CN117487018B - Rabbit monoclonal antibody against human epididymis secretory protein 4 and its application - Google Patents
Rabbit monoclonal antibody against human epididymis secretory protein 4 and its application Download PDFInfo
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Abstract
本发明属于抗体制备技术领域，尤其涉及抗人附睾分泌蛋白4的兔单克隆抗体及其应用。所述兔单克隆抗体为第一抗体或第二抗体，第一抗体轻链CDR1‑3的氨基酸序列分别如SEQ ID NO.3‑5所示，重链CDR1‑3的氨基酸序列分别如SEQ ID NO.8‑10所示；第二抗体轻链CDR1‑3的氨基酸序列分别如SEQ ID NO.13‑15所示，重链CDR1‑3的氨基酸序列分别如SEQ ID NO.18‑20所示。本发明兔单克隆抗体识别和结合人HE4蛋白的特异性好、亲和力高，以第一抗体和第二抗体配对建立的双抗夹心酶联免疫吸附检测体系，具有灵敏度高、准确性高、可靠性好和抗干扰能力优良等优点。
The present invention belongs to the technical field of antibody preparation, and particularly relates to rabbit monoclonal antibodies against human epididymal secretory protein 4 and their applications. The rabbit monoclonal antibody is a first antibody or a second antibody, and the amino acid sequences of the light chain CDR1-3 of the first antibody are shown as SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1-3 are shown as SEQ ID NO.8-10, respectively; the amino acid sequences of the light chain CDR1-3 of the second antibody are shown as SEQ ID NO.13-15, and the amino acid sequences of the heavy chain CDR1-3 are shown as SEQ ID NO.18-20, respectively. The rabbit monoclonal antibody of the present invention has good specificity and high affinity for recognizing and binding human HE4 protein, and the double antibody sandwich enzyme-linked immunosorbent assay system established by pairing the first antibody with the second antibody has the advantages of high sensitivity, high accuracy, good reliability, excellent anti-interference ability, etc.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody for resisting human epididymis secretion protein 4 and application thereof.
Background
Human epididymal secreted protein 4 (human epididymis protein, HE 4) is a secreted glycoprotein having a molecular weight of 22-26kDa, which belongs to the Whey acidic protein family (WAP) members, and has a structure comprising four disulfide core regions (WFDC) of Whey acidic protein, and thus is also called Whey acidic protein four disulfide core region 2 (WAP-four-disulfide core domain 2, WFDC2), which has a high homology with extracellular protease inhibitors. HE4 was originally found in distal epithelial tissue of male epididymis, closely related to sperm maturation, and later found to be expressed in airway epithelial cells, salivary gland mucous cells, ductal mammary epithelial cells, etc., and to act as a protease inhibitor in many family members. As a small secreted protein, HE4 is expressed less in normal tissues and is highly expressed in fibrosis and human cancers, which have been found to be highly expressed in malignant tumors such as ovarian cancer, lung cancer, etc. and detected early in the tumor. Positive expression rates of HE4 in ovarian tissue correlated with tissue types of ovarian cancer, with most significant increases in serous ovarian cancer and endometrioid ovarian cancer, and numerous studies have demonstrated that HE4 induces MMP-2 expression by activating the AKT signaling pathway, promoting ovarian cancer metastasis and upregulation of EMT. HE4 has higher sensitivity and specificity in serological diagnosis of ovarian cancer, can well screen early breast cancer, is considered as the most promising ovarian cancer marker following CA125, has better sensitivity and specificity to early ovarian cancer than CA125, and can be used for early diagnosis, identification, treatment monitoring and prognosis evaluation of ovarian cancer and the like.
Based on the outstanding characteristics of HE4 in clinical diagnosis and monitoring, it is important to develop a specific binding antibody for HE4, and a HE4 antibody with good specificity and high affinity is the basis for realizing high-sensitivity HE4 detection. At present, most of monoclonal antibodies for detecting HE4 are derived from mouse ascites, have generally low affinity and specificity with human HE4, cannot be well applied to detecting human HE4 protein, have low accuracy of detection results and poor detection sensitivity, and are difficult to be applied to detecting the level of human HE4 protein with low content in serum or cell tissues. Therefore, the development of a novel monoclonal antibody and a detection kit which are applicable to immunological detection and are used for resisting human epididymis secretory protein 4 has very important clinical value for HE4 level detection in related diseases, and particularly has important reference significance for the occurrence and development of ovarian cancer, prognosis judgment and curative effect monitoring.
