Anti-human-phosphorylated synuclein antibodies and uses thereof
By developing a monoclonal antibody that specifically binds to phosphorylated α-synuclein at serine position 129, the problem of insufficient diagnostic sensitivity and specificity in existing technologies has been solved, enabling effective detection and treatment of neurodegenerative diseases such as Parkinson's disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUIAN NEW DISTRICT KANGSHUN BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-02-13
- Publication Date
- 2026-06-16
Smart Images
Figure CN116102646B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-human phosphorylated synuclein antibody and its application. Background Technology
[0002] Parkinson's disease is a common neurodegenerative disease, clinically characterized by motor symptoms such as resting head vibration, bradykinesia, muscle rigidity, and postural instability. These symptoms are caused by the extensive death of dopamine neurons in the substantia nigra of the midbrain and the resulting significant reduction in dopamine neurotransmitters in the striatum. The main pathological feature of Parkinson's disease is the presence of eosinophilic inclusion bodies—Lewy bodies and Lewy neurites—with fibrotic α-synuclein being a major component. Numerous studies have shown that α-synuclein plays a crucial role in the pathogenesis of Parkinson's disease. Point mutations and polyploid variations in the α-synuclein gene can cause familial early-onset Parkinson's disease, and the polymorphism of its gene promoter is associated with the risk of developing Parkinson's disease. It has been demonstrated that α-synuclein is altered in the brain tissue of Parkinson's patients, manifested as abnormally increased expression levels and increased phosphorylation, nitration, ubiquitination, and glycosylation modifications. Abnormally expressed and modified α-synuclein causes its abnormal aggregation into true polymers, which then form fibers and deposit in Lewy bodies. Extensive evidence suggests that abnormally expressed and modified α-synuclein promotes its aggregation into neurotoxic oligomers, a key factor in neuronal degeneration and Parkinson's disease. Given the crucial role of α-synuclein in the pathogenesis and pathophysiology of Parkinson's disease, detecting changes in this protein in body fluids is considered diagnostically significant. Current cerebrospinal fluid (CSF) analysis shows decreased total α-synuclein levels and increased oligomeric α-synuclein levels in Parkinson's patients, demonstrating high diagnostic specificity and sensitivity. Currently, the detection of α-synuclein in the blood of Parkinson's patients is mainly limited to serum and plasma. However… Due to limitations in the sensitivity and stability of detection technologies, as well as the influence of other interfering factors, the detection results of α-synuclein in serum and plasma and its diagnostic significance in Parkinson's disease remain inconclusive. Normal values reported by different researchers vary by hundreds or even thousands of times. Reports of total α-synuclein levels in the plasma and serum of Parkinson's patients have varied, including higher levels than normal controls, lower levels than normal controls, and no significant changes. The level of phosphorylated α-synuclein in the plasma of Parkinson's patients is higher than that in normal controls.
[0003] Phosphorylation of α-synuclein can regulate its structure, membrane binding, aggregation, fibrillation, and neurotoxicity (Fujiwara et al. 2002; Anderson et al. 2006). α-synuclein is composed of phosphorylated proteins, with serine 87 and serine 129 being the major phosphorylation sites (Okochi et al. 2000; Pronin et al. 2000; Fujiwara et al. 2002; Kahle et al. 2002; Takahashi et al. 2003a; Chen and Feany 2005; Anderson et al. 2006; Kim et al. 2006; Ishii et al. 2007; Paleologou et al. 2010); a cycle of phosphorylation and dephosphorylation occurs in vivo. Phosphorylation at S129 and S87 has been shown to inhibit α-synuclein aggregation (Waxman and Giasson 2008; Paleologou et al. 2010). Similarly, tyrosine phosphorylation at Y125, Y133, and Y135 is associated with inhibition of α-synuclein aggregation and toxicity (Ellis et al. 2001; Nakamura et al. 2001; Ahn et al. 2002; Negro et al. 2002; Takahashi et al. 2003b; Chen and Feany 2005; Chen et al. 2009). Furthermore, phosphorylation of α-synuclein at S129 and Y125 affects protein-protein interactions (McFarland et al. 2008). The exact kinases and phosphatases mediating α-synuclein phosphorylation and dephosphorylation remain unknown.However, various in vitro and cell-based studies have identified several kinases capable of phosphorylating α-synuclein, including casein kinase I (Okochi et al. 2000) and Dyrk1A (Kim et al. 2006) at S87, phosphorylating α-synuclein at S129 via casein kinases I and II (Okochi et al. 2000), G protein-coupled receptors 1, 2, 5, and 6 (Pronin et al. 2000), LRRK2 (Qing et al. 2009), and polo-like kinases (Ingliset et al. 2009; Mbefo et al. 2010); and at Y125, via Fyn (Nakamura et al. 2001), Syk (Negro et al. 2002), Lyn (Negro et al. 2002), c-Frg (Negro et al. 2002), and Src tyrosine kinase (Elliset). (al.2001); at Y126 and Y133, Syk tyrosine kinase (Negro et al.2002).
