoclonal antibody directed against the human progestogen receptor of claim 1 wherein 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 rabbit monoclonal antibody to the human progestin receptor of claim 2, further comprising a light chain constant region and a heavy chain constant region, said light chain constant region and said light chain variable region comprising a light chain, said heavy chain constant region and said heavy chain variable region comprising a heavy chain;
The amino acid sequence of the light chain is shown as SEQ ID NO.1, and the constant region of the light chain is a kappa chain; the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the heavy chain is of an IgG type.
4. The rabbit monoclonal antibody directed against the human progestin receptor according to claim 1 or 2, wherein the rabbit monoclonal antibody is a full length antibody or an immunologically active antibody fragment;
the antibody fragment is selected from Fab fragment, F (ab) 2 Fragments, (Fv) fragments 2 Fragments, scFv fragments and sc (Fv) 2 At least one of the fragments.
5. The rabbit monoclonal antibody against human progestogen receptor according to claim 1, wherein the immunogen for preparing said rabbit monoclonal antibody is a human progestogen receptor 260-330 polypeptide fragment having the amino acid sequence shown in SEQ ID No. 11.
6. A nucleic acid molecule encoding a rabbit monoclonal antibody directed against a human progestin receptor according to any one of claims 1-5.
7. A recombinant vector or host cell comprising the nucleic acid molecule of claim 6.
8. Use of a rabbit monoclonal antibody directed against a human progestogen receptor according to any of claims 1-5, a nucleic acid molecule according to claim 6, a recombinant vector or host cell according to claim 7 for the preparation of a human progestogen receptor immunoassay kit.
9. A human progestogen receptor immunoassay kit comprising a rabbit monoclonal antibody directed against a human progestogen receptor according to any of claims 1-5 or an immunoconjugate of said rabbit monoclonal antibody conjugated to a detectable label.
10. The human progestin receptor immunoassay kit of claim 9, wherein the kit is one of an enzyme immunoassay kit, an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescent kit, an immunoblotting kit, or a flow cytometry kit;
immunodetection samples include cell lysates and naturally expressed progestogen receptors in tissue samples; wherein the cells comprise human breast ductal carcinoma cells, human breast cancer cells or human triple negative breast cancer cells, and the tissue comprises human breast invasive ductal carcinoma tissue, human cervical tissue or human triple negative breast cancer tissue.
CN202311633384.6A 2023-11-29 2023-11-29 Rabbit monoclonal antibody for human progestogen receptor and application thereof Pending CN117603357A (en)

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CN117603357A true CN117603357A (en) 2024-02-27

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