An antibody or antigen-binding fragment thereof against nse protein, a tumor diagnostic product and application thereof

Abstract

The application discloses an antibody or antigen-binding fragment thereof against NSE protein, a tumor diagnosis product and application thereof, and relates to the technical field of antibodies. The antibody is used for detecting NSE protein, and the sensitivity and specificity of detection can be improved. The antibody has good binding activity and affinity, and can be used for developing a detection product for detecting NSE protein. In addition, the binding protein can be used for diagnosing, assisting in diagnosing, evaluating the curative effect, or monitoring the recurrence and metastasis of a disease taking NSE protein as a marker. The application provides more protein selection for the detection of NSE protein and the diagnosis of a disease taking NSE protein as a marker.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to an antibody or antigen-binding fragment thereof against NSE protein, a tumor diagnosis product and application thereof. BACKGROUND

[0002] Neuron-specific enolase (NSE) is one of enolases involved in glycolysis pathway, and exists in neural tissue and neuroendocrine tissue. It is found in tumors related to the origin of neuroendocrine tissue, especially small cell lung cancer (SCLC), which has excessive expression of NSE, resulting in significant increase of NSE in serum.

[0003] The serum NSE of patients with small cell lung cancer (SCLC) is significantly increased, and the NSE concentration is increased in 60%-81% of small cell lung cancer cases. The diagnostic sensitivity of NSE reaches 80%, and the specificity reaches 80%-90%, while there is no significant increase in patients with non-small cell lung cancer (NSCLC), so it can be used for differential diagnosis of SCLC and NSCLC. The serum NSE level is positively correlated with the clinical stage of SCLC. Therefore, serum NSE detection has important clinical value for monitoring the condition, evaluating the efficacy and predicting the recurrence of SCLC.

[0004] In neuroblastoma, the NSE positive rate can reach 96%-100%, and the measured value is significantly increased. The serum NSE level is related to the stage and prognosis. Determination of serum NSE has high clinical value for early diagnosis and prognosis of such tumors.

[0005] In clinical practice, the condition of protein expression in tumor cells is often detected by immunohistochemical (IHC) pathological experiments, and the accuracy and sensitivity of IHC experiments are determined by the advantages and disadvantages of specific binding proteins monoclonal antibodies. Therefore, it is of great significance to develop a monoclonal antibody against NSE protein with high specificity for detecting the expression level of NSE protein.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide an antibody or antigen-binding fragment thereof against NSE protein, a tumor diagnosis product and application thereof to solve the above technical problems.

[0008] The present application is realized as follows:

[0009] In a first aspect, the present invention provides an antibody against NSE protein or an antigen-binding fragment thereof, comprising a heavy chain complementarity-determining region in the heavy chain variable region as shown in SEQ ID NO: 9, and a light chain complementarity-determining region in the light chain variable region as shown in SEQ ID NO: 10.

[0010] Secondly, the present invention also provides the use of antibodies or antigen-binding fragments thereof in the preparation of any of the following products:

[0011] (1) Tumor diagnostic products;

[0012] (2) Tumor auxiliary diagnostic products;

[0013] (3) Tumor efficacy assessment products;

[0014] (4) Products for monitoring tumor recurrence and metastasis;

[0015] (5) NSE protein detection products;

[0016] (6) NSE protein isolation or enrichment products;

[0017] Thirdly, the present invention also provides a product for tumor diagnosis, auxiliary diagnosis, efficacy evaluation, or recurrence and metastasis monitoring, the product comprising: an antibody or its antigen-binding fragment.

[0018] Fourthly, the present invention also provides an NSE protein detection product, an NSE protein isolation product, or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0019] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0020] In a sixth aspect, the present invention also provides a nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0021] The present invention has the following beneficial effects:

[0022] The antibody or its antigen-binding fragment provided by this invention can specifically bind to NSE protein, exhibiting good binding activity and affinity. Using the antibody of this invention to detect NSE protein can improve the sensitivity and specificity of detection. Based on the good binding activity and affinity of this antibody, it can be used to develop detection products for NSE protein, such as detection reagents and kits. Furthermore, this binding protein can be used for the diagnosis, auxiliary diagnosis, efficacy evaluation products, or recurrence and metastasis monitoring of diseases using NSE protein as a biomarker, such as small cell lung cancer. This facilitates early detection and early intervention. This invention provides more protein options for the detection of NSE protein and the diagnosis of diseases using NSE protein as a biomarker. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The results of immunohistochemical experiments on appendix tissue;

[0025] Figure 2 This is a diagram showing the results of an immunohistochemical experiment on pancreatic tissue. Detailed Implementation

[0026] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0027] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0028] Definition of noun

[0029] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0030] The term “antibody” in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to NSE proteins.

