CN117069848B - Anti-human CD146 rabbit monoclonal antibody and its application - Google Patents
Anti-human CD146 rabbit monoclonal antibody and its application Download PDFInfo
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human CD146 rabbit monoclonal antibody and application thereof. The anti-human CD146 rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise complementarity determining regions, the amino acid sequences of the light chain complementarity determining region 1, the light chain complementarity determining region 2 and the light chain complementarity determining region 3 are respectively shown in SEQ ID NO.5-7, and the amino acid sequences of the heavy chain complementarity determining region 1, the heavy chain complementarity determining region 2 and the heavy chain complementarity determining region 3 are respectively shown in SEQ ID NO. 8-10. The rabbit monoclonal antibody provided by the invention can specifically identify and combine human CD146 protein and only identify a target protein target of human, has good specificity and high target affinity, can be applied to the technical field of immunological diagnosis and detection, is especially suitable for the field of flow cytometry detection, and has good adaptability to various fluorescence.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to an anti-human CD146 rabbit monoclonal antibody and application thereof.
Background
CD146, also known as Melanoma Cell Adhesion Molecule (MCAM) or cell surface glycoprotein MUC18, is a single-chain membrane-penetrating glycoprotein belonging to the immunoglobulin superfamily (Igsf) members and having homology to a number of cell adhesion molecules. As a member of the adhesion molecule, CD146 mediates intercellular and intercellular interactions by interacting with its ligand, and upon activation, CD146 can form a complex with p59fyn through its cytoplasmic region. The activated p59fyn can further cause the tyrosine kinase p125PAK to phosphorylate and bind to the pilin (paxillon), which also suggests that CD146 can conduct and communicate extracellular to intracellular signals through its intracellular domain, helping in local adhesive aggregation, cytoskeletal reorganization, maintenance of cell structure and regulation of cell function. In addition to the adhesion molecular property, recent researches show that CD146 is active in various cell physiological processes, and the expression of the CD146 is found in vascular endothelial cells, activated T lymphocytes, smooth muscles, bone marrow interstitial cells and the like, and the CD146 participates in development, signal transduction, cell migration, mesenchymal stem cell differentiation, angiogenesis, immune response and the like, so that the CD146 has wide physiological and pathophysiological significance. CD146 is used as a surface receptor, can trigger tyrosine phosphorylation of FYN and PTK2/FAK1, enables the intracellular calcium concentration to be transiently increased, participates in signal transmission from endothelial extracellular signals to cells, promotes polarization of actin by activating a P125FAK channel, participates in actin cytoskeletal rearrangement, promotes melanoma metastasis by activating an Id-1 channel, and induces tumor angiogenesis by activating NF kappa B/P38. CD146 has been shown to be highly involved in the development of melanoma, prostate cancer, breast cancer, lung cancer, liver cancer, breast cancer, urothelial cancer, chorioapithelial cancer, osteosarcoma, etc., and in the development of these tumors, metastasis, therapy and prognosis, and has become a diagnostic marker molecule for various diseases and a candidate target for drug development.
By measuring human plasma soluble CD146 levels, diagnostic assessment of cancer response and prognosis of cancer treatment such as melanoma can be made. Related studies have demonstrated that normal human subjects aged 62.64 + -10.44 years have plasma soluble CD146 levels ranging from 165.52 + -98.47 ng/mL (male: 175.91 + -101.67 ng/mL, female: 96.70+ -40.20 ng/mL). Based on the diagnostic value of CD146, the specific CD146 monoclonal antibody is screened and prepared to specifically identify the human CD146 at the cellular molecular level, thereby having important significance for providing research basis for early diagnosis and treatment of cancers. Currently, there are patents disclosing antibody development concerning CD146, where the detection application mainly satisfies Immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB) and the like, and there are few antibodies satisfying the flow cytometry application, because in the flow cytometry detection process, the target antigen content is low and the interference factors are many, and the specificity and/or affinity of the antibodies are low, which easily causes non-specific binding, so there are few rabbit monoclonal antibodies satisfying the flow cytometry application on the market. Therefore, the development of the humanized rabbit monoclonal antibody with high specificity and high affinity, which can meet the application scene of flow cytometry analysis, has very important clinical significance.
Disclosure of Invention
Aiming at the problems of poor affinity and specificity of an anti-human CD146 antibody and the like in the prior art, the invention provides an anti-human CD146 rabbit monoclonal antibody and provides application of the rabbit monoclonal antibody in preparing a human CD146 protein detection reagent and/or a detection kit.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
In a first aspect, the invention provides an anti-human CD146 rabbit monoclonal antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise complementarity determining regions, the amino acid sequences of the light chain complementarity determining region 1, the light chain complementarity determining region 2 and the light chain complementarity determining region 3 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively, and the amino acid sequences of the heavy chain complementarity determining region 1, the heavy chain complementarity determining region 2 and the heavy chain complementarity determining region 3 are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.
Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4.
