CN117624367B - Anti-human CD141 protein rabbit monoclonal antibody and application thereof - Google Patents
Anti-human CD141 protein rabbit monoclonal antibody and application thereof Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody for resisting human CD141 protein and application thereof. The rabbit monoclonal antibody is a first antibody or a second antibody, the amino acid sequences of the light chain CDR1-3 of the first antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO. 8-10; the amino acid sequences of the light chain CDR1-3 of the second antibody are shown as SEQ ID NO.13-15 respectively, and the amino acid sequences of the heavy chain CDR1-3 are shown as SEQ ID NO.18-20 respectively. The antibody provided by the invention has high affinity and good specificity, is used for immune detection without non-specific binding, and can realize high-precision, high-specificity and high-sensitivity detection of trace CD141 protein in a sample by establishing a double-antibody sandwich enzyme-linked immunosorbent assay system by pairing the first antibody and the second antibody.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody for resisting human CD141 protein and application thereof.
Background
Thrombomodulin (thrombomodulin, TM), also known as CD141, THRM, THBD and Fetomodulin, is a single-chain transmembrane glycoprotein with anticoagulant effect, which is widely distributed on vascular and lymphatic endothelial cell membranes, and has been recently found to be synthesized by hematopoietic progenitor cells, dendritic cells, urothelial cells, vascular smooth muscle cells, mononuclear/macrophages, neutrophils, etc. The CD141 protein mainly acts as a thrombin receptor for the biological function of coagulation, and is capable of binding to thrombin to prevent the thrombin from participating in the coagulation cascade and inhibit the procoagulant activity of the thrombin, and can activate anticoagulant protein C into an active form (ACTIVATED PROTEINC, APC) through the CD 141/thrombin complex, and APC can participate in anticoagulation through inactivation of a coagulation factor, and can also act as a fibrinolysis promoting function through formation of a complex with a plasminogen activation inhibitor to promote plasminogen activation. In addition, CD141 plays an important role in anti-inflammatory, maintenance of epithelial cell morphology, anti-tumor cell proliferation, invasion, and the like.
The variation of the expression level of thrombomodulin in human body is related to a plurality of diseases, under normal physiological conditions, the level of CD141 protein in human serum is lower, the reference range is 3.8-13.3TU/mL, and when vascular endothelial cells are destroyed, CD141 falls into blood from the surface of endothelial cells, resulting in the rise of the concentration in blood plasma, so the value of thrombomodulin in blood plasma can be used as one of sensitive indexes for judging the damage degree and the severity of the disease. The higher the plasma CD141 protein value, the higher the thrombin concentration and the higher the risk of thrombosis. Therefore, detection of CD141 protein content is critical for the pre-disease diagnosis, treatment and prognosis evaluation of thrombotic patients. In addition, the generation or expression abnormality of CD141 can also be used for research of vascular tumors and differential diagnosis of mesothelioma and lung adenocarcinoma.
In view of the outstanding value of the activity or expression level of the CD141 protein in the aspects of diagnosis, monitoring, prognosis evaluation and the like of various diseases, the development of a thrombomodulin detection method with high sensitivity and strong specificity has important significance. The detection methods widely used in clinic at present are immunological detection methods such as an enzyme-linked immunosorbent assay based on antigen-antibody reaction, an immunohistochemical method and the like. However, the high-specificity anti-CD 141 protein antibodies on the market at present are few and have low affinity, so that the detection sensitivity is low, and the high-efficiency and accurate detection of trace proteins in a sample to be detected is difficult to realize. Therefore, developing a high affinity, high specificity monoclonal antibody is of great importance for improving the specificity, accuracy and sensitivity of immunodetection of CD141 protein.
Disclosure of Invention
In view of the above problems of the prior art, the present invention provides a rabbit monoclonal antibody against human CD141 protein and uses thereof, which aim to solve some of the problems of the prior art or at least to alleviate some of the problems of the prior art.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
In a first aspect, the present invention provides a rabbit monoclonal antibody against human CD141 protein, which is a first antibody or a second antibody, wherein: the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.
Further, the amino acid sequence of the first antibody 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; 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 amino acid sequence of the first antibody light chain 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.
Further, the first antibody or the second antibody is a full-length antibody or an antibody fragment; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
In a second aspect the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody as described above.
In a third aspect the invention provides a recombinant vector or host cell comprising a nucleic acid molecule encoding a first antibody and/or a second antibody as described above.
