A binding protein specifically binding to AFP polypeptide and tumor diagnosis kit

Abstract

The present invention discloses a binding protein that specifically binds to an AFP polypeptide and a tumor diagnostic kit, relating to the field of biotechnology. The binding protein can specifically bind to AFP and has good binding activity and affinity. Using the binding protein of the present invention to detect AFP can improve the sensitivity and specificity of AFP detection. The binding protein can be used for diagnosing or screening diseases that use AFP as a biomarker, and can be used to detect benign diseases that use AFP as a biomarker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a binding protein specifically binding to an AFP polypeptide and a tumor diagnosis kit. Background Art

[0002] AFP (alpha-fetoprotein) is a glycoprotein in the albumin family, primarily synthesized by fetal liver cells and the yolk sac. It is a relatively well-known and valuable marker for primary liver cancer and germ cell malignancies. Elevated AFP levels are detected in the blood of approximately 80% of patients with primary liver cancer, with high concentrations detected in 50% of these patients.

[0003] AFP is positive in the cytoplasm. Its concentration is high in the fetal circulation, but decreases after birth. By two to three months after birth, it is essentially replaced by albumin, making it difficult to detect in the blood. Consequently, its level in adult serum is extremely low. AFP has many important physiological functions, including transport, bidirectional regulation as a growth regulator, immunosuppression, and induction of T lymphocyte apoptosis.

[0004] AFP is closely associated with the development and progression of liver cancer and various other tumors. It is found at high concentrations in many tumors and can be used as a positive marker for a variety of tumors. Currently, it is primarily used clinically as a serum marker for primary liver cancer, both for diagnosis and treatment efficacy monitoring.

[0005] Clinically, immunohistochemistry (IHC) is commonly used to detect protein expression in tumor cells. The accuracy and sensitivity of IHC assays are determined by the quality of the monoclonal antibodies that specifically bind to the protein. Therefore, developing a monoclonal antibody with high binding specificity against AFP is crucial for improving the accuracy and sensitivity of IHC AFP detection.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide a binding protein that specifically binds to an AFP polypeptide. This binding protein can specifically bind to AFP and has good binding activity and affinity. Using the binding protein of the present invention to detect AFP can improve the sensitivity and specificity of AFP detection. This binding protein can be used for both diagnosing or screening for diseases that use AFP as a biomarker and detecting benign diseases that use AFP as a biomarker.

[0008] Definition of noun

[0009] The term "binding protein" refers to all proteins / protein fragments containing CDR regions, in particular antibodies or antibody functional fragments. "Antibody functional fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0010] The term "antibody" as used herein is used in the broadest sense and may 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 HRP-II antigen or a fragment thereof. "Antibody fragments" include portions of full-length antibodies, preferably antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), diabodies, or domain antibodies.

[0011] Typically, the variable regions VH / VL of the heavy and light chains of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0012] The present invention is achieved in that:

[0013] In a first aspect, the present invention provides a binding protein that specifically binds to an AFP polypeptide, wherein the binding protein comprises an antigen binding domain having six complementary determining regions as shown below:

[0014] CDR-VH1:SYGMS;

[0015] CDR-VH2: TISSSGGRNIYYPDSVKG;

[0016] CDR-VH3:QQYGVYVFDY;

[0017] CDR-VL1: KSSQSLLYSSNQKNYLA;

[0018] CDR-VL2: WASTRES;

[0019] CDR-VL3:QQYFSYPT.

[0020] The amino acid sequence of the complementarity-determining region described above, discovered and disclosed for the first time by the present invention, is a novel sequence that confers upon the binding protein the ability to bind to the AFP antigen. Furthermore, the binding protein exhibits good binding activity and affinity. Using the binding protein of the present invention to detect AFP can improve detection sensitivity and specificity. The binding protein can be used to diagnose diseases that use AFP as a marker. The present invention provides a wider range of protein options for the detection of AFP and the diagnosis of diseases that use AFP as a marker.

[0021] In a preferred embodiment of the present invention, the above-mentioned binding protein and AFP polypeptide are K D ≤3.126×10 9 The affinity binding is 0.1 L / mol, and the binding protein is an antibody or a functional fragment.

[0022] In an alternative embodiment, the binding protein and the AFP protein have a K 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 2×10 9 L / mol affinity.