[0004] In summary, many companies and research institutions internationally are actively conducting research in this area. However, there are currently few immunohistochemical detection and related diagnostic methods based on phosphorylation site-specific α-synuclein antibodies, and most remain within the research scope. Therefore, there is an urgent need to provide an antibody against Ser129 site amino acid phosphorylation α-synuclein. Summary of the Invention
[0005] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide an anti-human-phosphorylated synuclein antibody and its application.
[0006] The first aspect of the present invention provides an antibody comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises three complementarity-determining regions (CDRs) of amino acid sequences SEQ ID NO. 1, positions 26-33, 51-58, and 97-103, respectively. The light chain variable region comprises three CDRs of amino acid sequences SEQ ID NO. 2, positions 27-32, 50-52, and 89-97, respectively. The antibody binds to human α-synuclein.
[0007] Furthermore, the α-synuclein is a phosphorylated α-synuclein. Even further, the α-synuclein is a serine 129 phosphorylated α-synuclein.
[0008] Furthermore, the amino acid sequence of the heavy chain variable region is SEQ ID NO.1.
[0009] SEQ ID NO.1:
[0010] EIQLQQTGPELVKPGASVKISCKASGYSFTDYIMLWVKQSHGKSLEWIGNINPYYGSTS YNLKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARGDYLYWGQGTTLTVSS.
[0011] Furthermore, the amino acid sequence of the light chain variable region is SEQ ID NO.2.
[0012] SEQ ID NO.2:
[0013] DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKR FSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPPTFGGGTKLEIK.
[0014] Furthermore, both the heavy chain variable region and the light chain variable region contain a framework region derived from mice.
[0015] Furthermore, the antibody is a monoclonal antibody.
[0016] A second aspect of the present invention provides a biomaterial, said biomaterial being any one of A1) to A5):
[0017] A1) Nucleic acid molecules encoding the antibodies described in the first aspect above;
[0018] A2) An expression cassette containing the nucleic acid molecules described in A1);
[0019] A3) A recombinant vector containing the nucleic acid molecules described in A1);
[0020] A4) Recombinant microorganisms containing the nucleic acid molecules described in A1);
[0021] A5) Cell lines containing the nucleic acid molecules described in A1).
[0022] Furthermore, the nucleic acid molecule is a heavy chain variable region DNA molecule encoding SEQ ID NO.1 and / or a light chain variable region DNA molecule encoding SEQ ID NO.2.
[0023] The third aspect of this invention provides the use of the antibody described in the first aspect or the biomaterial described in the second aspect in the preparation of a medicament for the prevention, relief, and / or treatment of neurodegenerative disorders associated with α-synuclein pathology.
[0024] Furthermore, the neurodegenerative disorders are Parkinson's disease, Lewy body dementia, Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, or Creutzfeldt-Jakob disease.
[0025] Furthermore, the α-synuclein is a phosphorylated α-synuclein. Even further, the α-synuclein is a serine 129 phosphorylated α-synuclein.
[0026] A fourth aspect of the present invention provides a medicament for preventing, alleviating, and / or treating neurodegenerative disorders associated with α-synuclein pathology, the medicament comprising the antibody described in the first aspect or the biological material described in the second aspect.
[0027] Furthermore, the drug also includes a pharmaceutically acceptable carrier.
[0028] Furthermore, the neurodegenerative disorders are Parkinson's disease, Lewy body dementia, Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, or Creutzfeldt-Jakob disease.