Disclosure of Invention
The invention provides a rabbit monoclonal antibody for resisting human epididymal secretion protein 4 and application thereof, aiming at solving a part of problems in the prior art or at least relieving a part of problems in the prior art.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
The first aspect of the invention provides a rabbit monoclonal antibody against human epididymis secretion protein 4, comprising a first antibody and/or a second antibody, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region comprise 3 complementarity determining regions; wherein: the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the first antibody are respectively shown in SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 8-10; the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the second antibody are respectively shown in SEQ ID NO.13-15, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 18-20.
Further, the amino acid sequence of the light chain variable region of the first antibody 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; the amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17.
Further, the rabbit monoclonal antibody further comprises a light chain constant region and a heavy chain constant region, 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; wherein: the amino acid sequence of the light chain of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6; the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 16.
In a second aspect the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody as described above.
Further, the nucleotide sequence of the light chain variable region of the first antibody is shown as SEQ ID NO.21, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 22; the nucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.23, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 24.
Further, the nucleotide sequence of the light chain of the first antibody is shown as SEQ ID NO.25, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 26; the nucleotide sequence of the light chain of the second antibody is shown as SEQ ID NO.27, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 28.
In a third aspect the invention provides a recombinant vector comprising a nucleic acid molecule encoding a first antibody and/or a second antibody as described above.
In a fourth aspect, the present invention provides a kit for immunodetection of human epididymal secretion protein 4, said kit comprising a primary antibody and/or a secondary antibody as described above.
Further, the kit comprises the first antibody and the second antibody as described above, wherein the first antibody is used as a capture antibody, and the second antibody is used as a detection antibody or a labeled antibody.
Further, the immunodetection method includes one or more of an enzyme immunoassay, an enzyme-linked immunosorbent assay, an immunohistochemical method, an immunofluorescence method, an immunoblotting method and a flow cytometry.
Further, the immunodetection sample includes at least one of recombinant human HE4 protein and human HE4 protein secreted by the cell.
The invention has the advantages and positive effects that:
The rabbit monoclonal antibody provided by the invention has the advantages of good specificity for recognizing and combining human HE4 protein, high affinity, capability of recognizing and combining different antigen epitopes of human HE4 protein respectively by the first antibody and the second antibody, capability of establishing a double-antibody sandwich ELISA detection system by pairing the two, high sensitivity, high accuracy, good reliability, excellent anti-interference capability and the like, and has good application value in clinical diagnosis, prognosis judgment and curative effect monitoring of diseases related to HE4 expression level.
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 diagram of a vector used for constructing a rabbit monoclonal antibody expression vector according to example 1 of the present invention, from left to right, respectively pRB322 vector map carrying light chain constant regions and heavy chain constant regions in advance;
FIG. 2 is a graph showing the affinity of rabbit monoclonal antibody 2G4 for binding to human HE4 protein according to example 1 of the invention;
FIG. 3 is a graph showing the affinity of rabbit monoclonal antibody 2F6 for binding to human HE4 protein according to example 1 of the invention;
FIG. 4 is a graph showing the epitope recognition of rabbit monoclonal antibodies 2G4 and 2F6 of example 1 of the present invention;
FIG. 5 is a standard curve of the double antibody sandwich ELISA method established based on rabbit monoclonal antibodies 2G4 and 2F6 in example 2 of the present invention;
FIG. 6 is a graph showing the thermal stability of the ELISA method based on the rabbit monoclonal antibodies 2G4 and 2F6 according to example 3.
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. The terms "first," "second," and the like, are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order, it being understood that such uses may be interchanged where appropriate.
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 human epididymal secretion protein 4 (HE 4) protein. 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 with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining 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 and FR can be identified according to Kabat definition, chothia definition, a combination of both Kabat definition and Chothia definition, abM definition, contact definition, IMGT unique number definition and/or conformational definition, or any CDR determination method known in the art. As used herein, is 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.