[0031] In this invention, the terms "complementarity-determining region" or "CDR" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0032] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0033] Typically, the variable region (VH) of the antibody heavy chain is obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0034] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0035] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0036] In a first aspect, the present invention provides an antibody against NSE protein or an antigen-binding fragment thereof, comprising a heavy chain complementarity-determining region in the heavy chain variable region as shown in SEQ ID NO: 9, and a light chain complementarity-determining region in the light chain variable region as shown in SEQ ID NO: 10.

[0037] SEQ ID NO: 9:

[0038] EVQLVESGAELVRPGASVTLSKASGYTFTDYEMHWVKQTPVHGLEWIGGVDPETGGSASSQKFKDKATLTADKSSSTAYMEVRSLTSEDSAVYFCTRSGTAY WGQGTLVTVSA.

[0039] SEQ ID NO: 10:

[0040] DVVMTQTPLSLPVSLGDQASISCRSTQSLLHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQGTHVPWTFGGGTKLE.

[0041] Inputting the above SEQ ID NO:9 and SEQ ID NO:10 sequences into the CDR identification system will yield the corresponding CDR sequences.

[0042] The amino acid sequences of the heavy chain complementarity-determining regions and light chain complementarity-determining regions described above are newly discovered and disclosed in this invention, and can endow the binding protein with the ability to specifically bind to NSE protein antigens. Based on the good binding activity and affinity of this binding protein, it can be used to develop detection products for NSE protein, such as detection reagents and kits. Using the binding protein of this invention to detect NSE protein can improve the sensitivity and specificity of detection. Furthermore, this binding protein can be used to diagnose diseases that use NSE protein as a biomarker, which is beneficial for early detection and early intervention. This invention provides more protein options for the detection of NSE protein and the diagnosis of diseases using NSE protein as a biomarker.

[0043] In a preferred embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment binds to the NSE protein at a K0... D ≤4.69×10 8 Affinity binding at L / mol.

[0044] In a preferred embodiment of the present invention, the heavy chain complementarity-determining region includes CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 11-13, respectively, and the light chain complementarity-determining region includes CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 14-16, respectively.

[0045] CDR-H1: DYEMH; SEQ ID NO: 11;

[0046] CDR-H2: GVDPETGGSASSQKFKD; SEQ ID NO: 12;

[0047] CDR-H3: SGTAY; SEQ ID NO: 13;

[0048] CDR-L1: RSTQSLLHSDGNTYLH; SEQ ID NO: 14;

[0049] CDR-L2: KVSNRFP; SEQ ID NO: 15;

[0050] CDR-L3: SQGTHVPWT. SEQ ID NO: 16.

[0051] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region and / or a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 80% homology with the amino acid sequences shown in SEQ ID NO: 5-8; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO: 5-8.

[0052] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO: 1-4, for example, LFR1, LFR2, LFR3 and LFR4, which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO: 1-4.

[0053] The sequences of SEQ ID NO:1-8 are shown in the table below:

[0054]

[0055] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0056] In a preferred embodiment of the present invention, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0057] In a preferred embodiment of the present invention, the species source of the constant region is mice;

[0058] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of the antibody F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv.

[0059] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0060] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0061] Secondly, the present invention also provides the use of antibodies or antigen-binding fragments thereof in the preparation of any of the following products:

[0062] (1) Tumor diagnostic products;

[0063] (2) Tumor auxiliary diagnostic products;

[0064] (3) Tumor efficacy assessment products;

[0065] (4) Products for monitoring tumor recurrence and metastasis;

[0066] (5) NSE protein detection products;

[0067] (6) NSE protein isolation products or enrichment products.