Further, the anti-human CD146 rabbit monoclonal antibody also comprises a light chain constant region and a heavy chain constant region, wherein the light chain constant region and the light chain variable region form a complete light chain, the amino acid sequence of the light chain is shown as SEQ ID NO.1, the heavy chain constant region and the heavy chain variable region form a complete heavy chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2.
Further, the anti-human CD146 rabbit monoclonal antibody is a full-length antibody or an antibody fragment selected from at least one of a Fab fragment, a F (ab) 2 fragment, an Fv fragment, (Fv) 2 fragment, an scFv fragment, and an sc (Fv) 2 fragment.
In a second aspect the invention provides a nucleotide molecule for encoding an anti-human CD146 rabbit monoclonal antibody as described above.
In a third aspect the present invention provides a recombinant vector carrying a nucleotide molecule as described above.
Further, the starting vector of the recombinant vector is a mammalian expression vector pBR322.
In a fourth aspect the invention provides a host cell carrying a nucleotide molecule as described above or comprising a recombinant vector as described above.
Further, the host cell is a 293F cell.
In a fifth aspect, the invention provides the use of an anti-human CD146 rabbit monoclonal antibody as described above, a nucleotide molecule as described above, a recombinant vector as described above, a host cell as described above for the preparation of a human CD146 protein detection reagent and/or a detection kit.
Further, the detection method includes at least one of an enzyme immunoassay, an enzyme-linked immunosorbent assay, an immunohistochemical method, an immunofluorescence method, an immunoblotting method and a flow cytometry.
The invention has the advantages and positive effects that:
The rabbit monoclonal antibody with the complementarity determining region sequence provided by the invention can specifically identify and bind with human CD146 protein and only identify a target point of human target protein, has good specificity and high target affinity, can be applied to the technical field of immunological diagnosis and detection, is especially suitable for the field of flow cytometry detection, and has good adaptability to various fluorescence.
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 graph showing the results of ELISA detection of immune serum titer of New Zealand white rabbits according to example 1 of the present invention;
FIG. 2 is a flow cytometry analysis and detection chart of the combination of immune serum and HUVEC of New Zealand white rabbits of the invention, from left to right, with serum dilutions of 1:500, 1:1000 and 1:2000 respectively, wherein the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is the serum to be tested;
FIG. 3 is a flow cytometry analysis and detection chart of the combination of immune serum and positive cells Hela of New Zealand white rabbits according to the invention, from left to right, the serum dilutions are respectively 1:500, 1:1000 and 1:2000, wherein the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is the serum to be tested;
FIG. 4 is a flow cytometry analysis and detection chart of the combination of immune serum and positive cells A431 of New Zealand white rabbits in example 1 of the present invention, wherein the serum dilutions are respectively 1:500, 1:1000 and 1:2000 from left to right, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is the serum to be tested;
FIG. 5 is a diagram of a mammalian starting vector used for constructing a rabbit monoclonal antibody expression vector according to example 1 of the present invention, from left to right, respectively, a pRB322 vector map carrying a heavy chain constant region and an antibody light chain constant region in advance;
FIG. 6 is a graph showing the results of immunoblotting detection of the rabbit monoclonal antibody 1E1 of example 2 in combination with different samples;
FIG. 7 shows a flow cytometry analysis assay of binding of rabbit monoclonal antibody 1E1 to HUVEC as a positive cell in example 3 of the present invention (the fluorescent secondary antibody is 488 In the figure, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is antibody 1E1;
FIG. 8 is a flow cytometry analysis assay of the binding of rabbit monoclonal antibody 1E1 of example 3 to negative sample MCF-7 of the present invention (fluorescent secondary antibody is 488 In the figure, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is antibody 1E1;
FIG. 9 shows a flow cytometry analysis assay of binding of rabbit monoclonal antibody 1E1 to HUVEC as a positive cell in example 3 of the present invention (the fluorescent secondary antibody is 647 In the figure, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is antibody 1E1;
FIG. 10 is a flow cytometry analysis assay of the binding of rabbit monoclonal antibody 1E1 of example 3 to negative sample MCF-7 of the present invention (fluorescent secondary antibody is 647 In the figure, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is antibody 1E1.
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, the terms "comprising," "including," "containing," "having," and the like are intended to be non-limiting, as other steps and other ingredients may be added that do not affect the result.
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 include the cases of (i) A, (ii) B, and (iii) A and B.
In the context of the present invention, the terms "rabbit monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to Human (Human) CD146 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 and antibody fragments, as well as 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, examples of antibody fragments in particularly preferred embodiments include Fab, fab', (Fab) 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 classified into two types, kappa (kappa) and lambda (lambda) chains, respectively, heavy chains can be classified into five types, mu, delta, gamma, alpha and epsilon 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) through which the heavy and light chain CDRs are held closely together and cooperate to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, which determine the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. Framework regions are the more conserved parts of VH and VL, which are generally in the β -sheet configuration, connected 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 order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs may be identified according to Kabat definitions, chothia definitions, a combination of both Kabat and Chothia definitions, abM definitions, contact definitions, IMGT unique numbering definitions and/or conformational definitions, or any CDR determination method known in the art. As used herein, CDRs are defined by Kabat.
