CN117430700A - Anti-vimentin antibody or antigen binding fragment thereof, kit and application - Google Patents
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- CN117430700A CN117430700A CN202311474843.0A CN202311474843A CN117430700A CN 117430700 A CN117430700 A CN 117430700A CN 202311474843 A CN202311474843 A CN 202311474843A CN 117430700 A CN117430700 A CN 117430700A
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses an anti-vimentin antibody or an antigen binding fragment thereof, a kit and application, and relates to the technical field of antibodies. The antibody or the antigen binding fragment thereof provided by the invention can be specifically combined with the vimentin, has better combination activity and affinity, and can improve the sensitivity and the specificity of detection by using the antibody or the antigen binding fragment thereof to detect the vimentin. Therefore, the method can be used for developing detection products for detecting vimentin. In addition, the antibodies or antigen binding fragments thereof may be used to diagnose diseases that are labeled with vimentin. Is beneficial to early discovery and early intervention treatment of diseases. The invention provides more protein choices for the detection of vimentin and the diagnosis, prognosis, efficacy evaluation and recurrence monitoring of diseases with vimentin as a marker.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-vimentin antibody or an antigen binding fragment thereof, a kit and application.
Background
Vimentin, is expressed positive for immunohistochemical Vimentin, and indicates positive immunohistochemical staining for Vimentin, generally suggesting that there may be a tumor derived from mesenchymal tissue. The vimentin is a component of fibroblast, belongs to an important component of cytoskeleton, and can play roles in judging tissue sources and detecting antigen preservation conditions in clinical examination.
Since vimentin is mainly distributed in mesenchymal cells, and tumors of mesenchymal origin, it is pathologically mainly used for diagnosis of tumors of mesenchymal origin, such as hemangioma fibroma, neurofibroma, leiomyosarcoma, etc. Although vimentin antibodies are highly sensitive, they have low specificity, and clinically, it is not possible to determine what tumor is aberrantly expressed by only immunohistochemical vimentin is positive. Vimentin positive can be used as a marker for tumor diagnosis and treatment, and can also be used for identifying some diseases, such as endometrial adenocarcinoma positive expression, and cervical adenocarcinoma negative expression.
Clinical detection of protein expression in tumor cells is often performed by Immunohistochemical (IHC) pathology experiments, and the accuracy and sensitivity of the IHC experiments are determined by the quality of monoclonal antibodies that specifically bind the protein. Therefore, developing a monoclonal antibody with high binding specificity aiming at the Vimentin protein has important significance for detecting the expression level of the Vimentin protein.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an antibody for resisting vimentin or an antigen binding fragment thereof, a kit and application thereof so as to solve the technical problems.
The binding protein provided by the invention can specifically bind with the Vimentin protein, has better binding activity and affinity, and can improve the sensitivity and specificity of detection when used for detecting the Vimentin protein.
Noun definition
Vimentin is a structural protein encoded by VIM gene in human body and named Vimentin. The Vimentin protein is synonymous with the Vimentin protein herein.
The term "antibody or antigen-binding fragment thereof" refers broadly to all proteins/protein fragments comprising CDR regions, in particular antibodies or antigen-binding fragments thereof. "antigen binding fragments" include antigen compound binding fragments of these antibodies described above, including Fab, F (ab') 2, fd, fv, scFv, bispecific antibodies, multispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD, etc. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
The term "antibody" is used herein in its broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specifically binding to an HRP-II antigen or fragment thereof. An "antibody fragment" includes a portion of a full length antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.
Typically, the variable regions VH/VL of the heavy and light chains of an antibody are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
The invention is realized in the following way:
in a first aspect, the invention provides an antibody or antigen-binding fragment thereof against vimentin, the antibody comprising: a heavy chain variable region comprising CDR-VH1, CDR-VH2 and CDR-VH3 and a light chain variable region comprising CDR-VL1, CDR-VL2 and CDR-VL3; the amino acid sequence of each CDR region is as follows:
CDR-VH1：DYWID；
CDR-VH2：EISPGTGRISFNETFNG；
CDR-VH3：DDS；
CDR-VL1：KSSQSLLHSDGKTYLN；
CDR-VL2：LVSKLDP；
CDR-VL3：WQGTHFPT。
the amino acid sequence of the complementarity determining region is a novel sequence which is discovered and disclosed for the first time in the present invention and which can confer the ability of the binding protein to specifically bind to the antigen of the Vimentin protein. Based on the fact that the binding protein has good binding activity and affinity, the binding protein can be used for developing detection products for detecting the Vimentin protein, such as detection reagents and kits, and the sensitivity and the specificity of detection can be improved by using the binding protein to detect VINMENTIN protein. In addition, the binding protein can be used for diagnosing diseases such as cervical cancer and the like using the Vimentin protein as a marker. Is beneficial to early discovery and early intervention treatment of diseases. The invention provides more protein choices for the detection of the Vimentin protein and the diagnosis of diseases taking the Vimentin protein as a marker.