[0068] In the application of tumor diagnosis, auxiliary diagnosis, efficacy evaluation products, recurrence and metastasis monitoring, NSE protein detection products, NSE protein isolation products or enrichment products, the detection of NSE protein is achieved by utilizing the specific binding of anti-NSE protein antibodies or their antigen-binding fragments to NSE protein fragments.

[0069] In a preferred embodiment of the present invention, the tumor is a tumor that uses NSE protein as a tumor marker.

[0070] In a preferred embodiment of the present invention, the tumor is selected from tumors caused by nerve cells and / or neuroendocrine cells, pancreatic cancer, liver cancer, or appendiceal cancer.

[0071] In a preferred embodiment of the present invention, the tumors induced by nerve cells and / or neuroendocrine cells are selected from medullary thyroid carcinoma, small cell lung cancer, or neuroblastoma.

[0072] In a preferred embodiment of the present invention, the products used in (1)-(5) are reagents, kits, test strips, antibody chips, antibody probes or detectors; the separation or enrichment products used in (6) are magnetic beads, kits or separation columns.

[0073] In a preferred embodiment of the present invention, in applications (1)-(5), the antibody or its antigen-binding fragment is labeled with a detectable marker.

[0074] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0075] In optional embodiments, detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0076] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0077] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0078] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0079] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 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.

[0080] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0081] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0082] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes. In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0083] In a preferred embodiment of the present invention, in application (5), NSE protein detection is performed by binding protein-labeled tissues and / or cells, and the labeled tissues and / or cells are then detected.

[0084] Antibody chips are chips formed by immobilizing the aforementioned antibodies or their antigen-binding fragments on a carrier.

[0085] The antibody provided by this invention can be used as a coating antibody or a detection antibody.

[0086] Thirdly, the present invention also provides a product for tumor diagnosis, auxiliary diagnosis, efficacy evaluation, or recurrence and metastasis monitoring, the product comprising: an antibody or its antigen-binding fragment;

[0087] In a preferred embodiment of the present invention, the product is a reagent, reagent kit, test strip, antibody chip, antibody probe, or detector.

[0088] Fourthly, the present invention also provides an NSE protein detection product, an NSE protein isolation product or an enrichment product, the product comprising: the above-mentioned antibody or its antigen-binding fragment;

[0089] In a preferred embodiment of the present invention, the NSE protein detection product is a reagent, kit, test strip, antibody chip, antibody probe, or detector;

[0090] In a preferred embodiment of the present invention, the NSE protein separation product or enrichment product is a magnetic bead, a kit, or a separation column.

[0091] For example, the antibodies described above can be coated onto magnetic beads for the separation and enrichment of NSE proteins. In one embodiment, the antibodies are coated onto packing material and packed into a separation column for affinity separation and enrichment of NSE proteins. Therefore, the antibodies described above or their antigen-binding fragments have promising applications in the preparation of NSE protein enrichment products.

[0092] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned antibody or its antigen-binding fragment.

[0093] The host cells are selected from mammalian cells; the mammalian cells are selected from any one 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, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those skilled in the art.

[0094] In a sixth aspect, the present invention also provides a nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0095] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0096] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0097] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0098] Example 1

[0099] This embodiment describes the preparation of a monoclonal antibody specifically targeting the NSE protein.

[0100] 1. Animal (mouse) immunization

[0101] Mice were immunized with an immunogen using a standard method. The immunogen was human NSE protein, which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of P16 antigen. An antigen-protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the solution was emulsified thoroughly on an emulsifier to form a stable water-in-oil solution. The first tail blood serum titer was measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for final intraperitoneal immunization followed by cell fusion.

[0102] 2. Hybridoma cell fusion and screening

[0103] Preparations are required before cell fusion: (1) Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; (2) Sacrifice a negative mouse one day before fusion, inject mouse peritoneal trophoblast cells into the peritoneal cavity in a sterile environment using HAT medium, and plate them in 96-well plates at 100 μL per well. These cells promote the growth of hybridoma cells. Sacrifice the immunized mouse, and take the spleen in a sterile environment. Use PEG to chemically fuse spleen B cells and SP2 / 0 myeloma cells. Add appropriate HAT medium according to the number of cells to be plated, and finally plate the fused cells in a trophoblast cell culture plate at 100 μL per well.