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 CHl, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.
Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.
The term "Antigen binding fragment (Fab)" is a region of an antibody molecule that binds to an Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain structure (variable and one constant region fragment), whereby fragments such as Fab, F (ab ') 2, fab' can be obtained by proteolytic cleavage of the full-length antibody. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain, and into one F (ab ') 2 fragment and one pFC' fragment by pepsin. The F (ab ') 2 fragment may be further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has the same antibody-antigen affinity as a single-chain antibody (scF v), excellent tissue penetrability and the like, but also has a more stable structure, thereby playing a great role in clinical diagnosis and treatment.
The term "variable fragment (Fv)" refers to a minimum antibody fragment comprising only a variable region, located at the N-terminal portion of an antibody Fab fragment and consisting of a variable region of one light chain and one heavy chain, which is a dimer of one VH and one VL that is non-covalently bound (VH-VL dimer), the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than the whole antibody.
The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker) consisting of 10 to 25 amino acids, which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibody molecules, scfvs have the characteristic of small molecular weight, and thus have higher penetration and lower immune side reactions.
The full length sequences of the antibodies or antibody fragments of the invention may be from a single species, such as rabbit, or may be chimeric or humanized antibodies to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" antibody refers to an antibody in which a portion is derived from a particular source or species (rabbit) while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases the variable regions of the non-human antibody bind to the constant regions of a human antibody, such as a human rabbit chimeric antibody, and in other cases the CDR regions of the non-human antibody bind to the FR regions and constant regions derived from the human antibody sequence, i.e., the CDR region sequences of the non-human antibody are grafted onto a human antibody Framework (FR) sequence derived from a single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from the rabbit-derived CDR regions of the present invention.
The term "monoclonal antibody" refers to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the structure, source, or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides an anti-human CD146 rabbit monoclonal antibody, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise Complementarity Determining Regions (CDRs), the amino acid sequences of the light chain complementarity determining region 1 (LCDR 1), the light chain complementarity determining region 2 (LCDR 2) and the light chain complementarity determining region 3 (LCDR 3) are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the amino acid sequences of the heavy chain complementarity determining region 1 (HCDR 1), the heavy chain complementarity determining region 2 (HCDR 2) and the heavy chain complementarity determining region 3 (HCDR 3) are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.
The specificity of an antibody is the structural complementarity between the site at which the antibody recognizes and binds an antigen and an epitope, which consists of residues of amino acid sequences primarily from the Complementarity Determining Regions (CDRs), i.e., the affinity and specificity of an antibody depend in large part on the amino acid sequences of the Complementarity Determining Regions (CDRs) of the antibody. The rabbit monoclonal antibody with the CDR sequence can specifically identify and bind human CD146 protein, a positive cell sample (human umbilical vein endothelial cell HUVEC, human cervical cancer cell Hela and rat adrenal gland pheochromocytoma cell PC-12) for expressing the CD146 protein and positive tissue samples (Mouse Lung, mouse Testis and Rar Lung) of different species are detected by a Western blot method (Western blot), a single strip is detected in the human positive sample, the single strip is clear, and relevant cells or tissue samples of mice and rats are not identified, and the antibody is proved to only identify relevant targets of the species as human, so that the specificity is good, further fluorescent transition is good when the positive cell HUVEC and the negative cell human breast cancer cell MCF-7 are detected by a flow cytometry, fluorescent transition is not generated in the negative cell during the detection, namely, the monoclonal antibody for resisting the human CD146 has high affinity and high specificity with the human CD146 protein, and can be applied to the field of immunology antibody diagnosis and treatment of the field, and the application of the antibody is particularly applicable to the field of immunology cell diagnosis and the field.
Optionally, the light chain variable region and the heavy chain variable region each comprise a Framework Region (FR), 4 FR and 3 CDRs are sequentially staggered to form a variable region, and correspondingly, the amino acid sequence of the light chain variable region (VL) of the anti-human CD146 rabbit monoclonal antibody of the invention is shown in SEQ ID No.3, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID No. 4.
Optionally, the anti-human CD146 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 complete light chain, the heavy chain constant region and the heavy chain variable region comprising a complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, e.g., via IMGT online database (www.imgt.org), searching rabbit source IGG GAMMA CREIGN for heavy chain constant regions, and searching rabbit source IGG KAPPA C REIGN for light chain constant regions.
Illustratively, the amino acid sequence of the light chain comprising the light chain constant region is shown in SEQ ID NO.1 and the amino acid sequence of the heavy chain comprising the heavy chain constant region is shown in SEQ ID NO. 2.
Alternatively, the anti-human CD146 rabbit monoclonal antibody is a full length antibody or antibody fragment, including, but 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 of the hinge region, (iii) an Fv fragment consisting of one heavy chain variable region and one light chain variable region of the antibody, (iv) (Fv) 2 fragment, a Fv fragment consisting of two Fv fragments covalently linked together, (v) an scFv fragment consisting of a single polypeptide chain, one heavy chain variable region and one light chain variable region linked by a linker, (vi) a sc (Fv) 2 fragment, a linker, and the like. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.