The terms "monoclonal antibody" or "mab" and the like are used interchangeably and refer 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 being obtained from a substantially homogeneous population of antibodies, and is not to be construed as limiting the 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 for resisting human epididymis secretion protein 4, which comprises a first antibody and/or a second antibody, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs); wherein: the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the first antibody are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10; the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the second antibody are respectively shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20.
The invention uses human epididymis secretory protein 4 (HE 4) protein as immunogen, and develops a first antibody and a second antibody for resisting human HE4 protein by utilizing single B lymphocyte sorting and culturing technology. The first antibody and the second antibody provided by the invention can specifically identify the human HE4 protein (comprising recombinant human HE4 protein expressed by recombination and natural HE4 protein secreted by cells), have high binding affinity with the human HE4 protein, respectively identify and bind different antigen epitopes of the human HE4 protein through antigen epitope binding analysis, and a double-antibody sandwich enzyme-linked immunosorbent assay (double-antibody sandwich ELISA) system established by taking the first antibody as a capture antibody and the second antibody as a detection antibody or a labeled antibody is used for detecting the human HE4 protein, has high detection sensitivity and low detection limit of 4.1pg/mL, can be used for detecting the extremely low content of the human HE4 protein in a sample, has the advantages of high accuracy, good reliability, excellent anti-interference capability and the like, and has good application value in clinical diagnosis, prognosis judgment and curative effect monitoring of diseases related to HE4 level expression.
Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the first antibody 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. The amino acid sequence of the light chain variable region (VL) of the second antibody is shown as SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 17.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region and a heavy chain constant region, 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: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA C REIGN was searched for CL via IMGT online database (www.imgt.org).
Optionally, the amino acid sequence of the light chain (L chain) of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 6. The amino acid sequence of the light chain (L chain) of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 16.
Yet another embodiment of the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody 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.
Illustratively, the nucleotide sequence of the light chain variable region of the first antibody is shown as SEQ ID NO.21 and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 22. The nucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.23, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 24.
Illustratively, the nucleotide sequence of the light chain of the primary antibody is shown in SEQ ID NO.25 and the nucleotide sequence of the heavy chain is shown in SEQ ID NO. 26. The nucleotide sequence of the light chain of the second antibody is shown as SEQ ID NO.27, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 28.
It will be appreciated by those skilled in the art that nucleic acid molecules other than those exemplified above may likewise be encoded to produce the primary antibody and the secondary antibody due to the degeneracy of the genetic code, and thus the nucleic acid molecules exemplified above should not be construed as limiting the scope of the invention.
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 comprising a nucleic acid molecule encoding a first antibody and/or a second antibody as described above, e.g.comprising the nucleic acid molecules as shown in SEQ ID NOS.21-28.
The recombinant vector may be constructed by ligating the nucleic acid molecule provided by the present invention to various vectors by a method conventional in the art. Vectors are only capable of harboring the nucleic acid molecule and preferably include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and vectors are used interchangeably herein.
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 comprise a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector comprises 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 molecules of the invention. This is well known in the art and will not be described in detail herein.
The nucleic acid molecules encoding the heavy and light chains of the rabbit monoclonal antibodies of the invention can 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 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. In other embodiments, nucleic acid molecules encoding the heavy and light chains of an antibody of the invention may also 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. The choice of the aforementioned 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 capable of absorbing DNA can be obtained after the exponential growth phase, treated by CaCl 2 method or MgCl 2 method, and also can be prepared by microinjection, electroporation or liposome packaging. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome encapsulation, and the like.
The host cell is selected according to the type of recombinant vector, and 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: coli, streptomycete, salmonella typhimurium, fungal cells such as yeast, insect cells of drosophila S2 or Sf9, CHO, COS7, 293 series cells, and the like.
In a specific embodiment, when the recombinant vector is pBR322, the host cell is a human kidney epithelial cell (293F cell). After the host cell 293F transformed with the pBR322 recombinant vector containing the heavy chain gene and the light chain gene of the antibody is obtained, the 293F cell is cultured under proper conditions to obtain the supernatant containing the anti-human HE4 protein rabbit monoclonal antibody, and then the purified antibody is obtained by affinity purification.