[0104] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant from each well is collected, and hybridoma cells are screened using the human NSE-his protein antigen via ELISA.

[0105] The method is as follows: Coat the ELISA plate with 100 μL of PBS solution containing 2 μg / ml human NSE protein antigen and incubate at 37°C for two hours. After washing the plate three times with PBST, add 150 μL / well of PBS solution containing 3% skim milk powder and block at 4°C overnight. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:8000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, develop at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0106] Example 2

[0107] The ELISA-positive fusion wells from Example 1 were selected for immunohistochemical (IHC) experiments. The experimental steps are as follows:

[0108] The appendix cancer slices and pancreatic cancer tissue were baked in a 60°C oven for 60 minutes. The slices were soaked in xylene I for 15 minutes, then soaked in xylene II for 15 minutes. They were then soaked in anhydrous ethanol (1) for 5 minutes, anhydrous ethanol (2) for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes. They were also soaked in ddH2O for 5 minutes and washed 3 times. Antigen retrieval was performed using a pressure cooker (boiling method). 10 mmol / L citrate buffer (pH 6.0) was added to the pressure cooker to submerge the slices. The pressure was heated to boiling. The slices were placed on a heat-resistant slice rack and placed in the pot. The pot lid was closed and the pressure valve was closed. The pressure was continued and the pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the gas. After the pressure returned to zero, the pot lid was opened and the inner pot was removed and placed to cool at room temperature. After the solution cools to room temperature, remove the sections (about 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash 3 times; add one drop of goat serum blocking solution (about 25 μl) to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash 3 times.

[0109] Add NSE-8D11 purified antibody to the processed tissue sections. Incubate overnight in a humidified chamber at 4°C; remove from the 4°C freezer and incubate at room temperature for 60 minutes; gently rinse with PBST and soak for 5 minutes, washing 3 times; add 25 μL of HRP-labeled Long Island Biotechnology secondary antibody to each tissue group and incubate at room temperature for 45 minutes; wash; prepare DAB chromogenic solution, react in the dark for 10-15 minutes, then drop onto the sections and develop for 1-5 minutes; terminate the chromogenic reaction with distilled water; add 50 μL of hematoxylin staining solution to each tissue group and stain for 5-10 minutes, then rinse thoroughly with distilled water; decolorize the sections in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove and stop in distilled water, then incubate in PBST (pH 8.0) for 5-10 minutes; soak in 75% ethanol for 5 minutes; soak in 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin to seal the slide, then cover with a coverslip; take a microscope image.

[0110] Fusion cells showing positive binding were selected using ELISA and IHC assays and cloned using the limiting dilution method. Each positive cell line was seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA to identify hybridomas that specifically recognize NSE protein and can block NSE binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 8D11. This single-clone cell line was then expanded, and approximately 1 × 10⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against NSE protein was obtained.

[0111] Figure 1 , Figure 2 This is a figure showing the immunohistochemical results after incubation of NSE-8D11 purified antibody and tissue sections. Figure 1 The specimen tested was an appendix slice. Figure 2 The specimen tested was pancreatic tissue.

[0112] Depend on Figure 1 and Figure 2 The staining results show that the NSE-8D11 antibody has a strong binding ability to the corresponding antigen. This confirms that the NSE provided by this invention has the potential for application in the preparation of reagent kits.

[0113] Example 3

[0114] DNA cloning and sequencing were performed, including sequencing of the variable region gene of the anti-human NSE monoclonal antibody.

[0115] Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of Trizol reagent was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 min to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds and incubated at room temperature for 3 min. The mixture was centrifuged at 12000 rpm at 4°C for 10 min. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 min. The mixture was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The mixture was centrifuged at 12000 rpm at 4°C for 3 min, and the supernatant was discarded. The mixture was dried at room temperature for 5-10 min. 30-50ul of RNase-free ddH2O was added. The resulting RNA solution was stored at -70°C or used for subsequent experiments.