The antibody fragments of the invention have the CDR regions (SEQ ID NOS.5-10) as described above, more preferably the variable regions (SEQ ID NOS.3-4) as described above, leaving intact antigen recognition and binding sites capable of binding to the same antigen, in particular to the same epitope, as the full length antibody.
Alternatively, the immunogen of the anti-human CD146 rabbit monoclonal antibody is human recombinant CD146 protein, which is expressed by a mammalian cell expression system, the protein sequence of the human CD146 protein is referred to as NP-006491.2 (Uniprot ID: P43121), and the nucleic acid sequence is referred to as NM-006500.3.
Yet another embodiment of the present invention provides a nucleotide molecule for encoding an anti-human CD146 rabbit monoclonal antibody as described above.
The nucleotide molecule is typically DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA), the nucleic acid molecule may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand. The sequence of the nucleotide 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 nucleotide 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 carrying a nucleotide molecule as described above.
Typical vectors of the invention include plasmids, viruses, 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 nucleotide 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 nucleotide molecule inserted into the vector in a cell). Thus, a cloning vector may comprise a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector comprises regulatory elements (e.g., promoters, enhancers) for expression in a specified host cell. The nucleotide molecules of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleotide molecules of the invention. This is well known in the art and will not be described in detail herein.
It is noted that the nucleotide molecules encoding the heavy and light chains of an antibody of the invention may be cloned into a vector, each nucleotide sequence being linked downstream of a suitable promoter. For example, each of the nucleotide sequences encoding the heavy and light chains may be operably linked to a different promoter, or the nucleotide sequences encoding the heavy and light chains may be operably linked to a single promoter such that both the heavy and light chains are expressed from the same promoter. In other embodiments, the nucleotide molecules encoding the heavy and light chains of an antibody of the invention may also be separately constructed 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.
Alternatively, the recombinant vector comprises a prokaryotic vector (e.g., E.coli vector, a Bacillus subtilis vector), a eukaryotic vector (e.g., a yeast vector), or a viral vector (e.g., a lentivirus, an adenovirus).
In a preferred embodiment, the starting vector of the recombinant vector is the mammalian expression vector pBR322.
Yet another embodiment of the invention provides a host cell carrying a nucleotide molecule as described above or comprising a recombinant vector as described above.
Host cells include prokaryotic cells and eukaryotic cells, and are selected according to the type of recombinant vector, for example, when prokaryotic vectors are used, prokaryotic cells are selected as host cells, and commonly used prokaryotic host cells include E.coli, bacillus subtilis, and the like. In the case of eukaryotic vectors, eukaryotic cells are selected as host cells, and commonly used eukaryotic host cells include yeast, mammalian cells, and the like. After obtaining a host cell transformed with a nucleotide molecule as described above or comprising an expression vector as described above, the cell is cultured under suitable conditions to express the monoclonal antibody, and then the expressed monoclonal antibody is isolated.
In a preferred embodiment, the host cell is a 293F cell (human embryonic kidney cell).
The embodiment of the invention also provides application of the anti-human CD146 rabbit monoclonal antibody, the nucleotide molecule, the recombinant vector and the host cell in preparation of human CD146 protein detection reagents and/or detection kits.
The anti-human CD146 rabbit monoclonal antibody disclosed by the invention has high affinity and good specificity for human CD146 protein, can be used as an antigen binding antibody (capture antibody), can specifically identify and bind to target protein in a sample to be detected, can be connected with a luminescent marker or specifically bind to a detection antibody coupled with the luminescent marker, wherein the luminescent marker can be one of colloidal gold, chemical dye, fluorescent dye and the like, and is used for generating identifiable signal change, so that qualitative or quantitative detection of the human CD146 protein is realized. The anti-Human CD146 rabbit monoclonal antibody can be used for establishing an immunoassay method of the Human CD146 protein with good specificity and high sensitivity, in particular to multi-fluorescence flow type analysis.
Alternatively, immunodetection assays include, but are not limited to, enzyme immunoassays (Enzyme immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunospot (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (Immunohistochemistry, IHC), immunofluorescence (IF), immunoblotting (Western blot, WB) and Flow Cytometry (FC).
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
EXAMPLE 1 preparation of Rabbit monoclonal antibodies against human CD146 protein
The immunogen is Human recombinant CD146 protein, 24aa-559aa sequence of Human CD146 protein is constructed into pYURK-Chis vector, the nucleic acid sequence of Human CD146 protein is see NM_006500.3, the protein sequence is see NP_006491.2, uniprot is numbered as P43121, then pYURK-Chis vector is transferred into 293F cell, high quality recombinant Human CD146 mature protein 24aa-559aa with biological activity is expressed in the 293F cell, the recombinant Human CD146 mature protein is used as immunogen to immunize New Zealand white rabbits, and the rabbit monoclonal antibody against Human CD146 protein is obtained based on single B lymphocyte screening and monoclonal antibody development technology of culture. The foregoing CD146 protein expression and purification methods employ procedures conventional in the art.