In a fourth aspect, the invention provides the use of a rabbit monoclonal antibody against human CD141 protein as described above, a nucleic acid molecule as described above, a recombinant vector or a host cell as described above in the preparation of a kit for immunodetection of human CD141 protein.
In a fifth aspect the present invention provides a kit for the immunological detection of human CD141 protein, said kit comprising a first antibody and/or a second antibody as described above.
Further, the kit is 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 kit is an immunohistochemical kit or a flow cytometry kit, the kit comprises the first antibody, the first antibody is a primary antibody, and the anti-rabbit IgG antibody coupled with a detectable label is a secondary antibody.
Furthermore, the kit is a double-antibody sandwich ELISA kit, the kit comprises the first antibody and the second antibody, the first antibody is a primary antibody, and the second antibody coupled with a detectable label is a secondary antibody.
The invention has the advantages and positive effects that:
1. The first antibody and the second antibody can specifically identify and bind to the natural CD141 protein and the recombinant human CD141 protein expressed in vitro, the affinity constants of the antibodies and the CD141 protein are at the nM level, the affinity is high, the specificity is good, the antibodies and the CD141 protein are not specifically bound when being used for immune detection, false positive or false negative results can be avoided, and the accuracy, the reliability and the sensitivity of immune detection results are improved.
2. The first antibody and the second antibody can identify different epitopes of human CD141 protein, and the two antibodies are matched to establish a double-antibody sandwich enzyme-linked immunosorbent assay system, so that the detection limit of detecting human CD141 protein is as low as 1.33pg/mL, the high-precision, high-specificity and high-sensitivity detection of trace human CD141 protein in a sample to be detected can be realized, and the kit has important significance in clinical diagnosis and scientific research application and good application prospect.
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 electrophoresis of recombinant human CD141 protein purified in example 1 of the present invention;
FIG. 2 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, pRB322 vector map carrying light chain constant regions and heavy chain constant regions, respectively;
FIG. 3 is a graph showing the affinity of rabbit monoclonal antibody A of example 2 of the present invention for binding to human CD141 protein;
FIG. 4 is a graph showing the affinity of rabbit monoclonal antibody B of example 2 of the present invention for binding to human CD141 protein;
FIG. 5 is a graph showing the epitope recognition of rabbit monoclonal antibodies A and B of example 2 of the present invention;
FIG. 6 is a standard curve of the double antibody sandwich ELISA method established based on rabbit monoclonal antibodies A and B in example 3 of the present invention;
FIG. 7 is a graph showing the results of immunohistochemical detection of rabbit monoclonal antibody A of example 4 binding to samples of different tissues; wherein, figures A-D represent human lung, human tonsils, human lung squamous carcinoma and human lung adenocarcinoma tissues, respectively;
FIG. 8 is a graph showing the results of flow cytometry detection of rabbit monoclonal antibody A of example 5 binding to different cell samples of the present invention; wherein, panels A-B represent THP-1 cells and Jurkat cells, respectively.
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 "mab" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to human thrombomodulin (CD 141). 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 of anti-human CD141 protein, which is 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 adopts single B cell screening and culturing technology to develop the anti-human CD141 first antibody and the second antibody, the said antibodies can specifically identify and bind natural CD141 protein and recombinant human CD141 protein expressed in vitro, when used in immunological detection such as immune tissue method and flow cytometry, the positive signals are only specifically bound in positive cells and tissues expressing CD141 protein, the positive signals are obvious and the antibodies are positioned accurately, and no binding exists in negative cells and tissues not expressing CD141 protein, which shows that the antibody of the invention has high specificity and strong anti-interference capability, can avoid false positive or false negative results caused by non-specific binding, etc., and the affinity constant of the binding of the first antibody and recombinant human CD141 protein is 3.03X10 -9 (M), and the affinity constant of the binding of the second antibody and recombinant human CD141 protein is 3.02X10 -9 (M), and the high affinity and specificity ensure the accuracy, reliability and sensitivity of the immune detection result. In addition, the two antibodies can recognize different antigen epitopes of human CD141 protein, can be used for developing a double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) system, and the double-antibody sandwich ELISA established by taking the first antibody as a capture antibody and the second antibody as a detection antibody can realize high-precision, high-specificity and high-sensitivity detection of trace human CD141 protein in a sample to be detected, has important significance in clinical diagnosis and scientific research application and has good application prospect.
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 light chain and CH and VH comprising the 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).
Specifically, 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. Correspondingly, the light chain constant regions of the first and second antibodies are kappa chains and the heavy chain constant regions are of the IgG type.