[0116] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While in the ice bath, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds, and incubate at 42°C for 50 minutes and 85°C for 5 minutes on a PCR instrument to complete cDNA synthesis. Random primers are suitable for the synthesis of short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

[0117] PCR amplification of the light and heavy chains. For amplifying the variable region sequence of the antibody light chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VL + 1 μL RP-VL + 2 μL cDNA + 21 μL ddH2O. For amplifying the variable region sequence of the antibody heavy chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VH + 1 μL RP-VH + 2 μL cDNA + 21 μL ddH2O. The temperature cycling for PCR amplification of the variable regions of the heavy and light chains was as follows (steps 2 to 4 were repeated 35 times):

[0118] Step 1 - Pre-denaturation: 94℃, 4 min;

[0119] Step 2 - Denaturation at 94°C for 30 seconds;

[0120] Step 3 - Annealing at 55°C for 45 seconds;

[0121] Step 4 - Extend at 72°C for 60 seconds;

[0122] Step 5: 72℃, 10 min;

[0123] Step 6 - Store at 4℃.

[0124] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 375 bp for VH and approximately 325 bp for VL). DNA extraction was performed using the SanPrep DNA Gel Extraction Kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was incubated at 50°C for 5-10 minutes to dissolve; the solution was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the column was centrifuged at 9000g for 1 minute; the column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was purified and sequenced to obtain the variable region sequence of the antibody.

[0125] Experimental Example 1

[0126] This experimental example tests the affinity and sensitivity of the antibody (NSE-8D11 purified antibody) prepared in Example 2 above.

[0127] 1. Recombinant human NSE protein was plated at concentrations of 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L, respectively.

[0128] 2. Adjust the antibody concentration to 10. -7 mol / L level (1*10) -7 Up to 5*10 -7 (Mol / L is acceptable). Then serially dilute 1:2 to 1:256 and add to wells with different antigen coating amounts.

[0129] 3. Add secondary antibody and develop TMB colorimetric assay. Measure the absorbance at 450 nm; the data are shown in Table 1.

[0130] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This will result in four antibody concentrations (mol / L).

[0131] 5. Substitute the values ​​into the formula K=(N-1) / (N*AB'-AB) to calculate the affinity constant. AB' and AB are the antibody concentrations that produce the half-maximum absorbance at the corresponding antigen concentrations AG (3mg / L, 1.5ml / L, 0.75mg / L, 0.375mg / L). N=AG / AG' (AG>AG').

[0132] 6. When N=2, we get three K values: 0.425, 0.259, and 0.880. When N=4, we get two K values: 0.295 and 0.485. When N=8, we get one K value: 0.476. The average of the six K values ​​is 4.694 × 10⁻⁶. 8 L / mol.

[0133] Table 1. Statistical Table of Absorbance Values ​​at 450nm

[0134]

[0135] The results showed that the antibody (NSE-8D11 purified antibody) had good affinity for NSE protein and high detection sensitivity.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against NSE protein or an antigen-binding fragment thereof, characterized in that, It includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. The heavy chain complementarity-determining region includes CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 11-13. The light chain complementarity-determining region includes CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NO: 14-16.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region and / or a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:5-8; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:1-4.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO:

10.

4. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

6. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The species source of the constant region is mice.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, Fab'-SH, and scFv.

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-7 in the preparation of any one of the following products: (1) NSE protein detection products; (2) NSE protein isolate products.

9. The application according to claim 8, characterized in that, The product in application (1) is a reagent, kit, test strip, antibody chip or detector; the separation product in application (2) is a magnetic bead, kit or separation column.

10. The application according to claim 9, characterized in that, In the application (1), the antibody or its antigen-binding fragment is labeled with a detectable marker.

11. The application according to claim 8, characterized in that, In the application (1), the NSE protein detection is to label tissues and / or cells with the antibody or its antigen-binding fragment, and to detect the labeled tissues and / or cells.

12. An NSE protein detection product or an NSE protein isolation product, characterized in that, The product comprises: the antibody or antigen-binding fragment thereof as described in any one of claims 1-7.

13. The NSE protein detection product or NSE protein isolation product according to claim 12, characterized in that, The NSE protein detection products are reagents, kits, test strips, antibody chips, or detectors.

14. The NSE protein detection product or NSE protein isolation product according to claim 12, characterized in that, The NSE protein isolation product is a magnetic bead, a kit, or a separation column.

15. A cell characterized in that, The cells are non-plant cells that express the antibody or antigen-binding fragment of any one of claims 1-7.

16. A nucleic acid molecule, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-7.

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