In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof and the Vimentin protein are administered as K D ≤6.907×10 8 L/mol affinity binding.
In an alternative embodiment, the antibody or antigen binding fragment thereof has a K with a Vimentin protein D ≤4×10 9 L/mol、3×10 9 L/mol、2×10 9 L/mol、1×10 9 L/mol、9×10 9 L/mol、8×10 9 L/mol、7×10 9 L/mol、6×10 9 L/mol、5×10 9 L/mol、4×10 9 L/mol、3×10 9 L/mol or 2X 10 9 L/mol affinity.
In a preferred embodiment of the invention, the antibody or antigen binding fragment thereof further comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, which are shown in sequence SEQ ID NO. 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, which are shown in sequence SEQ ID NO. 5-8.
The sequences of SEQ ID NOS 1-8 are shown in the following table:
in preferred embodiments of the use of the invention, the antibody or antigen-binding fragment thereof further comprises a constant region;
in a preferred embodiment of the invention, the constant region comprises a light chain constant region and a heavy chain constant region, the light chain constant region being of the type of either a kappa (kappa) chain or a lambda (lambda) chain constant region; the heavy chain constant region is IgM, igD, igG, igA or IgE heavy chain constant region;
in a preferred embodiment of the invention, the IgG is selected from the heavy chain constant region of any one of IgG1, igG2, igG3, and IgG 4;
in a preferred embodiment of the invention, the constant region of the antibody is derived from bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human;
in a preferred embodiment of the invention, the constant region is derived from a cow, a chicken or a turkey;
in a preferred embodiment of the invention, the constant region is derived from a mouse;
in a preferred embodiment of the invention, the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 9, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 10.
In a preferred embodiment of the invention, the antigen binding fragment is selected from any one of the group consisting of Fab ', fab, F (ab') 2, scFv and Fv of an antibody;
in a preferred embodiment of the invention, the antibody is selected from at least one of a multispecific antibody, a fusion antibody, and a chimeric antibody.
The antigen binding fragments of the above antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that antigen binding fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds.
Antigen binding fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In a preferred embodiment of the invention, the antibody is selected from at least one of a multispecific antibody, a fusion antibody, and a chimeric antibody.
Noun interpretation:
multispecific antibodies are polymers of monoclonal antibodies that recognize different epitopes, which can bind different targets or different epitopes of the same target, and have higher antigen recognition capability than a single monoclonal antibody.
Fusion type antibody: the antibody or antigen binding fragment thereof provided by the invention can be easily combined with other structures (such as BSA, igG-Fc and the like) to form a novel fusion molecule, such as an enzyme, an antibacterial peptide or a developing substance and the like for prolonging the half-life period of the fusion molecule. In the novel fusion molecule, the antibody is bound to its target antigen in a targeted manner, and the moiety fused to the antibody performs the corresponding function. In clinicians, they want the drug to stay in the body long enough, however, the blood clearance of the antibody is fast, which is detrimental to the drug it carries. Therefore, the antibody and the long-life molecule are fused together by the gene technology, so that the existence time of the antibody in blood can be prolonged, namely the half life of the antibody is prolonged, and a better therapeutic effect is achieved.
The "chimeric antibody" according to the present invention includes, but is not limited to, an antibody in which a variable region of a non-human antibody is fused with a constant region or a framework region of a human antibody, and can reduce an immune response induced by the non-human antibody. In other embodiments, the constant regions may be adaptively recombined or optimized from different sources or types as desired.