[0023] In a preferred embodiment of the present invention, the above-mentioned binding protein includes the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are shown in SEQ ID NOs: 1-4, and / or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are shown in SEQ ID NOs: 5-8.

[0024] The sequences of SEQ ID NO: 1-8 are shown in the following table:

[0025]

[0026] In an alternative embodiment, the binding protein further comprises a constant region;

[0027] In an alternative embodiment, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

[0028] In an alternative embodiment, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or primate;

[0029] In an alternative embodiment, the primate is selected from a lemur, a macaque, an ape, or a human;

[0030] In an alternative embodiment, the constant region is of mouse origin.

[0031] In a preferred embodiment of the present invention, the binding protein is selected from any one of F(ab')2, Fab', Fab, Fv, scFv, and bispecific antibodies.

[0032] The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that the functional fragments of the above antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds.

[0033] Functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0034] In a second aspect, the present invention provides a reagent or kit for detecting alpha-fetoprotein, wherein the reagent or kit comprises the above-mentioned binding protein that specifically binds to the AFP protein.

[0035] This detection reagent or kit is used for the detection and determination of alpha-fetoprotein (AFP). Applications include, but are not limited to, detecting diseases with elevated AFP levels, detecting AFP levels in pregnant women, and detecting AFP in benign epithelial tumors. For example, AFP levels can be detected in benign diseases. The inventors have verified that the binding protein specifically binding to AFP provided by the present invention maintains a high affinity for AFP and exhibits good specificity, and can be used to prepare a detection reagent or kit for AFP.

[0036] In a preferred embodiment of the present invention, the binding protein is labeled with a detectable marker to facilitate detection.

[0037] In an optional embodiment, the above-mentioned detection of alpha-fetoprotein is performed by immunoassay, for example, labeling tissue cells with binding protein, and then detecting alpha-fetoprotein by binding to enzyme-labeled secondary antibody and performing color development reaction.

[0038] A detectable marker is a substance that has properties that can be observed directly by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0039] In an alternative embodiment, detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle labels.

[0040] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0041] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.60, Cy3.77, Cy3.78, Cy3.79, Cy3.80, Cy3.90, Cy3.91, Cy3.92, Cy3.93, Cy3.94, Cy3.95, Cy3.96, Cy3.97, Cy3.98, Cy3.99, Cy3.91, Cy3.91 5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0042] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphate glucose deoxidase.

[0043] In an alternative embodiment, radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re、 211 At125 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 Such as 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.

[0044] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0045] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids.

[0046] Nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0047] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0048] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0049] In a third aspect, the present invention also provides a reagent or kit for tumor diagnosis or tumor screening, which detects the level of AFP biomarker in a sample for tumor diagnosis or tumor screening, and the reagent or kit includes the above-mentioned binding protein that specifically binds to AFP protein.

[0050] In an alternative embodiment, the tumor is a primary tumor or a transplanted tumor.

[0051] In an alternative embodiment, the tumor is an epithelial tumor.

[0052] In an alternative embodiment, the type of tumor is selected from squamous cell carcinoma, testicular cancer, ovarian cancer, pancreatic cancer, gastric cancer, intestinal cancer, germline embryonal tumor or liver cancer.

[0053] The germ-derived embryonal tumor is selected from teratoma, undifferentiated blastoma, yolk sac carcinoma, choriocarcinoma, spermatoepithelioma or fetal carcinoma.

[0054] In a fourth aspect, the present invention further provides a use of a binding protein that specifically binds to an AFP protein in any of the following:

[0055] (1) Preparation of reagents or kits for tumor diagnosis or tumor screening;

[0056] (2) Prepare a detection reagent or kit for alpha-fetoprotein.

[0057] The above-mentioned tumor screening is used for tumor screening in a wide spectrum of people, including but not limited to physical examinations, hospital admission examinations, etc.

[0058] In an alternative embodiment, the detection of alpha-fetoprotein is an immunoassay of alpha-fetoprotein;

[0059] In an alternative embodiment, the immunoassay of alpha-fetoprotein is to label the sample to be tested with the binding protein.

[0060] In an optional embodiment, the sample to be tested is a cell or a tissue.