The embodiment of the invention also provides an application of the rabbit monoclonal antibody for resisting the human epididymis secretion protein 4, the nucleic acid molecule or the recombinant vector in preparation of a kit for immunodetection of the human epididymis secretion protein 4.
The application advantages of the rabbit monoclonal antibody, nucleic acid molecule or recombinant vector for resisting human epididymis secretion protein 4 in preparing the kit for immunodetection of human epididymis secretion protein 4 are the same as those of the rabbit monoclonal antibody for resisting human epididymis secretion protein 4 as described above in comparison with the prior art, and are not repeated here.
The primary antibody and the secondary antibody may be used alone, or in combination, or may be bound to or coupled to a detectable label (for detection purposes), respectively. Detectable labels for detection purposes 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.
In the case of immunological detection, if a first antibody and a second antibody are separately used, the first antibody or the second antibody is used as an antigen-binding antibody (capture antibody) which specifically recognizes and binds to the HE4 protein in the sample to be detected, and then a recognizable signal change is generated by a detectable label attached thereto, or a detection antibody such as IgG to which a detectable label is conjugated specifically binds to the antibody of the present invention to generate a recognizable signal change, thereby achieving qualitative or quantitative detection of the human HE4 protein.
In preferred embodiments, the immunoassay method includes, but is 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 (Immunohistochemistry, IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), flow Cytometry (FCM), and the like.
Optionally, the immunodetection sample includes, but is not limited to: recombinant human HE4, human HE4 secreted by cells.
Based on the fact that the first antibody and the second antibody of the invention recognize different antigen epitopes, the immune detection method is preferably a double-antibody sandwich enzyme-linked immunosorbent method, wherein in the double-antibody sandwich enzyme-linked immunosorbent method, the capture antibody is the first antibody, and the detection antibody is the second antibody marked by a detectable marker (such as biotin). The two have high sensitivity and specificity in pairing detection of human HE4, and the detection limit is as low as 4.1pg/mL.
Based on the same inventive concept, the embodiment of the invention also provides a kit for immunodetection of human epididymal secretion protein 4, which comprises the first antibody and/or the second antibody as described above.
The advantages of the kit for immunodetection of human epididymis secretion protein 4 are the same as those of the rabbit monoclonal antibody against human epididymis secretion protein 4 as described above in comparison with the prior art, and will not be described in detail herein.
Alternatively, the kit comprises a first antibody and a second antibody as described above, the kit being used for detecting human HE4 by a double-antibody sandwich enzyme-linked immunosorbent assay, wherein the first antibody serves as a capture antibody and the second antibody serves as a detection antibody or a labeled antibody.
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 antibody against human epididymis secretion protein 4 protein
This example was used to prepare immunogens for Human epididymal secretion Protein 4 (HE 4) rabbit monoclonal antibodies were recombinant Human HE4 Protein (from ABclonal self-produced Protein, product name: recombinant Human WAP/WFDC 2/HE4 Protein, cat# RP 00132). The antibody preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture, and specifically comprises the following steps:
1. Immunization of animals
4 New Zealand white rabbits are immunized by taking recombinant Human HE4 protein (goods No. RP 00132) as an immunogen; each white rabbit was immunized with 200. Mu.g of immunogen, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma Co.) to prepare an emulsifier prior to the first immunization, and injected subcutaneously in the abdomen and back of the rabbits at multiple points. 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare an emulsifier, which was subcutaneously injected at the abdomen and back of rabbits at multiple points to boost the immunization twice. After three immunizations, rabbit serum samples were collected, serum was taken at 1: titers of the Human HE4 protein are determined by ELISA after dilution by 243000 times, rabbits with OD 450nm exceeding 0.2 are taken, the immunization is enhanced by subcutaneous multipoint injection of 200 μg of immunogen once, and spleens are taken three days later.