In a second aspect, the invention also provides the use of an antibody or antigen binding fragment thereof in the preparation of a product for at least one use,
(1) A product for detecting vimentin; and applications are not for the purpose of diagnosis of disease;
(2) Products for diagnosing epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; epithelial tumors, autoimmune encephalitis or rheumatoid arthritis take vimentin as a diagnostic marker;
(3) Products for prognosis of epithelial tumors, autoimmune encephalitis or rheumatoid arthritis; epithelial tumors, autoimmune encephalitis or rheumatoid arthritis take vimentin as a diagnostic marker;
(4) Products for efficacy assessment of epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; epithelial tumors, autoimmune encephalitis or rheumatoid arthritis take vimentin as a diagnostic marker;
(5) Products for monitoring recurrence of epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; epithelial tumors, autoimmune encephalitis or rheumatoid arthritis take vimentin as a diagnostic marker;
the product is a reagent, a kit, a chip, a magnetic bead or a micro-pore plate.
Studies show that one of the Vimentin proteins is an important rheumatoid arthritis antigen, so that diagnosis, prognosis, efficacy evaluation or recurrence monitoring of rheumatoid arthritis can be realized by the anti-Vimentin antibody provided by the invention.
In a preferred embodiment of the invention, the epithelial tumor is papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, renal cancer, adenoma or squamous carcinoma;
in a preferred embodiment of the present invention, gastrointestinal cancer includes, but is not limited to, esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colorectal cancer, and anal cancer;
in preferred embodiments of the invention, the adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma, breast carcinoma, or pancreatic carcinoma.
In an alternative embodiment, breast cancer includes, but is not limited to, breast epithelial cancer;
preferably, the breast cancer is ductal breast cancer or lobular breast cancer.
Breast cancer, preferably stage II to IV and/or poorly differentiated invasive ductal cancer, acne cancer, and medullary cancer (preferably grade 2).
Ovarian cancer, serous and mucinous cancers (preferably stage Ic to stage IIIb), granulosa cell tumors, superficial epithelial-mesenchymal tumors (adenocarcinomas), cystic adenocarcinomas and endometrioid tumors.
Uterine cancer, preferably comprising endometrioid adenocarcinoma (preferably stage I to stage IIIc).
Bladder cancer, preferably comprising transitional cell carcinoma (preferably stage II to stage IV).
Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.
Squamous carcinoma is one or more of oral squamous carcinoma, pharyngeal squamous carcinoma, laryngeal carcinoma, esophageal squamous carcinoma (e.g., esophageal squamous carcinoma), squamous cell carcinoma of the lips, uterine squamous carcinoma, vaginal squamous carcinoma, and skin squamous carcinoma.
Squamous cell carcinoma of the oral cavity (oral squamous cell carcinoma, OSCC) is also known as oral squamous carcinoma. Oral squamous carcinomas include, for example, but are not limited to, tongue squamous carcinomas.
In another embodiment, the squamous cell carcinoma may be a squamous cell carcinoma formed by squamous metaplasia of a part such as bronchi, bladder, or renal pelvis.
In a preferred embodiment of the present invention, the product for detecting vimentin in use (1) is a product for vimentin immunoassay;
in a preferred embodiment of the invention, the vimentin immunoassay labels tissue cells with antibodies or antigen binding fragments thereof;
in a preferred embodiment of the invention, the tissue cells are at least one of the ovary, endometrium, cervix, lung, thyroid and mammary epithelium, bronchi, bladder and renal pelvis.
In a third aspect, the invention also provides a kit for detecting vimentin, a kit for diagnosing an epithelial tumor, an autoimmune encephalitis or a rheumatoid arthritis, a kit for prognosis of an epithelial tumor, an autoimmune encephalitis or a rheumatoid arthritis, a kit for efficacy assessment of an epithelial tumor, an autoimmune encephalitis or a rheumatoid arthritis, a kit for recurrence monitoring of an epithelial tumor, an autoimmune encephalitis or a rheumatoid arthritis, comprising an antibody or antigen-binding fragment thereof as described above;
in a preferred embodiment of the use of the invention, the antibody or antigen binding fragment thereof is labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In a preferred embodiment of the use of the invention, the detectable label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a colloid.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.
In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids; nanoparticles include, but are not limited to: organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In a preferred embodiment of the invention, the kit comprises a carrier, and the carrier carries the antibody or antigen-binding fragment thereof;
in a preferred embodiment of the invention, the carrier is a nanoparticle, a magnetic bead, an agarose gel microsphere, a silica gel microsphere, a latex microsphere or a porous material;
in a fourth aspect, the invention also provides a vector or cell, the vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof as described above; the cells secrete the antibodies or antigen binding fragments thereof or contain a vector.
Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibody amino acid sequence; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
The vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce the DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.
Cells include, but are not limited to, mammalian cells;
mammalian cells include, but are not limited to, any of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, vero, BHK cells, CV1 cells, heLa cells, MDCK cells, hep-2 cells, and Per6 cells. Among them, 293 series cells, per6 cells and CHO cells are common mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.
In a fifth aspect, the invention also provides a composition comprising an antibody or antigen-binding fragment thereof as described above. The composition includes, but is not limited to, a drug.
In addition, the present invention provides a method for producing the antibody or antigen-binding fragment thereof of the previous embodiment, comprising:
culturing the cells of the previous embodiments, and isolating and purifying the antibody or antigen-binding fragment thereof from the culture medium or from the cultured cells.
The production method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the antibody or antigen-binding fragment thereof, and culturing the host cell under suitable conditions to express the antibody or antigen-binding fragment thereof. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of an antibody or antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.
Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions, etc.
The invention also provides a method for detecting vimentin, which comprises the following steps: mixing the binding protein of any of the preceding embodiments with a test sample.
In alternative embodiments, the above-described methods are for the purpose of diagnosis of non-disease.
It should be noted that, those skilled in the art can perform qualitative or quantitative detection of vimentin in the sample to be detected based on the characteristics of the formation of immune complexes by antibody/antigen binding.
The invention has the following beneficial effects:
the antibody or the antigen binding fragment thereof provided by the invention can be specifically combined with the vimentin, has better combination activity and affinity, and can improve the sensitivity and the specificity of detection by using the antibody or the antigen binding fragment thereof to detect the vimentin.
The antibody or the antigen binding fragment thereof has better binding activity and affinity, and can be used for developing detection products for detecting vimentin, such as detection reagents, kits, chips and the like. In addition, the antibodies or antigen binding fragments thereof can be used to diagnose diseases that are labeled with vimentin, such as, for example, tumors of the upper, autoimmune encephalitis, rheumatoid arthritis, and the like. Is beneficial to early discovery and early intervention treatment of diseases. The invention provides more protein choices for the detection of vimentin and the diagnosis, prognosis, efficacy evaluation and recurrence monitoring of diseases with vimentin as a marker.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of an endometrial tissue immunohistochemical experiment;
FIG. 2 is a graph showing the results of immunohistochemical experiments on ovarian serous carcinoma tissues.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example demonstrates the preparation of monoclonal antibodies specific for vimentin.
1. Immunization of animals (mice)
Mice were immunized with the immunogen using a universal method. The immunogen is human vimentin, and is used as detection antigen to measure and screen serum titer and hybridoma, and the high-purity antigen can increase the opportunity of obtaining the required monoclonal antibody and reduce the screening amount. 5 mice were immunized, each with 50ug of human vimentin antigen. The antigen protein solution was prepared with PBS. And (3) placing a proper amount of antigen protein, PBS and Freund's adjuvant into a syringe, plugging a water outlet of the syringe by a plug, and placing the syringe on an emulsifying instrument for stirring and fully emulsifying to ensure that the antigen adjuvant forms a stable water-in-oil solution. The first tail blood serum titer detection is carried out 7-10 days after primary and secondary immunity, and good titer is obtained after 2-4 times of booster immunization. Cell fusion is carried out after the immune mice with high serum titer are selected for abdominal cavity termination.
2. Hybridoma cell fusion and screening
Readiness is required prior to cell fusion: (1) Culturing mouse myeloma cells SP2/0 to log phase of growth; (2) One negative mouse was sacrificed the day before fusion, and mouse peritoneal trophoblast cells were taken from the mouse peritoneal cavity with HAT medium injected into the mouse peritoneal cavity in a sterile environment and plated in 96-well plates, 100ul per well, with these cells promoting hybridoma cell growth. Immunized mice were sacrificed, spleens were removed under sterile conditions, splenocytes and SP2/0 myeloma cells were chemically fused with PEG, appropriate HAT medium was added according to the number of plates to be plated, and finally the fused cells were plated on trophoblast cell culture plates at 100ul per well.
The growth of surviving hybridoma cells was observed under a microscope after 7-10 days, and after two weeks of plating, the supernatants from each well were collected and hybridoma cells were screened by ELISA using human wave-his protein antigen.