[0061] In a fifth aspect, the present invention also provides a vector comprising a nucleic acid encoding the aforementioned binding protein that specifically binds to the AFP protein.

[0062] In a sixth aspect, the present invention also provides a host cell containing the above-mentioned vector.

[0063] The host can be a mammal suitable for antibody production. For example, the host can be rabbits, mice, rats, goats, camelids (such as camels and alpacas), or cartilaginous fish. Depending on the host, the anti-antibody produced can be double-chain (one light chain and one heavy chain) or single-chain (antibodies produced by camelids containing only heavy chains). This single-chain antibody is also called a nanobody.

[0064] The present invention also provides a method for producing the aforementioned binding protein, comprising:

[0065] The host cells of the aforementioned embodiments are cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cells.

[0066] The production method can be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein and culturing the host cell under appropriate conditions to allow the binding protein to be expressed. The host cell can also be transfected with one or more expression vectors, which can contain DNA encoding at least a portion of the binding protein, either alone or in combination. The binding protein can be isolated from the culture medium or cell lysate using conventional protein and peptide purification techniques, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.) and / or electrophoresis.

[0067] Construction of suitable vectors containing the desired coding and regulatory sequences can be performed using standard ligation and restriction techniques well known in the art. Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cut, tailed, and religated in the desired form. Mutations can be introduced into the coding sequence by any method to produce variants of the present invention, and these mutations can include deletions, insertions, or substitutions.

[0068] A method for detecting AFP comprises: mixing the binding protein of any one of the aforementioned embodiments with a sample to be tested.

[0069] In an alternative embodiment, the above method is for the purpose of non-disease diagnosis.

[0070] It should be noted that those skilled in the art can perform qualitative or quantitative detection of the AFP protein in the sample to be tested based on the characteristics of antibody / antigen binding to form immune complexes.

[0071] The present invention has the following beneficial effects:

[0072] The present invention provides a binding protein that specifically binds to an AFP polypeptide. The binding protein can specifically bind to AFP and has good binding activity and affinity. Using the binding protein of the present invention to detect AFP can improve the sensitivity and specificity of AFP detection. The binding protein can be used for both diagnosing or screening for diseases that use AFP as a biomarker and detecting benign diseases that use AFP as a biomarker. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0074] Figure 1 The following are the results of immunohistochemistry experiments on liver cancer samples (A is AFP-4E9 antibody, B is Roche's AFP antibody);

[0075] Figure 2 These are the results of immunohistochemistry experiments on placental tissue (A is AFP-4E9 antibody, B is AFP antibody from Roche). DETAILED DESCRIPTION

[0076] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may 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 may be used in another embodiment to produce further embodiments.

[0077] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0078] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0079] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0080] Example 1

[0081] This example is about the preparation of monoclonal antibodies specific to AFP protein.

[0082] 1. Animal (mice) immunization

[0083] Immunize mice with immunogens using a common method. The immunogen is human AFP protein, which is also used as a detection antigen to measure and screen serum titers and hybridomas. High-purity antigens can increase the chance of obtaining the desired monoclonal antibody and reduce the amount of screening. Immunize 5 mice, each with 50ug of AFP antigen. Prepare the antigen protein solution with PBS. Put an appropriate amount of antigen protein, PBS, and Freund's adjuvant into a syringe, plug the water outlet of the syringe with a stopper, and place it on an emulsifier to stir and emulsify it fully, so that the antigen adjuvant forms a stable oil-in-water solution. The first tail blood serum titer test is performed 7-10 days after the primary and secondary immunizations, and a good titer is obtained after 2 to 4 booster immunizations. Select immunized mice with high serum titers and perform cell fusion after the final intraperitoneal immunization.

[0084] 2. Hybridoma Cell Fusion and Screening

[0085] Before cell fusion, preparations are required: 1. Culture mouse myeloma SP2 / 0 cells until the logarithmic growth phase. 2. One day before fusion, sacrifice a negative mouse. In a sterile environment, inject HAT medium into the mouse's peritoneal cavity to obtain mouse peritoneal trophoblast cells. Plate 100 μl per well of these cells in a 96-well plate. These cells promote hybridoma cell growth. Sacrifice the immunized mouse, remove the spleen under a sterile environment, and chemically fuse the splenic B cells and SP2 / 0 myeloma cells using PEG. Add the appropriate amount of HAT medium based on the number of cells to be plated, and finally plate the fused cells onto trophoblast cell culture plates at 100 μl per well.