[0029] Furthermore, the α-synuclein is a phosphorylated α-synuclein. Even further, the α-synuclein is a serine 129 phosphorylated α-synuclein.
[0030] The fifth aspect of the present invention provides the use of the antibody described in the first aspect above in the detection of α-synuclein or in the preparation of products for the detection of α-synuclein.
[0031] Furthermore, the α-synuclein is a phosphorylated α-synuclein. Even further, the α-synuclein is a serine 129 phosphorylated α-synuclein.
[0032] Furthermore, the method for detecting α-synuclein includes contacting the antibody described in the first aspect above under conditions that allow the formation of a complex between the antibody and α-synuclein, and detecting the formation of the complex.
[0033] Numerous studies have shown that α-synuclein is a protein that plays a crucial role in the pathogenesis of Parkinson's disease. Point mutations and polyploid variations in the α-synuclein gene can cause familial early-onset Parkinson's disease, and the Dogelian nature of its gene promoter is associated with the risk of developing Parkinson's disease. It has been demonstrated that α-synuclein is altered in the brain tissue of Parkinson's patients, exhibiting abnormally increased expression levels and increased phosphorylation, nitration, ubiquitination, and glycosylation modifications. Abnormally expressed and modified α-synuclein causes its abnormal aggregation into true aggregates, which then form fibers and deposit in Lewy bodies. Extensive evidence suggests that abnormally expressed and modified α-synuclein promotes its aggregation into neurotoxic oligomers, a key factor contributing to neuronal degeneration and Parkinson's disease. Phosphorylated α-synuclein antibodies reduce the abnormal aggregation caused by phosphorylated α-synuclein by binding to it, thereby reducing the toxic effects of this aggregation on neurons and slowing the progression of Parkinson's disease. Therefore, this monoclonal antibody has a certain therapeutic effect on Parkinson's disease.
[0034] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows:
[0035] This invention provides an anti-α-synuclein antibody that can bind to α-synuclein phosphorylated at serine 129. This antibody can be used not only to measure the level of abnormally phosphorylated α-synuclein, but also to prevent, alleviate, and / or treat neurodegenerative disorders (such as Parkinson's disease, Lewy body dementia, Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, or Creutzfeldt-Jakob disease) associated with α-synuclein pathology. Attached Figure Description
[0036] Figure 1 A schematic diagram of the antigen layout on small squares cut from an NC membrane;
[0037] Figure 2 This is a diagram showing the initial screening results of hybridoma cells;
[0038] Figure 3 This is a diagram showing the results of secondary screening of hybridoma cells;
[0039] Figure 4 Image showing the results of hybridoma cell subclonal screening;
[0040] Figure 5 The figure shows the results of Western blot identification of the specificity of the 3B3 antibody against P-α-syn and W-α-syn proteins. Detailed Implementation
[0041] The following detailed description is exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof.
[0043] Unless otherwise specified, the experimental methods in the following examples all employ conventional techniques in this technical field or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available products.
[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0045] Example 1: Obtaining mouse anti-human phosphorylated synuclein antibodies
[0046] 1. Preparation of antigen proteins:
[0047] A human α-synuclein peptide containing serine phosphorylation at position 129 (amino acid sequence of the peptide: EAYEMP(pS)EEGYQD) was synthesized as an antigen.
[0048] 2. Immunized animals
[0049] Immunization was performed using female BALB / c mice. 250g of the antigen protein prepared in step 1 was dissolved in 500μL of PBS buffer to obtain an antigen solution. The antigen solution was mixed with Freund's complete adjuvant at a 1:1 volume ratio, and emulsified in a syringe to obtain the emulsion, which was the immunogen. The immunogen was administered subcutaneously to the abdomen of BALB / c female mice at a dosage of 200μL / mouse (i.e., 50μg of antigen protein / mouse).
[0050] Female BALB / c mice were boosted with immunization every two weeks. On day 5 after the third booster immunization, 50-100 μL of blood was collected from the tail vein, and serum was separated. Serum antibody titers and specificities were detected by Dot blot and Western blot methods, respectively. Mice with high serum titers were selected for cell fusion.