It should be noted that the rabbit monoclonal antibody of the present invention may be a full-length antibody (having a typical Y-type molecular structure) 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, specifically, an antibody fragment includes CDR regions as described above, more preferably has variable regions as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, particularly 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) A F (ab) 2 fragment, a bivalent fragment comprising 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 fragment 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) The sc (Fv) 2 fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via 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 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.
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 encoding a first antibody and/or a second antibody as described above.
The starting vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. 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 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 are capable of absorbing DNA, can be obtained after the exponential growth phase and treated with CaCl 2 or MgCl 2. 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: coli, streptomycete, salmonella typhimurium, fungal cells such as yeast, insect cells of drosophila S2 or Sf9, CHO, COS7, 293 series cells, and the like. After obtaining a host cell transformed with the 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 against human CD141 protein 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 kit for immunodetection of human CD141 protein.
The advantages of the application in preparing the kit for immunodetection of human CD141 protein are the same as those of the rabbit monoclonal antibody against human CD141 protein described above with respect to the prior art, and are not described in detail herein.
Based on the same inventive concept, the embodiment of the invention also provides a kit for immunodetection of human CD141 protein, which comprises the first antibody and/or the second antibody as described above.
The advantages of the kit for immunodetection of human CD141 protein are the same as those of the rabbit monoclonal antibody against human CD141 protein described above with respect to the prior art, and will not be described in detail herein.
The first antibody and the second antibody may be used alone, or may be used together, or may be used in combination. In the case of immunoassay, for example, when the primary antibody and/or the secondary antibody are used separately or in combination, the primary antibody and/or the secondary antibody are used as primary antibodies or capture antibodies, and a sample to be examined is contacted with the primary antibody and/or the secondary antibody, followed by detection of the antibodies. In some embodiments, the first antibody and/or the second antibody may be conjugated to a detectable label, and qualitative or quantitative detection of CD141 protein may be achieved by analyzing the change in the identifiable signal produced by the detectable label. In other embodiments, the primary and/or secondary antibodies (primary or capture antibodies) to the human CD141 protein are not labeled, but rather a detectable label is conjugated to a secondary antibody (detection antibody) or other molecule that can bind to the primary and/or secondary antibodies, e.g., if the anti-human CD141 protein antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, whereby the primary antibody is specifically bound by the conjugated secondary antibody to a detectable label to produce a change in a recognizable signal. When the antibodies are paired, one of the first antibody and the second antibody is used as a primary antibody or a capture antibody, and the other is used as a secondary antibody or a detection antibody.
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 (Immunohistochemistry, IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), flow Cytometry (FCM), and the like, 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.
Alternatively, the immunodetection samples include naturally expressed CD141 protein and recombinantly expressed CD141 protein in plasma, serum, urine, cell lysates, cell culture fluids, and tissue samples.
In a preferred embodiment, the kit is an immunohistochemical kit or a flow cytometry kit, the first antibody is a primary antibody, and the anti-rabbit IgG antibody coupled with a detectable label is a secondary antibody.
Based on the fact that the first antibody and the second antibody recognize different antigen epitopes, in a preferred embodiment, the kit is a double-antibody sandwich enzyme-linked immunosorbent kit, comprising the first antibody and the second antibody, wherein the capture antibody (first antibody) is the first antibody, and the detection antibody (second antibody) is the second antibody coupled with a detectable label.
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 anti-human CD141 protein rabbit monoclonal antibody
The immunogen for preparing the rabbit monoclonal antibody against human CD141 protein in this example is a human CD141 protein which is verified to have biological activity by recombinant expression in vitro by 293F cells, specifically, a coding gene (nucleic acid sequence see NCBI accession number NM-000361.3) corresponding to the amino acid sequence comprising 19 th to 515 th AA of CD141 protein (amino acid sequence see NCBI accession number NP-000352.1) is constructed between BamHI and NheI cleavage sites of pXY A (IL 2ss+C-6 x His) plasmid, then HEK293 cells are used for expression to obtain recombinant human CD141 protein, and the recombinant protein is obtained by affinity purification by nickel column to obtain a soluble recombinant protein with a concentration of 1.17mg/mL and a purity of 90%, SDS-PAGE map of the obtained protein is shown in FIG. 1, wherein BSA represents a bovine serum albumin standard, and lanes represent Marker, lanes 5. Mu.L and 10. Mu.L represent recombinant human CD141 protein with loading amounts of 5. Mu.L and 10. Mu.L, respectively. Plasmid pXY A (IL 2ss+C-6 His) was obtained by amplification synthesis from a purchased gene library using the gene library as a template; the gene Library was purchased from Thermo FISHER SCIENTIFIC CDNA/ORF Library-Plate Map(s); the gene library catalog number is: MHS6278-202800780.