[0086] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, supernatants from each well were collected and screened for hybridomas using an ELISA assay using the human AFP-his protein antigen. The procedure is as follows: Coat the microplate with 100 μl of a 2 μg / ml solution of human AFP peptide antigen in PBS at 37°C for two hours. Wash the plate three times with PBST, then block overnight with 150 μl / well of 3% skim milk powder in PBS at 4°C. Wash the plate three times, add 80 μl / well of hybridoma supernatant, incubate at 37°C for one hour, and then wash the plate three times. Add 100 μl / well of a 1:8000 dilution of horseradish peroxidase-conjugated goat anti-mouse secondary antibody, incubate at 37°C for 45 minutes, wash the plate three times, and pat dry. Add 100 μl / well of TMB colorimetric solution, develop at room temperature for 5-10 minutes, stop with 2 M sulfuric acid, and measure the absorbance of each well at 450 nm. Positive hybridoma cells were selected.

[0087] Select the ELISA-positive fusion wells and perform immunohistochemistry (IHC), and select the positive wells for subsequent experiments.

[0088] The immunohistochemistry (IHC) experimental steps are as follows:

[0089] Bake the liver cancer slices in a 60°C constant temperature oven for 60 minutes, soak the slices in xylene I for 15 minutes, then replace with xylene II and soak for 15 minutes, then soak 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; soak in ddH2O for 5 minutes and wash three times; use a pressure cooker for antigen repair (boiling method) to add 10mL / mol citrate buffer (pH 6.0) enough to submerge the slices into the pressure cooker, heat to boiling, place the slices on a heat-resistant material slice rack, put them into the pot, cover the pot, buckle the pressure valve, continue heating, set the pressure to maintain for 4 minutes, open the vent valve to release the air after the time is up, open the pot lid after the pressure returns to zero, remove the inner pot and let it cool at room temperature. After the solution cools to room temperature, remove the slices (about 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, and wash twice; place the slices 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 and wash three times; add one drop of goat serum blocking solution (about 25 μl) to each tissue group and incubate in a wet box at room temperature for 45 minutes; soak in PBST for 5 minutes and wash three times.

[0090] The treated tissue sections were added with Roche's AFP antibody for comparison, and the remaining sections were added with the AFP-4E9 antibody secreted by the above-mentioned positive hybridoma cells. Incubate overnight in a 4°C wet box; remove from the 4°C refrigerator and incubate at room temperature for 60 minutes; gently rinse with PBST, soak for 5 minutes, and wash three times; add 25ul of HRP-labeled Changdao Company secondary antibody (CAT#:) to each tissue group and incubate at room temperature for 45 minutes; wash; prepare DAB color development solution, react in the dark for 10-15 minutes, then add it dropwise to the slice and color for 1-5 minutes; terminate the color development reaction with distilled water; add 50ul of hematoxylin stain to each tissue group, stain for 5-10 minutes, and rinse with distilled water; decolorize the slice in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove it and place it in distilled water to terminate, and then place it in PBST (pH8.0) for anti-blueing 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 change to xylene and soak for another 10 minutes; add neutral gum to seal the slide, then cover with a glass slide; photograph the slide under a microscope.

[0091] The fusion cells that were positive for binding were selected by ELISA and IHC experiments, cloned by limiting dilution, and each well of the positive strain was plated on a 48 / 96-well plate and continued to be cultured. A second round of screening was performed by ELISA to screen out hybridomas that specifically recognized the AFP peptide and could block AFP binding. The hybridomas were subcloned by limiting dilution to obtain a monoclonal cell line 4E9. The monoclonal cell line was expanded and about 1×10 6 The cells are injected into selected mice (paraffin oil is injected into the peritoneal cavity one week in advance). After 7-10 days, the mice develop ascites, which is collected for antibody purification. After purification, mouse monoclonal antibodies specific for the AFP polypeptide are obtained.

[0092] Example 2

[0093] DNA cloning and sequencing were performed on the mouse monoclonal antibody specific for anti-AFP polypeptide screened in Example 1, and the variable region gene of the anti-human AFP monoclonal antibody was sequenced.