[0051] 3. Cell fusion
[0052] (1) Preparation of myeloma cells: One week before fusion, Sp2 / 0 mouse myeloma cells were resuscitated to ensure that at least 5 × 10⁶ cells were prepared on the day of fusion. 7 One healthy myeloma cell.
[0053] (2) Final immunization: Three days before fusion, mice with high serum titers were injected with antigen via the tail vein or five days before fusion, mice with high serum titers were injected with antigen via the peritoneum.
[0054] (3) Preparation of thymocytes:
[0055] ① After removing the cones from two prepared BalB / c mice (SPF grade) that are about 4 weeks old, immerse them in 75% alcohol for several tens of seconds for disinfection and wipe the mice with alcohol cotton balls; after surgery, thorax opening and thymus removal of the mice, wash them in 4℃ basic culture medium to remove impurities (excess tissue, membranes, etc.); transfer the washed thymus to a new 4℃ basic culture medium, and grind and squeeze the thymus with a sterile ground glass slide to squeeze out the thymic cells from the capsule.
[0056] ② Filter the basic culture medium containing cells using a sterile 200-mesh nylon mesh to remove impurities; transfer the cells to a 50ml centrifuge tube, centrifuge at 2000rpm for 5 minutes, and remove the supernatant; resuspend the cells in 30ml of 4℃ basic culture medium, centrifuge at 1500rpm for 5 minutes, and remove the supernatant; add another 30ml of 4℃ basic culture medium to resuspend the cells, take 0.1ml of the suspension, dilute it 100 times, and count the cells using a hemocytometer.
[0057] ③ Add 37℃ HAT medium to 100ml (5×10⁻⁶) 6 (each vial / ml), take 50ml into two 75cm containers respectively. 2 Incubate overnight in a CO2 incubator in culture flasks. On average, each mouse yields 1–2 × 10⁻⁶ cells / year. 8 Each cell.
[0058] (4) Preparation of spleen cells
[0059] ① Select mice with high serum titers, remove the cones, immerse them in 75% alcohol for several tens of seconds for disinfection, and wipe the mice with alcohol cotton balls; after surgery, thoracotomy and spleen removal, wash the mice in 4℃ basic culture medium and remove impurities (excess tissue, membranes, etc.); transfer the washed spleen to a new 4℃ basic culture medium, grind and squeeze the spleen with a sterile ground glass slide to squeeze the spleen cells out of the capsule.
[0060] ②Filter the basic culture medium containing cells using a sterile 200-mesh nylon mesh to remove impurities; transfer the cells to a 50ml centrifuge tube, centrifuge at 1600rpm for 5 minutes, and remove the supernatant; add 10ml of 4℃ basic culture medium to suspend the cells in the centrifuge tube, then add more to 40ml, centrifuge at 1600rpm for 5 minutes, remove the supernatant, add another 10ml of 4℃ basic culture medium to suspend the cells, take 0.1ml of the suspension and dilute it 100 times, and count the cells using a hemocytometer.
[0061] ③ Add 900 μL of basal culture medium to each of the two EP tubes. Add 100 μL of cell suspension to the first EP tube, mix thoroughly, and then add another 100 μL from the first EP tube to the second EP tube. Mix thoroughly and then count the cells (an average of 1–3 × 10⁶ cells per mouse can be obtained). 8 (Spleen cells).
[0062] (5) Cell fusion
[0063] ① After suspending the prepared myeloma cells, centrifuge at 1200 rpm for 5 min and discard the supernatant. Resuspend the cells in 10 ml of basal culture medium in a centrifuge tube and count the cells using a hemocytometer.
[0064] ② Add 900 μL of basic culture medium to each of the two EP tubes. Take 100 μL of cell suspension and add it to the first EP tube. Mix thoroughly. Then take another 100 μL from the first EP tube and add it to the second EP tube. Mix thoroughly and then count the cells.
[0065] ③ In a 50ml centrifuge tube, add 1×10 8 10 spleen cells and 2×10 7 Mix 5:1 myeloma cells, centrifuge at 1600 rpm for 6 min, discard the supernatant, and then gently tap the centrifuge tube wall with your finger to loosen the cell pellet; add 1 ml of preheated PEG (mol wt. 1300-1600) solution at 37°C to the centrifuge tube while stirring (timing required), add the solution within 1 min, and then stir for another 1 min.