The preparation method of the antibody is a monoclonal antibody development technology based on single B lymphocyte screening and culture, firstly, single antigen specific B lymphocytes are separated from immunized rabbit spleen, then, light chain and heavy chain coding genes of the antibody are extracted through specific primers, the light chain and heavy chain coding genes are constructed on an expression vector, the expression vector is transfected into cells and cultured, cell culture solution is affinity purified through protein A, candidate rabbit monoclonal antibodies are obtained, and 2 strains of rabbit monoclonal antibodies are obtained through screening through methods such as affinity test, antigen epitope identification and the like, and are named as rabbit monoclonal antibody A and rabbit monoclonal antibody B respectively. The antibodies A and B were sequenced, and the sequencing was performed by Jin Kairui Biotechnology Inc., the Amino Acid (AA) sequences of which are shown in tables 1-2, respectively. 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 sequence information for Rabbit monoclonal antibody A of this example
TABLE 2 sequence information of rabbit monoclonal antibody B of this example
The preparation method of the rabbit monoclonal antibodies A and B specifically comprises the following steps:
1. Animal immunization: recombinant human CD141 protein is used as immunogen to immunize New Zealand white rabbits, each of which is immunized by 200 mug, the immunogen is mixed with the equivalent amount of complete Freund's adjuvant to prepare an emulsifier before the first immunization, and the emulsifier is injected subcutaneously at the abdomen and back of the rabbits 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, serum titers against the immunogen were determined by ELISA, rabbits with high serum titers were boosted by subcutaneous multipoint injection of 200. Mu.g of the immunogen for three days, and spleens were obtained.
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 (RPMI Medium 1640basic+1% Pen Strep; RPMI 1640 is purchased from Gibco, product number C11875500BT; pen Strep is purchased from Gibco, product number 15140-163), placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissues and fat on rabbit spleen tissues, shearing spleen tissues, placing spleen tissues in the cell screen for grinding, taking a clean grinding rod, grinding the tissues by rolling the tail end of the pressed part until the whole spleen tissues are nearly white as far as possible, and filtering the ground cells into the culture Medium through the cell screen; pipetting the cell-containing medium into a sterile 50mL centrifuge tube, pipetting 10mL of medium, washing the culture dish again, and pipetting the cells remaining in the culture dish into the centrifuge tube as much as possible; centrifuging at room temperature for 5min with a centrifugal force of 400g, sucking and discarding the supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate (purchased from BioGems company) at room temperature, gently blowing off cell clusters with a pipette, timing for 1min, performing erythrocyte lysis, adding 37mL of basic culture medium after timing is completed, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min with a centrifugal force of 400g, sucking and discarding the supernatant, reserving cells, adding 40mL of basic culture medium placed at room temperature, gently blowing off cell clusters with a pipette, resuspending cells, centrifuging at room temperature for 5min with a centrifugal force of 400g, sucking and discarding the supernatant, reserving cells; adding 20mL of basal medium placed at normal temperature, and gently blowing off cell clusters by using a pipette to resuspend 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 single 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 application (publication No. CN111518765A, publication No. 2020-08-11)".
4. Cloning of the genes encoding the rabbit monoclonal antibodies: the cultured B lymphocyte supernatant was subjected to antigen-coated ELISA to identify positive clones capable of binding to recombinant human CD141 protein. Cells of positive clones were collected and lysed, and RNA was extracted according to the Quick-RNA TM MicroPrep kit instructions (purchased from ZYMO, cat. No. R1051) and reverse transcribed into cDNA. The cDNA is taken as a template, the VL and VH genes of a naturally paired rabbit monoclonal antibody are amplified from the cDNA of the corresponding positive clone by PCR, and 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 Gloria HiFi (from ABclonal self-produced under the trade designation RK 20717) and 6.5. Mu.LH 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') nucleic acid sequences are shown below, with F and R representing forward and reverse primers, respectively:
VL-F:5'-tgaattcgagctcggtacccatggacacgagggcccccac-3' (see SEQ ID NO. 21);
VL-R:5'-cacacacacgatggtgactgttccagttgccacctgatcag-3' (see SEQ ID NO. 22);
VH-F:5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3' (see SEQ ID NO. 23);
VH-R:5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3' (see SEQ ID NO. 24).