1. Immunization of animals
The recombinant Human CD146 mature protein is used as an immunogen to respectively immunize 4 New Zealand white rabbits, 200 mug of the immunogen is immunized on each white rabbit, the immunogen is mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifier before the first immunization, the emulsifier is subcutaneously injected into the abdomen and the back of the rabbit, 100 mug of the immunogen is mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare the emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of the rabbit for two times. Serum samples of rabbits were collected after three immunizations, titers against Human CD146 were determined by ELISA, titers were determined by ELISA after dilution of serum at 1:243K, rabbits with OD450nm exceeding 0.2 were boosted by 200 μg immunogen subcutaneous multipoint injection once, animals were sacrificed three days later, and spleens were taken. And (3) determining the specificity of the serum and the positive sample by using an FC method, diluting the serum, and detecting the affinity of the serum and the sample to be tested by using the FC.
Immune serum titer is measured by indirect enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), and the specific procedures include:
1) Coating recombinant Human CD146 protein diluted with 1 XPBS buffer (final concentration 1. Mu.g/mL), adding coating solution, 25. Mu.L/well into 384-well plate, balancing 384-well plate, and centrifuging briefly (stopping rpm reaching 1000) to obtain plate, and standing at 4deg.C overnight;
2) Blocking, taking out overnight plates at 4℃and washing 5 times with a plate washer with 75. Mu.L of wash buffer (PBS containing 0.05% (v/v) Tween-20) per well, adding 50. Mu.L of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) per well to plates using a row gun, and incubating for 1h at room temperature for blocking irrelevant binding sites;
3) The serum to be tested is subjected to gradient dilution and sample addition, wherein the plate washing process is repeated, the serum to be tested is subjected to gradient dilution in a 96-well plate, the serum is subjected to triple dilution with a dilution buffer (PBS contains 1% BSA) at a ratio of 1:1000, 8 gradients are added, and the dilution ratio can be adjusted according to actual conditions;
4) Secondary antibody incubation, namely washing a pore plate in a repeated plate washing process, diluting horseradish peroxidase (HRP) -coupled goat anti-rabbit IgG with a dilution buffer solution at a dilution ratio of 1:5000, adding diluted goat anti-rabbit IgG-HRP into a plate by using a row gun according to 25 mu L/hole, combining target proteins in the plate, and incubating for 1h at room temperature in a dark place;
5) And (3) stopping the reaction and developing, namely cleaning the pore plate by repeating the plate washing process, adding TMB into the pore plate according to 100 mu L/hole for developing, keeping out of the sun for 10min at 37 ℃, adding 100 mu L/hole of 0.5M oxalic acid solution for stopping the reaction, measuring the absorption value of 450nm, taking the serum of the rabbit before immunization as a negative control, and judging the titer of the immune serum by taking the ratio of the measured value to the control value as positive, wherein the ratio is more than or equal to 2.1.
Serum titers are shown in fig. 1, which shows that the rabbits developed a stronger immune response after the third and fourth booster immunizations, with higher titers of neutralizing antibodies.
The specificity of immune serum and a sample to be tested adopts a Flow Cytometry (FC) method, and the method comprises the following steps:
1) The experiment preparation comprises the steps of irradiating ultraviolet to a disinfection ultra-clean workbench for 15-20min, and starting a fan for 5min to prepare aseptic work;
2) The staining method comprises collecting and washing cells, determining total number of cells, checking whether cell viability is about 95% and not less than 90%, resuspending cells to about 3×10 6-5×106 cells/mL with 1×PBS solution, dispensing cells into 96-well V-plates, washing 100 μl per well with 1×PBS, staining with Live/head (Zombie NIR Fixable Viability Kit, available from Biolegend, cat No. 423106), diluting L/D staining solution (L/D STAINING solution) with 1×PBS at a ratio of 1:1500, dispensing L/D staining solution into 96-well V-plates according to design, dispensing 100 μl per well, resuspending cells in each well, mixing; coating with aluminum foil, gently mixing on a microplate shaking mixer for 15min, centrifuging 400g for 5min, discarding supernatant, washing with FACS buffer for 2 times, coating with 1X INTRACELL ULAR fixative (Fixation buffer) 100 μl per well into 96-well V-shaped plates, mixing, coating with aluminum foil, gently mixing with a microplate shaking mixer for 30min, centrifuging 400g for 5min, discarding supernatant, washing with 1X Permeablization buffer for 2 times, coating with immune serum diluted with 1X Perm buffer into 96-well V-shaped plates, suspending cells in each well for 100 μl per well for 2 times, coating with aluminum foil, gently mixing with 400g for 30min on a microplate shaking mixer, centrifuging for 5min, discarding supernatant for 2 times, washing with 1X Permeablization buffer, coating with Fluorescein (FITC) APure F (ab') 2 Fragment Goat Anti-20-Rabbit diluted with 1:200 (IgG # 6-6 t 100 μl per well for 100 μl of anti-fluorescent cells per well for 096-96V-6V-shaped plates per well for 30 μl of 100%, mixing, wrapping with aluminum foil paper, mixing gently on a microplate shaking mixer for 30min, washing with 1× Permeablization buffer for 2 times, re-suspending each well of cells with 200 μl FACS buffer, and preserving in dark.