The method comprises the following steps: the ELISA plate was coated with 100ul 2ug/ml of human vimentin antigen in PBS for two hours at 37 ℃. After 3 PBST washes, 150 ul/well of 3% nonfat dry milk in PBS was added and blocked overnight at 4 ℃. Plates were washed three more times, 80 ul/well hybridoma supernatant was added, incubated for 1 hour at 37℃and plates were washed three more times. Add 1 at 100 ul/well: 8000 dilution of horseradish peroxidase-labeled goat anti-mouse secondary antibody, incubation at 37 ℃ for 45 minutes, plate washing for 3 times, and beating. 100 ul/well TMB color development was added, developed for 5-10 minutes at room temperature, stopped with 2M sulfuric acid solution, and absorbance was measured for each well at 450 nm. Positive hybridoma cells were selected.
Example 2
Fusion cells that bind positively were selected by ELISA and IHC experiments.
Specifically, ELISA-positive fusion wells of example 1 were selected for Immunohistochemical (IHC) experiments. The experimental procedure was as follows:
baking ovarian serous carcinoma slices and endometrial tissues in a 60 ℃ incubator for 60 minutes, soaking the slices in xylene I for 15 minutes, replacing xylene II, and then soaking for 15 minutes, wherein the slices are soaked in absolute ethyl alcohol (1) for 5 minutes, and soaked in absolute ethyl alcohol (2) for 5 minutes, 95% ethyl alcohol for 5 minutes, 85% ethyl alcohol for 5 minutes and 75% ethyl alcohol for 5 minutes respectively; ddH 2 Soaking in O for 5 minutes, and cleaning for 3 times; adding 10mmol/L citrate buffer solution (pH 6.0) which is enough to submerge slices into a pressure cooker for antigen retrieval (boiling method), heating to boiling, placing the slices on a heat-resistant material slice frame, placing into the cooker, covering a cooker cover, fastening a pressure valve, continuing heating, setting pressure maintaining for 4 minutes, opening a deflation valve for deflation after the time is up, opening the cooker cover after the pressure is zeroed, taking out the inner cooker, and cooling at room temperature. Taking out the slice (about 40 min) after the solution is cooled to room temperature; ddH 2 O is soaked for 5 minutes, washed for 2 times, PBST is soaked for 5 minutes, and washed for 2 times; the sections were placed in 20ml of 3% H 2 O 2 -in methanol solution, protected from light, treated at room temperature for 10 minutes; PBST is soaked for 5 minutes and is washed for 3 times; one drop of goat serum blocking solution was added to each tissue group and incubated in a wet box for 45 minutes at room temperature; PBST was soaked for 5 minutes and washed 3 times.
The treated ovarian serous carcinoma sections and endometrial tissue sections were added to the wave-shaped hybridoma cell supernatant. Incubation overnight in a wet box at 4 ℃; taking out from the refrigerator at the temperature of 4 ℃ and incubating for 60 minutes at room temperature; PBST is gently washed and then soaked for 5 minutes, and the PBST is washed for 3 times; adding 25ul of HRP-labeled secondary antibody of Changdao company into each tissue group, and incubating for 45 minutes at room temperature; washing; preparing DAB color development liquid, reacting for 10-15 min in dark, then dripping the DAB color development liquid onto a slice, and developing for 1-5 min; terminating the color reaction with distilled water; dropping 50ul hematoxylin dye solution into each tissue group, dyeing for 5-10 minutes, and washing with distilled water; the slices are put into 1 percent hydrochloric acid-ethanol for decoloration for 2 to 3 seconds, then are quickly taken out and put into distilled water for termination, and then are put into PBST (pH 8.0) for blue reflection for 5 to 10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min: soaking in absolute ethanol for 5 minutes. Soaking in xylene for 10min, and soaking for 10min after changing xylene; dripping a neutral resin sealing piece, and covering a glass slide sealing piece; microscope imaging.
Fusion cells which are positive in combination are selected through ELISA and IHC experiments, cloned through a limiting dilution method, and plated with 48/96 well plates for each positive strain, and cultured continuously. And performing secondary screening by ELISA method, screening out hybridomas which specifically recognize vimentin and can block wave form binding, and subcloning by limiting dilution method to obtain a monoclonal cell strain 8D11.