2. Isolation of B lymphocytes from spleen
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 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 (purchased from BioGems company) at room temperature, gently blowing off cell clusters by using a pipettor, 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 pipettor, 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 basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
3. B lymphocyte sorting and culturing
See patent "method for efficiently isolating individual antigen-specific B lymphocytes from spleen cells (publication No. CN 110016462A)" and patent "an in vitro culture system for B lymphocytes and application (publication No. CN 111518765A)".
4. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B lymphocyte supernatant was used to identify positive clones capable of binding to the Human HE4 protein by antigen coated ELISA. Cells of positive clones were collected and lysed, and RNA was extracted according to the Quick-RNA TM Micro Prep kit instructions (available from ZYMO, cat. No. R1051) and reverse transcribed 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. The PCR reaction system is as follows: 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 2X GloriaHiFi (from ABclonal self-produced under the trade designation 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 ℃.
Amplification VL and VH primer pair (5 '-3') nucleotide sequences are shown below, with F and R representing forward and reverse primers, respectively:
VL-F (see SEQ ID NO. 29): 5'-tgaattcgagctcggtacccatggacacgagggcccccac-3';
VL-R (see SEQ ID NO. 30): 5'-cacacacacgatggtgactgttccagttgccacctgatcag-3';
VH-F (see SEQ ID No. 31): 5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3';
VH-R (see SEQ ID No. 32): 5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'.
5. Production and purification of Rabbit monoclonal antibodies
The VL gene and VH gene (with signal peptide upstream) of the rabbit monoclonal antibody were respectively loaded on the expression vector pBR322 carrying the light chain constant region and the heavy chain constant region in advance, and the expression pattern of the expression vector pBR322 carrying the light chain constant region and the heavy chain constant region in advance was used as shown in fig. 1, wherein pBR322Ori and f1Ori are replication initiation sites in e.coli, AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a transcription promoter, SV40 PA terminator is a tailing signal, LIGHT CHAIN constant is a nucleic acid sequence of the light chain constant region (CL) (left panel), HEAVY CHAIN constant is a nucleic acid sequence of the heavy chain constant region (CH) (right panel). Specifically, the mammalian cell expression vector pBR322 containing antibodies CL and CH is subjected to conventional linearization treatment by NheI and XbaI restriction enzymes respectively (note: light chain corresponding vector is subjected to XbaI digestion treatment, heavy chain corresponding vector is subjected to NheI digestion treatment), the amplified VL gene and VH gene are purified, the VL gene and VH gene with signal peptide coding genes at the upstream are respectively constructed into the expression vector by adopting a homologous recombination mode, and whether the construction of the expression vector is successful is verified by sequencing.
Transfecting the successfully constructed expression vector carrying the antibody light chain gene and the heavy chain gene into 293F cells; culturing for 72-96h after transfection to obtain supernatant containing rabbit monoclonal antibody recognizing human HE 4. The rabbit monoclonal antibodies recognizing the human HE4 Protein are purified from the supernatant of the transfected culture medium by using Protein A affinity gel resin, the concentrations of the rabbit monoclonal antibodies 2G4 and 2F6 are respectively 4.11mg/mL and 4.22mg/mL, and the rabbit monoclonal antibodies are split after being qualified in antibody verification and are preserved at a low temperature of-20 ℃ for standby. Protein a affinity chromatography uses procedures conventional in the art and will not be described in detail herein.
It should be noted that, the signal peptide upstream of the VL gene and VH gene in this example may be expressed by using an antibody commonly used in the art, and specifically, see "rabbit monoclonal antibody against Human interferon α2" and its application (publication No. CN116063487a, publication No. 2023-05-05) "or" high affinity Human IL-5 rabbit monoclonal antibody and its application (publication No. CN115819578a, publication No. 2023-03-21) ", a signal peptide" MDTRAPTQLLGLLLLWLPGATF "or" MDTRAPTQLLGLLLLWLPGARC "is provided upstream of the light chain variable region, and a signal peptide" METGLRWLLLVAVLKGVQC "is provided upstream of the heavy chain variable region.