[0094] Total RNA was extracted from the mouse monoclonal cell line 4E9 using Trizol reagent. Cells cultured in a 9 cm dish were collected and transferred to a 1.5 ml centrifuge tube. The supernatant was aspirated. 1 ml of Trizol reagent was added and the cells were lysed by pipetting. The lysed sample or homogenate was allowed to stand at room temperature for 5-10 minutes to completely separate the nuclear proteins from the nucleic acids. 0.2 ml of chloroform was added, the mixture was shaken vigorously for 15 seconds, and the mixture was allowed to stand at room temperature for 3 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a clean centrifuge tube, an equal volume of isopropanol was added, the mixture was mixed, and the mixture was allowed to stand at room temperature for 20 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes and the supernatant was discarded. The pellet was washed with 1 ml of 75% ethanol. Centrifuge at 12,000 rpm at 4°C for 3 minutes and the supernatant was discarded. The pellet was dried at room temperature for 5-10 minutes. 30-50 μl of RNase-free ddH2O was added. The resulting RNA solution was stored at -70°C or used for subsequent experiments.

[0095] Total RNA was reverse transcribed into cDNA using the AMV First-Strand cDNA Synthesis Kit. The experimental system was configured as follows: 6ul of total RNA + 1ul of Oligo dT + 4ul of RNase-free water (a total of 11ul). Gently mix and centrifuge for 3-5 seconds. The reaction mixture was pre-denatured by incubating at 65°C for 5min, then placed on ice for 30s, centrifuged for 3-5s, and then placed on ice for 2min. While still on ice, 4ul of 5x buffer + 1ul of dNTP mix + 1ul of RNase inhibitor + 1ul of reverse transcriptase (a total of 20ul system) were added. Gently mix and centrifuge for 3-5 seconds. cDNA synthesis was completed in a PCR instrument at 42°C for 50 minutes and 85°C for 5 minutes. Random primers are suitable for the synthesis of short-chain cDNAs under 500bp. The transcribed RNA template does not require a poly(A) tail, and the 5' end region can be transcribed.

[0096] PCR amplification of light and heavy chains. To amplify the antibody light chain variable region sequence, configure the PCR reaction system: 25ul of 2x Taq enzyme buffer + 1ul of FP-VL + 1ul of RP-VL + 2ul of cDNA + 21ul of ddH2O. To amplify the antibody heavy chain variable region sequence, configure the PCR reaction system: 25ul of 2x Taq enzyme buffer + 1ul of FP-VH + 1ul of RP-VH + 2ul of cDNA + 21ul of ddH2O. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (steps 2 to 4 are repeated 35 times):

[0097] Step 1: Pre-denaturation at 94°C for 4 min;

[0098] Step 2 - denaturation at 94°C for 30 seconds;

[0099] Step 3: Annealing at 55°C for 45 seconds.

[0100] Step 4 - extension at 72°C for 60 seconds;

[0101] Step 5-72°C, 10 min;

[0102] Step 6 - Store at 4°C.

[0103] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA segments of corresponding sizes were cut out (approximately 375 bp for VH and approximately 325 bp for VL). DNA was extracted using the SanPrep column-based DNA gel extraction kit. The procedure is as follows: Cut the gel containing the target fragment from the agarose gel and weigh it. Add 3-6 times the weight of the gel in buffer B2 and incubate in a 50°C water bath for 5-10 minutes to dissolve the gel. Transfer the gel solution to an adsorption column and centrifuge at 8000g for 30 seconds. Discard the liquid in the collection tube. Add 500ul of wash solution to the column and centrifuge at 9000g for 30 seconds. Discard the liquid in the collection tube. Repeat the addition of wash solution once and discard the liquid. Centrifuge the adsorption column at 9000g for 1 minute. Place the adsorption column in a clean 1.5ml centrifuge tube and add 15-40ul of Elution Buffer to the center of the adsorption membrane. Let it stand at room temperature for 1 minute and then centrifuge for 1 minute. The prepared DNA solution is obtained, and the variable region sequence of the antibody is obtained by sequencing the purified PCR product.

[0104] Experimental Example 1

[0105] This experiment was conducted to test the affinity and sensitivity of mouse monoclonal antibodies specific for AFP polypeptide.