Expanding the monoclonal cell line to collect about 1×10 6 Individual cells are injected into selected mice (the mice need to be injected with paraffin oil in the abdominal cavity in advance for one week), and after waiting for 7-10 days, the mice generate ascites, and the ascites is collected for antibody purification. After purification, a mouse monoclonal antibody of the specific anti-vimentin is obtained.
Example 3
The mouse monoclonal antibodies specific for vimentin in example 2 were selected for Immunohistochemical (IHC) experiments. The experimental procedure was as follows:
ovarian serous carcinoma sections and endometrial tissue sections were treated as in example 2. The treated ovarian serous carcinoma sections and endometrial tissue sections were then compared with Rogowski waveform antibody (CAT#: MAB-2673), and the remaining sections were added to the purified antibodies obtained after purification of 8D11 ascites in example 2. Incubation overnight in a wet box at 4 ℃; taking out from the refrigerator at the temperature of 4 ℃ and incubating for 60 minutes at room temperature; PBST is gently washed and then soaked for 5 minutes, and the PBST is washed for 3 times; adding 25ul of HRP-labeled secondary antibody of Changdao company into each tissue group, and incubating for 45 minutes at room temperature; washing; preparing DAB color development liquid, reacting for 10-15 min in dark, then dripping the DAB color development liquid onto a slice, and developing for 1-5 min; terminating the color reaction with distilled water; dropping 50ul hematoxylin dye solution into each tissue group, dyeing for 5-10 minutes, and washing with distilled water; the slices are put into 1 percent hydrochloric acid-ethanol for decoloration for 2 to 3 seconds, then are quickly taken out and put into distilled water for termination, and then are put into PBST (pH 8.0) for blue reflection for 5 to 10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min: soaking in absolute ethanol for 5 minutes. Soaking in xylene for 10min, and soaking for 10min after changing xylene; dripping a neutral resin sealing piece, and covering a glass slide sealing piece; microscope imaging.
Fig. 1 and 2 are diagrams of the results of immunohistochemical experiments of waveforms, wherein the detection sample in fig. 1 is ovarian serous carcinoma tissue, and the detection sample in fig. 2 is endometrial tissue. In FIG. 1, A is a waveform antibody prepared in example 1, and B is a waveform antibody of Roche. In fig. 2, a is a waveform antibody prepared in example 1, and B is a waveform antibody of roche company.
As can be seen from the staining results of FIGS. 1 and 2, there was no significant difference between the staining sites and staining intensity of the antibodies to be stained with the wavy antibodies and the Roche antibodies. The waveform antibody provided by the invention has application prospect in preparing a kit.
Example 4
DNA cloning and sequencing was performed and the variable region gene of the anti-human waveform monoclonal antibody was sequenced.
Total RNA was extracted from the mouse monoclonal cell line using Trizol reagent. Cells cultured in 9cm dishes were collected in 1.5ml centrifuge tubes and the supernatant was aspirated to dryness. 1ml of Trizol reagent was added and the cells were blown up uniformly to lyse the cells, and the lysed sample or homogenate was left at room temperature for 5-10min to allow complete separation of nucleoprotein from nucleic acid. 0.2ml of chloroform was added thereto, and the mixture was vigorously shaken for 15sec and left at room temperature for 3min. Centrifuge at 12000rpm at 4℃for 10min. The upper aqueous phase is sucked and transferred into a clean centrifuge tube, added with isopropyl alcohol with equal volume, evenly mixed and placed for 20min at room temperature. Centrifuge at 12000rpm at 4℃for 10min, discard supernatant. The precipitate was washed with 1ml of 75% ethanol. Centrifuge at 12000rpm at 4℃for 3min, discard supernatant. Drying at room temperature for 5-10min. Adding 30-50ul RNase-free ddH 2 O. The resulting RNA solution was stored at-70℃or used in subsequent experiments.
The total RNA is reverse transcribed into cDNA by using AMV first-strand cDNA synthesis kit. The experimental system was configured as follows, with 6ul total RNA+1ul Oligo dT+4ul RNase-free water (11 ul total). After gentle mixing, the mixture was centrifuged for 3-5s, and after 5mi of pre-denaturation in a warm bath at 65℃the reaction mixture was ice-cooled for 30s, and then centrifuged for 3-5s followed by 2min in an ice-cooled bath. Under ice bath, 4ul of 5 Xbuffer+1 ul of dNTP mixture+1 ul of RNase inhibitor+1 ul of reverse transcriptase (total 20ul system) were added, and after gentle mixing, the mixture was centrifuged for 3-5s, and on a PCR instrument, the mixture was subjected to 42℃for 50 minutes and 85℃for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-chain cDNA below 500bp, and the transcribed RNA template can transcribe 5' terminal regions without poly (A) tail.