6. Monoclonal antibody screening and identification
The obtained multi-strain anti-human HE4 rabbit monoclonal antibody is primarily identified and screened, and the specific method is as follows:
1) Antibody affinity assay: the affinity of the obtained rabbit monoclonal antibody is primarily determined by using a Gator biomolecular interaction analyzer of Probe Life company, wherein the antigen is recombinant Human HE4 protein, the working concentration is 150nM and 75nM, and the working concentration of the rabbit monoclonal antibody is 2 mug/mL; by comparing the affinities of the respective antibodies, antibodies having affinity constants of.ltoreq.1X10 -9 were selected.
2) Identification of antigen recognition epitopes: the rabbit monoclonal antibody with the affinity constant less than or equal to 1X 10 -9 is subjected to pairing reaction by using a Gator biomolecule interaction analyzer of Probe Life company to test the identified epitope determinant, the antigen is recombinant Human HE4 protein, the working concentration is 5 mug/mL, and the working concentrations of the first antibody and the second antibody are respectively 2 mug/mL and 5 mug/mL. By analyzing the pairing data between the two antibodies, two antibodies recognizing different epitopes were selected, and named rabbit monoclonal antibody 2G4 and rabbit monoclonal antibody 2F6, respectively.
Further, the affinity of the rabbit monoclonal antibody 2G4 and the rabbit monoclonal antibody 2F6 is measured, the antibody concentration is 2 mug/mL, the antigen concentration is 10 mug/mL, the detection results of the 2G4 and the 2F6 are respectively shown in figures 2-3, wherein the ordinate represents the thickness change of the conjugate after the probe is combined with the antibody and the protein, the abscissa represents the combination time, the dark gray curve is a real-time combination numerical curve, the light gray curve is a fitting average value curve, the result shows that the antibody is combined with the antigen to reach a saturated state in a short time, no obvious dissociation exists in a long time, and the overall affinity condition is good. Affinity constants obtained by curve fitting and calculation are shown in table 1, dissociation coefficient K off is used for representing the constant of the dissociation speed of the antibody and the antigen, binding coefficient K on is used for representing the constant of the binding speed of the antibody and the target thereof, and affinity constant K D is the ratio of K off/Kon and represents the equilibrium dissociation constant between the antibody and the antigen thereof. As can be seen from Table 1, the affinity constants K D of the rabbit monoclonal antibodies 2G4 and 2F6 to recombinant Human HE4 were 5.37X10 -10 (M) and 9.44X10 -11 (M), respectively, with high affinity to HE4 protein.
TABLE 1 determination of affinity-related parameters for Rabbit monoclonal antibodies 2G4 and 2F6
| Rabbit monoclonal antibodies | Koff(1/s) | Kon(1/Ms) | KD(M) |
| 2G4 | 1.43×10-4 | 2.65×105 | 5.37×10-10 |
| 2F6 | 2.38×10-5 | 2.52×105 | 9.44×10-11 |
FIG. 4 shows the results of epitope recognition by rabbit monoclonal antibodies 2G4 and 2F6, wherein the ordinate indicates the change in thickness of the conjugate after binding of the probe to the antibody and protein, and the abscissa indicates the change in time of binding of the screening antigen to the protein. As can be seen from FIG. 4, the rabbit monoclonal antibodies 2G4 and 2F6 respectively recognize different epitopes and the recognition sites do not interfere with each other, so that the two antibodies can be used as paired antibodies for double-antibody sandwich ELISA detection.
The selected antibodies 2G4 and 2F6 were sequenced, and the sequencing work was completed by Jin Kairui Biotechnology Inc., the amino acid and nucleotide sequences of the resulting 2G4 and 2F6 are shown in tables 2-3, respectively, and the light chain variable region VL and heavy chain variable region VH sequence identity of the rabbit monoclonal antibodies 2G4 and 2F6 were 64.3% and 80.2%, respectively. For convenience of description, light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively, AA denotes an amino acid sequence, and DNA denotes a nucleotide sequence.