5. Production and purification of rabbit monoclonal antibodies: the heavy chain and light chain genes of the rabbit monoclonal antibody are respectively loaded on an expression vector, and the construction process is as follows: mammalian cell expression vector pBR322 containing rabbit monoclonal antibody light chain constant region (CL) and heavy chain constant region (CH) genes is treated by conventional linearization treatment with NheI and XbaI restriction endonucleases respectively, VL and VH genes with signal peptide coding genes at the upstream obtained by amplification are purified, and then constructed to the upstream of CL and CH genes of the expression vector pBR322 by adopting a homologous recombination mode, and whether the construction of the expression vector is successful is verified by sequencing. The expression pattern of the mammalian expression vector pBR322 carrying the CL and CH genes is shown in FIG. 2, in which pRB322 origin and f1 origin are replication promoters in E.coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a promoter in eukaryotes, SV40 PA terminator is a tailing signal, LIGHT CHAIN constant is the nucleic acid sequence of the light chain constant region (left panel), HEAVY CHAIN constant is the nucleic acid sequence of the heavy chain constant region (right panel). CL, CH genes CL is obtained by searching IMGT online database (www.imgt.org), searching rabbit source IGG GAMMA C REIGN for CH, searching rabbit source IGG KAPPA C REIGN.
The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as the "high affinity Human IL-5 rabbit monoclonal antibody and its use (publication No. CN115819578A, publication No. 2023-03-21)", the light chain variable region has the signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream and the heavy chain variable region has the signal peptide "METGLRWLLLVAVLKGVQC" upstream.
The expression vector containing the light chain gene and the heavy chain gene of the corresponding rabbit monoclonal antibody which is verified to be correct is transfected into 293F cells together, and the supernatant containing the recombinant human CD141 protein-recognizing antibody is obtained after culturing for 72-96 hours after transfection. Then, the rabbit monoclonal antibodies were purified from the culture supernatants using protein A affinity gel resin (from Tiandi Kong, cat. SA 023100), and the purification protocol was followed by the affinity gel resin instructions, which are not described in detail.
6. Screening of monoclonal antibodies: the antibody affinity determination is carried out on the obtained multi-strain Anti-human CD141 rabbit monoclonal antibody by using a Gator biological molecule interaction analyzer of Probe Life company, wherein the antigen is recombinant human CD141 protein, the working concentration is 1.3mg/mL, the working concentration of the antibody is 1.0mg/mL, the antigen epitope recognition determination is further carried out on the screened out high-affinity antibody by using the analyzer, the Probe is an Anti-His Probe (purchased from Probe Life company, product number is 20-5066), and the antibody pairs with different antigen recognition epitopes are screened out, so that the antibody A and the antibody B of the invention are obtained.
Example 2 Performance test of Rabbit monoclonal antibodies A and B
1. Antibody affinity assay: accurate measurement is carried out by using a Probe Life company Gator biomolecule interaction analyzer, and the concentration of the antibody A and the concentration of the antibody B are 3 mug/mL; antibodies were immobilized on Protein a probes, and then binding was performed with recombinant human CD141 Protein at 75nM and 150nM concentrations for antibody a and antibody B, respectively, to obtain affinity curves, which were shown in fig. 3-4 for antibodies a and B, respectively, wherein the ordinate represents the change in the thickness of the conjugate after binding of the probe to the antibody and Protein, the abscissa represents the binding time, the dark gray curve was a real-time binding numerical curve, and the light gray curve was a fitted average curve. The affinity constants calculated by curve fitting are shown in table 3.
TABLE 3 determination of affinity-related parameters for Rabbit monoclonal antibodies A and B
| Rabbit monoclonal antibodies | Koff(1/s) | Kon(1/Ms) | KD(M) |
| A | 7.18×10-4 | 2.37×105 | 3.03×10-9 |
| B | 3.50×10-4 | 1.02×105 | 3.42×10-9 |
Dissociation coefficient K off is a constant characterizing the rate of dissociation of an antibody from an antigen, binding coefficient K on is a constant characterizing the rate of binding of an antibody to its target, and affinity constant K D is a ratio of K off/Kon, representing the equilibrium dissociation constant between an antibody and its antigen. As is clear from Table 3, the affinity constants K D of the rabbit monoclonal antibodies A and B to human CD141 protein were 3.03X10 -9 M and 3.42X10 -9 M, respectively, and the affinity of the antibodies to antigen was high.