PCR amplification of light and heavy chains. For amplifying antibody light chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VL 1ul+RP-VL 1ul+cDNA 2ul+ddH 2 And O21 ul. For amplifying antibody heavy chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VH1ul+RP-VH1ul+cDNA 2ul+ddH 2 And O21 ul. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (wherein steps 2 to 4, 35 cycles are repeated):
step 1, pre-denaturation at 94 ℃ for 4min;
step 2-denaturation 94℃for 30sec;
step 3-annealing at 55 ℃ for 45sec;
step 4-extending at 72 ℃ for 60sec;
step 5-72 ℃ for 10min;
step 6-storing at 4 ℃.
The PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA segments of the corresponding sizes (approximately 325bp for VH and approximately 325bp for VL) were excised and DNA extracted using the SanPrep column DNA gel recovery kit. The following is a brief description: cutting off a gel block containing the target fragment from agarose gel, and weighing; adding buffer B2 with the weight 3-6 times of that of the rubber block, and carrying out water bath at 50 ℃ for 5-10 minutes to obtain sol; transferring the sol into an adsorption column, and centrifuging 8000g for 30 seconds; pouring out the liquid in the collecting pipe; adding 500ul wash Solution,9000g into the column, centrifuging for 30 seconds, and pouring out the liquid in the collecting pipe; repeatedly adding the wash solution once, and pouring out the liquid; centrifuging the hole adsorption column at 9000g for 1 min; the column was placed in a clean 1.5ml centrifuge tube, 15-40 g ul Elution Buffer g was added to the center of the adsorption membrane, and after 1 min of standing at room temperature, it was centrifuged for 1 min. The prepared DNA solution is obtained, and the variable region sequence of the antibody is obtained by purifying the PCR product and sequencing.
Experimental example 1
This experimental example was conducted to examine the affinity and sensitivity of the antibody (anti-vimentin purified antibody) obtained in example 2 above.
1. Recombinant human vimentin was wrapped in 3mg/L, 1.5mg/L, 0.75mg/L, 0.375mg/L, respectively.
2. Adjusting the antibody concentration to 10 -7 mol/L level (1X 10 -7 To 5x 10 -7 mol/L). Then diluted 1:2-1:256 in a multiple ratio and added to wells with different antigen coating amounts.
3. Adding secondary antibody and developing TMB. The absorbance at 450nm was measured and the data is shown in Table 1.
4. And (3) according to the antigen-antibody combination S curve, determining the antibody concentration of half-absorbance values under different antigen concentrations. Thus there are four antibody concentrations (mol/L).
5. The affinity constant is calculated by substituting the formula k= (N-1)/(n×ab' -AB). AB', AB is the concentration of antibody that gave half-absorbance at the corresponding antigen concentrations AG (3 mg/L, 1.5ml/L, 0.75mg/L, 0.375 mg/L). n=ag/AG '(AG > AG').
6. When n=2, three K values of 1.300, 0.580, 0.481 can be obtained. When n=4, two K values of 0.712, 0.511 can be obtained. A K value of 0.599 can be obtained when n=8. The average value of 6K values was determined to be 9.822 ×10 8 L/mol。
Table 1 shows statistics of absorbance at 450nm
Experimental example 2
This experimental example was subjected to immunohistochemical neutralization experiments.
The (antigen: antibody=10; 1) vimentin was mixed with the purified antibody against vimentin prepared in example 2 and subjected to neutralization reaction at 37℃for 1 hour. The immunohistochemical procedure was followed, as a primary antibody, by dropping on endometrium sections overnight at 4℃and then by performing immunohistochemical staining and shooting in the same manner.