3) Flow analysis the analysis was performed using AND maintaining SOP-105-AND-CA-008 operations with Beckman cytoflex flow analyzers.
The cells used in this example include positive cells, human umbilical vein endothelial cells, HUVEC, human cervical cancer cells, hela, and human epidermal cancer cells, A431. The FC detection diagrams of rabbit immune serum diluted by the Human CD146 recombinant protein are shown in figures 2-4, wherein figures 2-4 are respectively flow cytometry detection diagrams of positive cells HUVEC, hela and A431, the serum dilutions are respectively 1:500, 1:1000 and 1:2000 from left to right, the abscissa in the diagrams represents the fluorescence brightness of APC (647), the ordinate represents the cell number, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is serum to be tested. As can be seen from the figure, the fluorescence transition of each serum dilution is better, the animal is determined to be immunized better, antibodies which can be used for flow detection are generated in vivo, and the animal can enter the stage of separating spleen cells.
2. Isolation and culture of B lymphocytes in spleen
Separating spleen cells, namely 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 excessive connective tissue and fat on spleen tissues of rabbits, shearing spleen tissues, placing the spleen tissues into the cell screen for grinding, taking a clean grinding rod, and grinding the tissues by rolling the tail end of the pressed part. The cells in the membrane slowly come out, pass through a cell sieve, are suspended in a culture dish solution, and a cell washing screen is washed by 10mL of basal medium, and the basal medium outside the cell screen is collected. Centrifuging at room temperature for 5min with a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate (BD PHARM LYSETMLYSING Buffer, product number BD 555899) at room temperature, gently blowing off cell clusters with a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min with a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters with a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min with a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters with a pipettor, resuspending cells by a cell screen, filtering the resuspension cells again, removing clustered cells, and counting the cells.
The method for sorting and culturing B lymphocytes in spleen is disclosed in patent 'method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication No. CN 110016462A)' and patent 'an in vitro B lymphocyte culture system and application (publication No. CN 111518765A)'.
3. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected and lysed, and RNA was extracted using Quick-RNA TM Micro Prep kit (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 a plurality of clones are selected for sequencing. The PCR reaction was performed using 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 (available from Eboltag Biotechnology Co., ltd.) and 6.5. Mu. L H 2 O. The PCR amplification procedure consisted of a pre-denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally holding at 72℃for 5min, and the resulting reaction solution was kept at 4 ℃.
The nucleotide sequences of the forward primer and the reverse primer are as follows:
VL-F5'-tgaattcgagctcggtacccatggacacgagggcccccac-3' (see SEQ ID NO: 11);
VL-R5'-cacacacacgatggtgactgttccagttgccacctgatcag-3' (see SEQ ID NO: 12);
VH-F5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3' (see SEQ ID NO: 13);
VH-R5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3' (see SEQ ID NO: 14).
Sequencing the amplified cDNA, completing the sequencing work by Jin Kairui Biotechnology Inc to obtain a light chain variable region (VL) amino acid sequence shown as SEQ ID NO. 3 and a heavy chain variable region (VH) amino acid sequence shown as SEQ ID NO.4, and then inquiring an IMGT online database (www.imgt.org) to obtain a constant region sequence to obtain a light chain amino acid sequence shown as SEQ ID NO.1 and a heavy chain amino acid sequence shown as SEQ ID NO.2, wherein the obtained rabbit monoclonal antibody against Human CD146 protein is named as 1E1. The specific sequences involved in 1E1 are shown in Table 1.
TABLE 1 summary of amino acid sequence information of Rabbit monoclonal antibody 1E1 of the invention
4. Production and purification of Rabbit monoclonal antibody 1E1
In order to obtain a plurality of rabbit monoclonal antibodies recognizing Human CD146 protein, the heavy chain genes and the light chain genes of the plurality of rabbit monoclonal antibodies selected above are respectively loaded on an expression vector, and a mammal expression vector pBR322 used carries light chain and heavy chain constant regions in advance, wherein the expression patterns are shown in FIG. 6, pRB322origin and f1origin are replication promoters in E.coli (E.Coli), AMPCILLIN is plasmid resistance gene, CMV IMMEARLY promotor is promoter in eukaryote, SV40 PA ter minator is tailing signal, HEAVY CHAIN constant is nucleotide sequence of rabbit heavy chain constant region (left graph), LIGHT CHAIN constant is nucleotide sequence of rabbit monoclonal antibody light chain constant region (right graph). The method comprises the steps of carrying out conventional linearization treatment on 2 expression vectors by using NheI and XbaI restriction endonucleases respectively, purifying the amplified PCR products, respectively constructing heavy chain variable region genes and light chain variable region genes with signal peptide coding genes at the upstream into corresponding mammal expression vectors by adopting a homologous recombination mode, carrying out sequencing verification, transfecting the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies into 293F cells together, culturing for 72-96h after transfection, and culturing to obtain supernatant containing the Human CD146 rabbit monoclonal antibodies. Purifying rabbit monoclonal antibody 1E1 recognizing human CD146 protein from culture supernatant by using protein A affinity gel resin, sub-packaging after the antibody is verified to be qualified, and preserving at low temperature of-20 ℃ for standby, wherein the concentration of the obtained antibody is 2mg/mL, and the purity is more than 95%.