2. Identification of antigen recognition epitopes: antigen recognition epitope identification was performed using a Probe Life company Gator biomolecular interaction analyzer, comprising the steps of: 1) Vibrating and wetting the Anti-His probe in a matched buffer, and centrifuging at 1000rpm for 300s; 2) Placing the Anti-His probe in a buffer solution for initial point calibration, ensuring that the probe is initially in a stable state, and centrifuging at 1000rpm for 60s; 3) Solidifying recombinant human CD141 protein on the Anti-His probe with solidification concentration of 10 mug/mL, centrifuging at 1000rpm for 150s; 4) Placing the probe after curing the protein in a buffer solution for vibration washing, and centrifuging at 1000rpm for 60s; 5) The probe after solidification of the protein is firstly placed in 5 mug/mL of the primary antibody diluent, so that the primary antibody and the CD141 protein are combined to saturation, and the primary antibody and the CD141 protein are centrifuged at 1000rpm for 180s; 6) Placing the immobilized protein and the probe after the primary antibody in 5 mug/mL of secondary antibody diluent, enabling the secondary antibody to be combined with other epitopes of CD141 protein different from the primary antibody, and centrifuging at 1000rpm for 180s; the results of the epitope measurement of antibody A and antibody B are shown in FIG. 5, in which the ordinate indicates the thickness change of the conjugate after the probe binds to the antibody and the protein, and the abscissa indicates the binding time.
As can be seen from the analysis of the pairing data between the two antibodies, after the antibody A and the human CD141 protein are combined, the antibody B can still be combined with the human CD141 protein, which shows that the antibody A and the antibody B recognize and combine different antigen epitopes on the surface of the CD141 protein and the combining sites are not interfered with each other, so that the antibody A and the antibody B can be used as paired antibodies for double-antibody sandwich ELISA detection.
Example 3 method for establishing double antibody sandwich ELISA adsorption based on rabbit monoclonal antibodies A and B
The double-antibody sandwich ELISA method established based on the antibodies A and B specifically comprises the following steps: 1) Coating the capture antibody A: diluting the antibody A into 2 mug/mL by using 1 XPBS, uniformly mixing by using a vortex instrument, adding into a 96-well micro-pore plate by using 100 mug/well, covering a cover plate film, and placing in a refrigerator at 4 ℃ for incubation for 16-20h; 2) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed once by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 3) Closing: adding E013 blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween and 0.1%proclin 300,pH 7.2% in 1 XPBS) into the plate holes at a ratio of 200 μl/hole, covering with cover plate film, blocking at 37deg.C for 2 hr, discarding blocking solution after blocking, drying the ELISA plate, baking at 37deg.C for 0.5-2 hr, and taking out; 4) Adding CD141 protein: human CD141 protein was diluted with a dilution (phosphate buffer containing 2% bovine serum albumin, 0.05% Tween-20, 0.1% proclin 300) to a concentration of: 2000. 1000, 500, 250, 125, 62.5, 31.25 and 0pg/mL, then sequentially adding 100 mu L/hole into an ELISA plate, covering a cover plate film, and incubating at 37 ℃ for 2h; 5) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 6) Adding detection antibody B: diluting the biotin-labeled rabbit monoclonal antibody B (B-biotin) to 0.0125 mu g/mL, sequentially adding 100 mu L/hole into an ELISA plate, covering a cover plate film, and incubating at 37 ℃ for 1h; wherein the method for labeling the antibody B by the biotin comprises the following steps: preparing an antibody B 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 antibody B solution is taken, and 10 mu L of 60mg/mL of NHS-LC-biotin solution is added; After mixing, the mixture was left at room temperature for 30min, and then 50. Mu.g of 500mM Tris solution (pH 9.0) was added to stop the reaction; finally adding a large amount of 1 XPBS buffer solution with pH of 7.4, centrifuging by using a centrifugal column with the exclusion limit of 30KD, and removing redundant biotin molecules and balancing a buffer solution system; 7) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 8) Adding SA-HRP: 100 xSA-HRP (horseradish peroxidase labeled streptavidin, available from Wuhan Sanying biotechnology Co., ltd., product No. SA 00001-0) concentrate is diluted 100 times, and added into an ELISA plate sequentially at 100 μl/hole, covered with a cover plate film, and incubated at 37deg.C for 0.5 hr; 9) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 10 Adding TMB color development liquid: adding 3,3', 5' -tetramethyl benzidine (TMB) color development liquid into an ELISA plate at a concentration of 100 mu L/hole, covering a cover plate film, and incubating at 37 ℃ for 15min;11 Standard curve is drawn: after the incubation is completed, the ELISA plate is taken out, 50 mu L of stop solution (1 mol/L hydrochloric acid) is added into each hole, and reading is immediately carried out at 450nm and 600nm by an ELISA reader; The graph is plotted with the human CD141 protein concentration on the abscissa and the absorbance correction (OD 450-OD630) on the ordinate, and the curve obtained by fitting is shown in fig. 6.