[0106] The experimental steps are as follows:

[0107] 1. Coat the plate with AFP antigen at 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L.

[0108] 2. Adjust the antibody concentration to 10 -7 mol / L level (1*10 -7 Up to 5*10 -7 Then dilute the sample in a serial ratio of 1:2-1:256 and add it to the wells with different antigen coating amounts.

[0109] 3. Add secondary antibody and develop color with TMB. Measure absorbance at 450 nm. The measured data are shown in Table 1.

[0110] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration that gives the half-maximum absorbance at different antigen concentrations. This gives you four antibody concentrations (mol / L).

[0111] 5. Substitute the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB' and AB are the antibody concentrations that produce half-maximal absorbance at the corresponding antigen concentrations AG (3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L). N = AG / AG' (where AG > AG').

[0112] Table 1

[0113]

[0114]

[0115] In summary, the mouse monoclonal antibody specific for AFP peptide binds to AFP protein with a KD≤3.126×10 9 The affinity binding is 0.1 L / mol, and the binding protein is an antibody or a functional fragment.

[0116] Figure 1 、 Figure 2 For the immunohistochemical assay of AFP, Figure 1 The specimen was liver cancer. Figure 2 The test specimen is placental tissue. Figure 1 A is the purified AFP-4E9 antibody produced by the present invention, and B is the AFP antibody produced by Roche. Figure 2 Figure A represents the purified AFP-4E9 antibody produced by the present invention, and Figure B represents the AFP antibody produced by Roche. The staining results in Figures A and B show no significant difference in staining location or intensity between the AFP-4E9 and Roche antibodies. This demonstrates the promising application of the AFP provided by the present invention in the preparation of kits.

[0117] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to AFP protein, characterized in that: The antibody comprises an antigen binding domain having six complementarity determining regions as shown below: CDR-VH1:SYGMS; CDR-VH2: TISSSGGRNIYYPDSVKG; CDR-VH3:QQYGVYVFDY; CDR-VL1: KSSQSLLYSSNQKNYLA; CDR-VL2: WASTRES; CDR-VL3:QQYFSYPT.

2. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 1, wherein: The antibody and AFP protein are K D ≤3.126×10 9 L / mol affinity binding.

3. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 1 or 2, characterized in that: The antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, whose sequences are sequentially shown in SEQ ID NOs: 1-4, and / or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, whose sequences are sequentially shown in SEQ ID NOs: 5-8.

4. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 3, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

5. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 4, characterized in that: The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

6. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 4, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or primate.

7. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 6, characterized in that: The primate is selected from a lemur, a macaque, an ape or a human.

8. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 4, characterized in that: The constant region is derived from mouse.

9. The antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to claim 1, characterized in that: The antigen-binding fragment of the antibody is selected from any one of F(ab')2, Fab', Fab, Fv and scFv.

10. A detection reagent or kit for alpha-fetoprotein, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof that specifically binds to the AFP protein according to any one of claims 1 to 9.

11. The reagent or kit according to claim 10, characterized in that The antibody is labeled with a detectable marker.

12. A reagent or kit for tumor diagnosis or tumor screening, characterized in that: It is used for tumor diagnosis or tumor screening by detecting the level of AFP biomarker in the sample to be tested, and the reagent or kit includes the antibody or antigen-binding fragment thereof that specifically binds to the AFP protein according to any one of claims 1-9.

13. Use of the antibody or antigen-binding fragment thereof that specifically binds to AFP protein according to any one of claims 1 to 9 in any of the following: (1) Preparation of reagents or kits for tumor diagnosis or tumor screening; the tumor uses alpha-fetoprotein as a biomarker; (2) Prepare a detection reagent or kit for alpha-fetoprotein.

14. The use according to claim 13, characterized in that The detection of alpha-fetoprotein is an immunoassay of alpha-fetoprotein.

15. The use according to claim 14, characterized in that The immunoassay of alpha-fetoprotein is to label the sample to be tested with the antibody; The sample to be tested is a cell or a tissue.

16. A carrier, characterized in that The invention contains a nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to an AFP protein as claimed in any one of claims 1 to 9.

17. A host cell, characterized in that It contains the vector according to claim 16.

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