The signal peptide of this example may be an antibody expression signal peptide sequence commonly used in the art, and specifically, see "rabbit monoclonal antibody against human interferon. Alpha.2 and its use (publication No. CN116063487A, publication No. 2023-05-05)", wherein the light chain variable region has the signal peptide "MDTRAPTQLLGLLLLWLPGATF" upstream and the heavy chain variable region has the signal peptide "METGLRWLLLVAVLKGVQC" upstream.
Example 2 immunoblot analysis (Western blot, WB) against rabbit monoclonal antibody 1E1
Cell culture, namely collecting the adherent cells, removing the culture solution of the adherent cells, washing the cells with PBS, NS or a serum-free culture medium for 1 time, centrifuging at a low speed, discarding the supernatant, and collecting the sediment. Suspension culture cell collection, namely, centrifuging suspension cells at a low speed, discarding supernatant, collecting sediment, and flicking the cells by fingers to loosen the cells. Tissue sample collection, which is to cut the tissue into small fragments, the smaller the better. Freezing the tissue in liquid nitrogen or ultralow temperature refrigerator for over 30min, grinding with liquid nitrogen, and controlling the grinding process within 1-2min to reduce protein degradation.
The cell samples used in this example include positive cells HUVEC, hela and PC-12, and the tissue samples include positive tissues Mouse Lung, mouse Testis and Rar Lung, wherein positive means that the corresponding cells or tissues express CD146 protein.
Total protein extraction 1) cell/tissue lysis the vessel containing the cell pellet or tissue fragment was fully inserted into ice. The cell pellet was added to the corresponding volume of lysate (3 mL when the cell mass was sufficient, calculated on the basis of the cell mass) at a ratio of 1mL lysate/10 7 cells (1T 75 flask cell mass), lysed for 20min, and the centrifuge tube was placed on a vortex shaker at 5min intervals for 10s. Adding protein lysate to the homogenizer according to 0.5mL lysate/100 mg tissue, grinding every 3min, repeating for 5 times to grind the tissue as much as possible (protease inhibitor is optionally added or not added in the lysate as required), 2) centrifuging, balancing the cracked sample, placing in a precooled high-speed refrigerated centrifuge, centrifuging at 12000rpm for 15min, and 3) protein denaturation, wherein after centrifuging, the supernatant is the protein extract. A small amount of protein extract was aspirated and the protein concentration was determined by BCA method. 5X Loading Buffer (final working solution 1X) of 1/5 supernatant volume was added to the centrifuge tube of the remaining protein extract, after the temperature of the dry thermostat had risen to 95 ℃, 1.5mL centrifuge tube was inserted into the heating well, and the mixture was denatured by heating at 95℃for 10min, and after the liquid had cooled completely, the mixture was stored at-20 ℃.