The lowest human recombinant CD141 concentration with the average light absorption value being greater than the average light absorption value of the three-time blank control is the sensitivity of the double-antibody sandwich method ELISA method, and the result shows that the detection sensitivity of the established double-antibody sandwich method ELISA method based on the anti-human CD141 protein rabbit monoclonal antibodies A and B reaches 1.33pg/mL, and the detection sensitivity can meet the high-sensitivity and high-reliability detection of the very low content human CD141 protein in the sample to be detected.
EXAMPLE 4 immunohistochemical analysis of Rabbit monoclonal antibodies (Immunohistochemistry, IHC)
Human CD141 protein is mainly expressed in vascular and lymphatic endothelial cells in human samples, protein subcellular localization is carried out on cell membranes, and positive samples (expressing CD141 protein) are selected to be human conventional tonsil, conventional lung, lung squamous cell carcinoma and high-grade urothelial carcinoma; negative samples (which do not express CD141 protein) are lung adenocarcinoma, conventional liver, glioblastoma samples.
IHC staining and analysis included 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 5 min; 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 repairing of 0.01M sodium citrate repairing liquid (pH 6.0); 3) Inactivation of endogenous peroxidases: 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) Incubation resistance: removing the blocking solution, dripping rabbit monoclonal antibody A diluted by antibody diluent-PBS working solution (primary antibody dilution ratio is 1:1000, primary antibody working concentration after dilution is 1 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 company, 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) Returning blue: 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 Dewatering: 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 Sealing plate): 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).
Immunohistochemical staining results were divided into: positive (tan staining in antigen expressing cells of a specific tissue, such as endothelial cells, with low background) and negative (no tan staining in cells of a specific tissue). The rabbit monoclonal antibody A is used for combining with a sample to be detected, and the positive sample has specific brown staining on human tonsils, human lungs, high-grade urothelial cancers and human lung squamous cancers, the brown staining is accurate in positioning, clear in staining and free of non-specific staining, and the background is clean; the detection sensitivity of 4 positive samples was 4/4=100%. The staining is negative in negative samples of human lung adenocarcinoma, human liver and human glioblastoma, and the detection specificity of 3 negative samples reaches 3/3=100%. The partial staining results are shown in FIG. 7, wherein graphs A-D are the staining results of human lung, human tonsils, human lung squamous carcinoma, and human lung adenocarcinoma tissues in that order. The results show that the antibody A provided by the invention can identify the naturally expressed CD141 protein in the tissue cell sample, has high antibody specificity and strong anti-cell component interference capability, has no non-specific binding, ensures the accuracy and reliability of the detection result, and is beneficial to avoiding false positive and false negative results.