TABLE 2 summary of sequence information relating to monoclonal antibody 2G4 of this example
TABLE 3 summary of sequence information relating to monoclonal antibody 2F6 of this example
Example 2 method for establishing double antibody sandwich ELISA based on rabbit monoclonal antibodies 2G4 and 2F6
The embodiment provides a double-antibody sandwich ELISA detection method established based on anti-human HE4 rabbit monoclonal antibodies 2G4 and 2F6, which specifically comprises the following steps:
2.1, coated capture antibody 2G4: the rabbit monoclonal antibody 2G4 is diluted to 4 mug/mL by 1 XPBS, and after being uniformly mixed by a vortex meter, 100 mug/hole is added into a 96-well ELISA plate, a cover plate film is covered, and the mixture is placed in a refrigerator at 4 ℃ for incubation for 16-20h.
2.2, Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.3, Sealing: adding blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween 20 and 0.1%proclin 300,pH 7.2% in 1 XPBS) into plate hole at 200 μl/hole, covering with cover plate film, sealing at 37deg.C for 2 hr, discarding blocking solution after sealing, drying ELISA plate, baking at 37deg.C for 0.5-2 hr, and taking out.
2.4, Adding protein: diluting the recombinant Human HE4 protein, wherein the concentration after dilution is as follows: 8000pg/mL, 4000pg/mL, 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 0pg/mL, and then sequentially adding 100. Mu.L/well to the ELISA plate, covering the cover plate membrane, and incubating at 37℃for 2 hours.
2.5, Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.6, Adding detection antibody 2F6: after dilution of the biotin-labeled rabbit monoclonal antibody 2F6 (2F 6-biotin) to 0.028. Mu.g/mL, the mixture was sequentially added to an ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 1 hour. The preparation method of the 2F6-biotin comprises the following steps: biotin labeling treatment method: preparing rabbit monoclonal antibody 2F6 into a solution with the concentration of 1mg/mL, and preparing NHS-LC-biotin into a solution with the concentration of 60mg/mL by using DMSO; 200 mu L of 1mg/mL of anti-rabbit monoclonal antibody 2F6 solution is taken, and 10 mu L of 60mg/mL of NHS-LC-biotin solution is added; after mixing, standing at room temperature for 30min, adding 50 μg 500mM Tris solution with pH 9.0 to stop the reaction; finally, a large amount of 1 XPBS buffer, pH 7.4, was added and centrifuged with a 30KD exclusion limit for removing excess biotin molecules and allowing the buffer system to equilibrate.
2.7, Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.8, Adding SA-HRP: 100 XSA-HRP (horseradish peroxidase labeled streptavidin, available from Wuhan Sanying Biotechnology Co., ltd., product No. SA 00001-0) concentrate was diluted 100 times, and then added to an ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.
2.9, Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.10, Adding TMB color development liquid: the 3,3', 5' -Tetramethylbenzidine (TMB) color development solution was added to the ELISA plate at 100. Mu.L/well, covered with a cover plate film, and incubated at 37℃for 15min.
After incubation was completed, the microplate was removed, 50. Mu.L of stop solution (1 mol/L hydrochloric acid) was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
And (5) according to a standard curve graph 5 drawn by the detection result, the standard curve equation is as follows: y=0.0003x+0.1717, r 2 =0.9949. The detection limit of the established double-antibody sandwich ELISA method for detecting the human HE4 protein by using the rabbit monoclonal antibody 2G4 as a capture antibody and 2F6 as a detection antibody can be as low as 4.1pg/mL, and the method has the advantages of high sensitivity and good reliability.
EXAMPLE 3 thermostable detection of rabbit monoclonal antibodies 2G4 and 2F6
The detection system formed by the ELISA plate coated with the capture antibody, the freeze-dried recombinant Human HE4 protein and the 100X concentrated detection antibody is respectively placed at-20 ℃,4 ℃ and 37 ℃ for sealing and preservation, and then taken out after 7 days, and tested according to the double-antibody sandwich ELISA method established in the example 2.
The results are shown in FIG. 6, wherein the light gray curve represents the results of antibody stability at-20℃and the dark gray curve represents the results of antibody stability at 37 ℃. The results show that the stability variation coefficient of the antibody pair is smaller than 15% after 7d is destroyed at 37 ℃, and the stability reaches the standard, which indicates that the stability of the antibody pair formed by the rabbit monoclonal antibodies 2G4 and 2F6 is better.
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.
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