Immunoblotting experiment 1) preparation of separating gel, namely selecting proper separating gel according to the size of protein, injecting the prepared gel solution into a pre-installed gel preparation device, and adding isopropanol sealing gel. The mixture is horizontally placed at room temperature for about 30min. The preparation method comprises the steps of taking care of preventing glue solution from generating bubbles and injecting the bubbles into a glue making device, adding TEMED before glue injection to prevent solidification before glue injection, slowly dragging and adding the glue along one side of a glass plate when injecting isopropanol, and 2) preparing concentrated glue, namely pouring the isopropanol along one side of the glass plate after separating glue solidification, and sucking the isopropanol by using filter paper. And preparing concentrated gel according to the required volume. And (3) injecting the prepared glue solution into a glue preparation device, slowly inserting the prepared sample comb into the glue solution along one end of the glass plate, and horizontally placing for 20-30min at room temperature. And 3) loading samples, namely after the concentrated glue is solidified, pulling out the sample comb vertically by both hands, and injecting the sample comb into the inner and outer grooves by using Tris-GLYCINE SDS Running Buffer to form a closed loop. The samples were pipetted into vertical comb wells with a protein content of 25 ug/well. The inner tank Tris-GLYCINE SDS Running Buffer needs to be filled, the outer tank Tris-GLYCINE SDS Running Buffer is 3-5cm above the bottom, 4) electrophoresis, after sample loading is finished, the power supply of an electrophoresis apparatus is connected, proper electrophoresis parameters are set, the electrophoresis parameters of the concentrated gel are constant pressure 80V, and the voltage is regulated to 120V when a sample enters the separation gel. When bromophenol blue electrophoreses to the bottom of the gel, stopping electrophoresis, closing the power supply of an electrophoresis apparatus, 5) transferring a membrane, namely, immersing a 0.45 mu mNC membrane in WESTERN TRANSFER Buffer for standby, transferring a protein band on the gel to a mNC membrane in an electrotransfer system according to a conventional method, 6) sealing, namely, after electrotransfer, putting the mNC membrane into a proper antibody incubation tank, adding Western Blocking Buffer room temperature to incubate 1mL, 7) HRP-labeled primary antibody, namely, diluting the primary antibody 1E1 with the HRP label by WESTERN TRANSFER Buffer for 1:217000, pouring diluted primary antibody working solution (the final concentration of the antibody is 0.01 mu g/mL) into the incubation tank, and standing at room temperature for 2 hours or 4 ℃, 8) washing by adding TBST Buffer for 4 times after the primary incubation is finished, 5 min/time, 9) exposing, namely, using 1-2mL working solution per 10cm 2 membrane, sucking volume SolutionI and evenly mixing the working solution with SolutionII in proportion to prepare the luminescence detection working solution. The membrane was removed with flat-headed forceps and the lower edge of the membrane was gently contacted with absorbent paper to remove excess liquid from the membrane. The working solution is added on the transfer film by a liquid transfer device so as to cover the transfer film, and the transfer film is incubated for 1-2min at room temperature, and the step can be finished on a clean preservative film or in a plastic box. And (3) tabletting detection, namely fixing the film in a tablet clamp, pressing the tablet in a darkroom for 1min, immediately developing and fixing, and adjusting the tabletting time according to the result. Or directly tabletting for 30s, 1,3 and 5min respectively, and then developing and fixing the observation results together, and 10) detecting by a fluorescence imager, namely placing the film into the fluorescence imager and detecting by referring to the instruction manual of the instrument.
The results of the WB detection of rabbit monoclonal antibody 1E1 against the Human CD146 protein are shown in fig. 6, where 72KD and 120KD refer to 72KD and 120KD as references, 1:217000 refers to an antibody dilution ratio of 1:217000, working concentration is the final diluted antibody concentration, t=30s, and exposure 30s. In this embodiment, the detection samples HUVEC and Hela are human-related samples, the detection samples Mouse Lung and Mouse Testis are Mouse-related samples, and the detection samples PC-12 and Rar Lung are rat-related samples. From the figure, only the relevant positive sample of human has a single clear band, which proves that the antibody 1E1 recognizes the relevant target of the species as human, the mouse and the rat are not recognized, and the impurity band is not arranged on the lanes, thus proving that the rabbit monoclonal antibody 1E1 has high specificity and good affinity.
Example 3 Flow cytometric analysis (FC) against rabbit monoclonal antibody 1E1
The cell samples of this example include a positive sample HUVEC and a negative sample MCF-7, wherein positive means that the corresponding cell or tissue expresses CD146 protein, and negative means that the corresponding cell or tissue does not express CD146 protein. The assay is described in example 1, wherein the primary antibody is rabbit monoclonal antibody 1E1 of the invention, the concentration of the antibody is 0.2. Mu.g/mL, and the fluorescent secondary antibody is488 (Fluorescein (FITC) AffiniPure F (ab') 2Fragment Goat Anti-Mouse IgG, fcgamma FRAGMENT SPECIFIC, available from jackson under the accession number 115-096-071) and647(Alexa647AffiniPure Goat Anti-Mouse IgG, fcγ FRAGMENT SPECIFIC, available from jackson under accession number 115-605-071) to verify the suitability of antibodies for different fluorescence. The antibody 1E1 of the invention recognizes CD146 protein, the fluorescent secondary antibody 1E1 can judge the specificity and affinity of the primary antibody by recognizing the fluorescence intensity (fluorescence transition) and the fluorescence cell ratio, and the results are shown in figures 7-10, wherein figures 7-8 are HUVEC and MCF-7 respectivelyFC detection of 488 fluorescence, FIGS. 9-10 are HUVEC and MCF-7, respectivelyFC detection plot of 647 fluorescence.
The graph shows that the fluorescence transition is better, which indicates that the antibody recognition target human CD146 protein has high affinity, a unique peak exists positively, the specificity of the antibody is high, and the two fluorescence detection results of 488 and 647 are better, which indicates that the antibody can meet the detection characteristic of multiple fluorescence.
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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| CN116396384B (en) * | 2023-01-17 | 2023-11-17 | 武汉爱博泰克生物科技有限公司 | Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit |
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| CN102936283A (en) * | 2008-01-31 | 2013-02-20 | 中国科学院生物物理研究所 | Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method |
| CN104395470A (en) * | 2012-04-27 | 2015-03-04 | 米伦纽姆医药公司 | Anti-GCC antibody molecules and use of same to test for susceptibility to GCC-targeted therapy |
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