EXAMPLE 5 Flow cytometric analysis of Rabbit monoclonal antibodies (FC)
In this example, positive cells (human monocytic leukemia cell line THP-1) with high expression of CD141 and negative cells (human T lymphocyte leukemia cell line Jurkat) without expression of CD141 are selected as samples to be tested, and the detection method comprises the following steps: 1) Ultraviolet irradiation sterilization super clean bench for 15-20min, and blower fan for 5min, preparing for aseptic work; 2) Collecting and washing cells, and then determining the total number of cells, and checking whether the cell viability is about 95% and not less than 90%; 3) Cells were resuspended to about 3X 10 6-5×106 cells/mL with 1 XPBS solution, dispensed into 96-well V-plates, 100 μl per well, washed once with 1 XPBS; 4) L/D staining was performed with a Zombie NIR (Live/DEAD STAINING solution), which was first performed with 1 XPBS at 1:1500, and then distributing the diluted L/D staining solution into a 96-well V-shaped plate according to 100 mu L of each well, re-suspending cells in each well, and mixing; 5) Wrapping with aluminum foil paper, and gently mixing on a microplate vibration mixer for 15min; 6) 400g was centrifuged for 5min, the supernatant was discarded and washed 2 times with FACS buffer; 7) 1X INTRACELL ULAR fixative (Fixation buffer) was dispensed into 96-well V-plates at 100. Mu.L per well, cells in each well resuspended, and mixed; 8) Wrapping with aluminum foil paper, and gently mixing on a microplate vibration mixer for 30min; 9) 400g for 5min, discarding supernatant, and washing with 1× Permeablization buffer for 2 times; 10 100. Mu.L of antibody A diluted with 1 XPerfer (working concentration of primary antibody 2. Mu.g/mL) was dispensed into a 96-well V-plate per well, and the cells in each well were resuspended and mixed; 11 Wrapping with aluminum foil paper, and gently mixing on a microplate vibration mixer for 30min;12 400g for 5min, discarding the supernatant, and washing with 1X Permeablization buffer times; 13 100 μl per well would be measured with 1 x Permeablization buffer at 1:200 diluted Fluorescein (FITC) AffiniPure F (ab') 2Fragment Goat Anti-Rabbit IgG fluorescent secondary antibodies (purchased from jackson, cat# 111-096-046) were dispensed into 96-well V-plates, cells in each well resuspended, and mixed; 14 Wrapping with aluminum foil paper, and gently mixing on a microplate vibration mixer for 30min;15 1× Permeablization buffer washes 2 times; 16 Resuspension each well of cells with 200 μl FACS buffer, and preserve in the dark; 17 Using AND maintaining SOP-105-AND-CA-008 operations with Beckman cytoflex flow analyzers. Antibodies of identical species origin, identical subtype and sub-chain as the streaming primary antibody (i.e., antibody a) were used as isotype controls.
FIG. 8 shows the results of flow cytometry after binding of rabbit monoclonal antibody A to positive cells THP-1 (panel A) and negative cells Jurkat (panel B), with the abscissa representing relative fluorescence intensity, the ordinate representing cell number, the red curve as negative control, the blue curve as isotype control, and the green curve as rabbit monoclonal antibody A. Flow cytometry detection results are divided into: positive (specific signal transitions on cells) and negative. As can be seen from the graph, the anti-human CD141 antibody A has specific binding on THP-1 cells and specific signal transition of 1.8 orders of magnitude, and has no signal transition on Jurkat cells, which proves that the antibody can specifically recognize CD141 protein in a natural state and has no non-specific recognition, further proves that the antibody has high specificity and strong anti-interference capability, and is beneficial to ensuring the precision, the accuracy, the reliability and the like of immunodetection.
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 human CD141 protein, wherein the rabbit monoclonal antibody is a first antibody or a second antibody, wherein:
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively;
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.
2. The rabbit monoclonal antibody against human CD141 protein according to claim 1, characterized in that the amino acid sequence of the light chain variable region of the first antibody 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;
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.
3. The rabbit monoclonal antibody against human CD141 protein according to claim 2, characterized in that the amino acid sequence of the first antibody light chain is shown in SEQ ID No.1 and the amino acid sequence of the heavy chain is shown in 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.
4. The rabbit monoclonal antibody against human CD141 protein according to claim 1, wherein the first or second antibody is a full length antibody or antibody fragment;
The antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
5. A nucleic acid molecule encoding the first antibody and/or the second antibody of any one of claims 1-4.
6. A recombinant vector or host cell comprising a nucleic acid molecule encoding the first antibody and/or the second antibody of any one of claims 1-4.
7. Use of a rabbit monoclonal antibody against human CD141 protein according to any one of claims 1-4, a nucleic acid molecule according to claim 5 or a recombinant vector or host cell according to claim 6 for the preparation of a kit for immunodetection of human CD141 protein.
8. A kit for immunodetection of human CD141 protein, comprising the first antibody and/or the second antibody of any one of claims 1-4.
9. The kit for immunodetection of human CD141 protein of claim 8, wherein the kit is an enzyme immunoassay kit, an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescent kit, an immunoblotting kit, or a flow cytometry kit.
10. The kit for immunodetection of human CD141 protein of claim 9, wherein said kit is an immunohistochemical kit or a flow cytometry kit, said kit comprising said first antibody, and said first antibody is a primary antibody, and an anti-rabbit IgG antibody conjugated with a detectable label is a secondary antibody;
Or the kit is a double-antibody sandwich ELISA kit, the kit comprises the first antibody and the second antibody, the first antibody is a primary antibody, and the second antibody coupled with a detectable label is a secondary antibody.
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