[0066] ④ Add preheated 37℃ basic culture medium (first time): 1 ml / min, while stirring. Add preheated 37℃ basic culture medium (second time): 1 ml / min, while stirring. Add preheated 37℃ basic culture medium (third time): 8 ml / 3 min, while stirring.
[0067] ⑤ Centrifuge at 1200 rpm for 5 minutes, remove the supernatant (containing PEG) to loosen the cell pellet; add 10 ml of thymocyte suspension, disperse the spleen cells, and then add another 90 ml of thymocyte suspension to bring the final volume to 100 ml. The spleen cell density is 1 × 10⁻⁶ cells / ml. 6 / ml, thymocyte density 5×10 6 / ml. Assuming a total volume of 50ml, the spleen cell density is 2×10⁻⁶. 6 / ml, thymocyte density 2.5×10 6 / ml.
[0068] ⑥ After thoroughly suspending the cells, seed them into 96-well plates (100 μL / well) * 10 plates. The typical hybridoma formation rate is 1 hybridoma / 4 × 10⁻⁶. 4 Each well contains 2.5 hybridomas (for a total volume of 100 ml) or 5 hybridomas (for a total volume of 50 ml). Incubate at 37°C with 5% CO2. On day 2, add approximately 100 μL (about 2 drops) of HAT medium to each well. On day 4, replace half the medium (aspirate half the medium and add 2 drops of fresh HAT medium). On day 7, replace half the medium again. On day 10, replace half the medium again, and perform screening on days 12-13.
[0069] 4. Hybridoma cell screening
[0070] (1) Initial screening
[0071] The Dot blot method was used to perform initial screening of all fusion cells in the 96-well plate.
[0072] 1) The experimental method is as follows:
[0073] ① Coating of NC membrane: The NC membrane is coated with a coating solution containing 0.1% gelatin;
[0074] ② Preparation of antigen solution: Prepare an antigen solution with a concentration of 0.4 mg / ml by preparing the human α-synuclein peptide containing serine phosphorylation at position 129 synthesized in step 1;
[0075] ③ Spotting antigen: Spot the antigen onto the NC membrane, 1 μL / spot. After fumigation with paraformaldehyde, cut each spot on the NC membrane into a small square (example of antigen layout on the cut squares of the NC membrane is shown below). Figure 1 (As shown in A) is pending.
[0076] ④ After numbering the fusion cell 96-well plate and the prepared 48-well plate accordingly, add PBST to the 48-well plate at a rate of 200 μL / well.
[0077] ⑤ Transfer 100 μL of the supernatant from hybridoma cells cultured for 12-13 days into a new, numbered 96-well plate. Add fluid to the cells.
[0078] ⑥ Primary antibody incubation: Add 50 μL of cell supernatant from a new 96-well plate to the PBST solution in the corresponding numbered 48-well plate. Add the corresponding numbered NC membrane to the 48-well plate and incubate overnight at room temperature with shaking. The next day, wash the membrane with PBST.
[0079] ⑦ Incubation with secondary antibody: Dilute goat anti-mouse IgG secondary antibody 1 / 1000 with PBST, add the NC membrane to the antibody solution, and incubate at room temperature with shaking for 2 hours. Then, wash the membrane with PBST.
[0080] ⑧ Triple antibody incubation: Dilute the horseradish peroxidase-labeled triple antibody 1 / 1000 with PBST, add the NC membrane to the antibody solution, and incubate at room temperature with shaking for 2 hours. Then, wash the membrane with PBST.
[0081] ⑨ DAB color development: Add the NC membrane to the DAB color development solution and stop the color development with tap water.
[0082] 2) Experimental Results
[0083] Positive hybridoma cells were screened based on the colorimetric results of the NC membrane. The screening results for positive hybridoma cells are as follows: Figure 2 As shown.
[0084] Depend on Figure 2 It can be seen that a total of 48 hybridoma cells with good positive results were screened.
[0085] (2) Secondary screening:
[0086] Dot blot was used to specifically identify 48 positive hybridoma cells obtained from the initial screening.