The neutralization test results are shown as C in FIG. 2. In the neutralization assay, the antigen binds to the wavy antibody, and the antibody cannot bind to the antigen in the tissue, so that the tissue is hardly stained. It can be seen that the waveform of the anti-vimentin monoclonal antibody provided by the embodiment of the invention can identify and combine with vimentin in human cells, and the cytoplasm of the cells in endometrial tissues is brown in the subsequent IHC staining experiment.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An antibody against vimentin, or an antigen-binding fragment thereof, said antibody comprising: a heavy chain variable region comprising CDR-VH1, CDR-VH2 and CDR-VH3 and a light chain variable region comprising CDR-VL1, CDR-VL2 and CDR-VL3; the amino acid sequence of each CDR region is as follows:
CDR-VH1：DYWID；
CDR-VH2：EISPGTGRISFNETFNG；
CDR-VH3：DDS；
CDR-VL1：KSSQSLLHSDGKTYLN；
CDR-VL2：LVSKLDP；
CDR-VL3：WQGTHFPT。
2. the anti-vimentin antibody or antigen-binding fragment thereof according to claim 1, further comprising light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H and having sequences shown in sequence SEQ ID NOs 5-8.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a constant region;
preferably, the constant regions include a light chain constant region and a heavy chain constant region, the type of light chain constant region being either a kappa-type or lambda-type light chain constant region; the heavy chain constant region is IgM, igD, igG, igA or IgE heavy chain constant region;
preferably, the IgG is selected from the heavy chain constant region of any one of IgG1, igG2, igG3 and IgG 4;
preferably, the constant region of the antibody is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin;
preferably, the constant region is of species origin of dairy cow, chicken or turkey;
preferably, the constant region is derived from a mouse;
preferably, the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 9, and the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 10.
4. An antibody or antigen-binding fragment thereof according to claim 3, wherein the antigen-binding fragment is selected from any one of Fab ', fab, F (ab') 2, scFv and Fv of the antibody;
preferably, the antibody is selected from at least one of a multispecific antibody, a fusion antibody, and a chimeric antibody.
5. The use of an antibody or antigen-binding fragment thereof according to any one of claim 1 to 4 for the preparation of a product for at least one of the following uses,
(1) A product for detecting vimentin; and said use is not for the purpose of diagnosis of a disease;
(2) Products for diagnosing epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; the epithelia tumor, autoimmune encephalitis or rheumatoid arthritis takes vimentin as a diagnostic marker;
(3) Products for prognosis of epithelial tumors, autoimmune encephalitis or rheumatoid arthritis; the epithelia tumor, autoimmune encephalitis or rheumatoid arthritis takes vimentin as a diagnostic marker;
(4) Products for efficacy assessment of epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; the epithelia tumor, autoimmune encephalitis or rheumatoid arthritis takes vimentin as a diagnostic marker;
(5) Products for monitoring recurrence of epithelial tumors, autoimmune encephalitis, or rheumatoid arthritis; the epithelia tumor, autoimmune encephalitis or rheumatoid arthritis takes vimentin as a diagnostic marker;
the product is a reagent, a kit, a chip, a magnetic bead or a micro-pore plate.
6. The use according to claim 5, wherein the epithelial tumor is papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, renal cancer, adenoma or squamous carcinoma;
preferably, the gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colorectal cancer and anal cancer;
preferably, the adenocarcinoma is selected from lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma, breast carcinoma or pancreatic carcinoma.
7. The use according to claim 5, wherein the product for detecting vimentin in use (1) is a product for vimentin immunoassay;
preferably, said vimentin immunoassay tags tissue cells with said antibody or antigen binding fragment thereof;
preferably, the tissue cells are at least one of the ovary, endometrium, cervix, lung, thyroid and mammary epithelium, bronchi, bladder and renal pelvis.
8. A kit for detecting vimentin, a kit for diagnosing an epithelial tumor, an autoimmune encephalitis, or a rheumatoid arthritis, a kit for prognosis of an epithelial tumor, an autoimmune encephalitis, or a rheumatoid arthritis, a kit for efficacy assessment of an epithelial tumor, an autoimmune encephalitis, or a rheumatoid arthritis, a kit for recurrence monitoring of an epithelial tumor, an autoimmune encephalitis, or a rheumatoid arthritis, comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4;
preferably, the antibody or antigen binding fragment thereof is labeled with a detectable label;
preferably, the kit comprises a carrier, and the carrier carries the antibody or antigen-binding fragment thereof;
preferably, the carrier is a nanoparticle, a magnetic bead, an agarose gel microsphere, a silica gel microsphere, a latex microsphere or a porous material;
preferably, the detectable label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a colloid.
9. A vector or cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4; the cell secretes the antibody or antigen-binding fragment thereof of any one of claims 1-4 or contains the vector.
10. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4.
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