[0087] 1) The experimental method is as follows:
[0088] ① Coating of NC membrane: The NC membrane is coated with a coating solution containing 0.1% gelatin;
[0089] ② Preparation of antigen solutions: The human α-synuclein peptide containing phosphorylated serine at position 129 (denoted as P-α-syn, human phosphorylated α-synuclein Ser129), wild-type α-syn peptide (denoted as W-α-syn, peptide sequence EAYEMPSEEGYQD), and full-length wild-type α-syn protein (denoted as F-α-syn) synthesized in step 1 were used as antigens, and the above three antigens were prepared into antigen solutions with a concentration of 0.4 mg / ml.
[0090] The F-α-syn amino acid sequence is:
[0091] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVA EKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMP VDP DNEAYEMPSE EGYQDYEPEA.
[0092] ③ Antigen Spotting: Spot the antigen onto the NC membrane, 1 μL / spot. After fumigation with paraformaldehyde, cut the NC membrane into small squares for later use. An example of the layout of the three antigens on the small squares cut from the NC membrane is shown below. Figure 1 As shown in B.
[0093] ④ Add PBST to the 24-well plate, 320 μL / well.
[0094] ⑤ Primary antibody incubation: Transfer 80 μL of the supernatant from 48 positive hybridoma cells into the corresponding numbered PBST solution. Add the corresponding numbered NC membrane to a 24-well plate and incubate overnight at room temperature with shaking. The next day, wash the membrane with PBST.
[0095] ⑥ Secondary antibody incubation: Dilute goat anti-mouse IgG secondary antibody 1 / 1000 with PBST, add the NC membrane to the antibody solution, and incubate at room temperature with shaking for 2 hours. Then, wash the membrane with PBST.
[0096] ⑦ Triple antibody incubation: Dilute the horseradish peroxidase-labeled triple antibody 1 / 1000 with PBST, add the NC membrane to the antibody solution, and incubate at room temperature with shaking for 2 hours. Then, wash the membrane with PBST.
[0097] ⑧ DAB color development: Add the NC membrane to the DAB color development solution and stop the color development with tap water.
[0098] 2) Experimental results:
[0099] Based on the color development results of the NC film, as follows: Figure 3 As shown.
[0100] Depend on Figure 3 It can be seen that the 3B3 hybridoma cell line has a stronger specific recognition ability for P-α-syn protein.
[0101] (3) Hybridoma cell subcloning
[0102] Subcloning experiments were performed on the positive hybridoma cells with good specificity results screened by the Dot blot method to obtain purer hybridoma cells. The specific method is as follows:
[0103] 1) First, use a hemocytometer to count the selected hybridoma cells: After fully suspending the positive hybridoma cells with good specificity results screened by the Dot blot method, use HT medium to serially dilute the cells to 1 / 10 and 1 / 100 in two EP tubes respectively, and then take the 1 / 10 cell dilution for cell counting.
[0104] 2) Take 150 cells from each 96-well culture plate. Calculate and take 150 cells from the 1 / 100 cell dilution and add them to 20 ml of HT medium. After fully suspending them, plate them at 200 μL / well. Place them in a CO2 incubator and incubate for about 12 to 14 days.
[0105] (4) Hybridoma cell subcloning screening
[0106] All subcloned tumor cells were again subjected to Dot blot analysis for specific identification. The specific experimental method for Dot blot analysis for specific identification is the same as step (2), and will not be repeated here.
[0107] The results of hybridoma cell subclonal screening are as follows Figure 4 As shown. By Figure 4 It can be seen that the positive cell line obtained after subcloning 3B3 hybridoma cells has been purified.
[0108] 5. Hybridoma cell cryopreservation
[0109] From 3B3 positive hybridoma cells, select wells containing a single clone. After thoroughly resuspending the cloned cells in the wells, transfer them from a 96-well plate to a 24-well plate for amplification culture. Transfer the hybridoma cells from the 24-well plate to culture flasks and amplify them for several days. After thoroughly resuspending the cells in the culture flasks, transfer them to centrifuge tubes and centrifuge at 1200 rpm for 5 min, then remove the supernatant. Resuspend the cells using cell cryopreservation buffer and aliquot them into cryovials, approximately 1 ml per cryovial. First, place the cells in a gradient cryopreservation box at -80°C for approximately 24 hours, then transfer them to a liquid nitrogen tank for long-term storage.
[0110] 6. Sequencing
[0111] RNA extraction, reverse transcription, antibody variable region PCR amplification, and sequencing were performed on positive hybridoma cells 3B3 to obtain the variable region cDNA sequence of the monoclonal antibody secreted by the mouse hybridoma cell line 3B3.
[0112] The cDNA sequence of the heavy chain variable region is as follows:
[0113] GAGATCCAGCTGCAGCAGACTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGACTACATCATGCTCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAATATTAATCCTTACTATGGTAGTACTAGCTACAATCTGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATGCAGCTCAACAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGATTACCTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGAGATCCAGCTGCAGCAGACTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGACTACATCATGCTCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAATATTAATCCTTACTATGGTAGTACTAGCTACAATCTGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATGCAGCTCAACAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGATTACCTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.3);
[0114] The cDNA sequence of the light chain variable region is:
[0115] (SEQ ID NO.4).
[0116] The corresponding amino acid sequences are as follows:
[0117] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1. Among them, the CDR (complementarity-determining region) 1 of the heavy chain variable region...
[0118] The amino acid sequences of the heavy chain variable region are shown in positions 26-33 of SEQ ID NO.1, CDR 2 of the heavy chain variable region is shown in positions 51-58 of SEQ ID NO.1, and CDR 3 of the heavy chain variable region is shown in positions 97-103 of SEQ ID NO.1. The amino acid sequences of FR (backbone region) 1 of the heavy chain variable region are shown in positions 1-25 of SEQ ID NO.1, the amino acid sequences of FR2 of the heavy chain variable region are shown in positions 34-50 of SEQ ID NO.1, the amino acid sequences of FR3 of the heavy chain variable region are shown in positions 59-96 of SEQ ID NO.1, and the amino acid sequences of FR4 of the heavy chain variable region are shown in positions 104-114 of SEQ ID NO.1.
[0119] The amino acid sequences of the light chain variable regions are shown in SEQ ID NO.2. The amino acid sequences of CDR1 in the light chain variable regions are shown at positions 27-32 of SEQ ID NO.2, the amino acid sequences of CDR2 in the light chain variable regions are shown at positions 50-52 of SEQ ID NO.2, and the amino acid sequences of CDR3 in the light chain variable regions are shown at positions 89-97 of SEQ ID NO.2. The amino acid sequences of FR (backbone region)1 in the light chain variable regions are shown at positions 1-26 of SEQ ID NO.2, the amino acid sequences of FR2 in the light chain variable regions are shown at positions 33-49 of SEQ ID NO.2, the amino acid sequences of FR3 in the light chain variable regions are shown at positions 53-88 of SEQ ID NO.2, and the amino acid sequences of FR4 in the light chain variable regions are shown at positions 98-107 of SEQ ID NO.2.
[0120] The positive hybridoma cell line 3B3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 6, 2023. The cell line is classified and named as "mouse anti-human phosphorylated synuclein antibody hybridoma cell line" with accession number CGMCC No. 45345. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0121] Example 2: Preparation of Monoclonal Antibodies
[0122] The mouse hybridoma cell line 3B3 preserved in Example 1 was used to prepare a monoclonal antibody against human α-synuclein phosphorylated at serine position 129. The specific operation steps are as follows:
[0123] 1. Cell resuscitation
[0124] Quickly remove the cryovials from the -80°C freezer or liquid nitrogen tank; immediately place them in a 42°C water bath to thaw the cryopreservation solution completely within 2 minutes. Add basal culture medium to the centrifuge tube, aspirate the cryopreservation solution into the centrifuge tube, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Add more basal culture medium, mix well, and centrifuge (1200 rpm) for 5 minutes, discarding the supernatant; finally, resuspend the cells in complete culture medium and transfer to a 25 cm³ centrifuge tube. 2 The culture flasks were placed in a CO2 incubator and cultured for one week for amplification.
[0125] 2. Preparation of ascites
[0126] The cells obtained from the cell resuscitation treatment in step 1 were resuspended and diluted, and counted using a hemocytometer. The cells were then centrifuged (1200 rpm) for 5 minutes, the supernatant was discarded, and the cells were resuspended in sterile 0.01M PBS buffer. The suspended cells were then intraperitoneally injected into mice (8-week-old male Balb / c mice, who had received an intraperitoneal injection of 0.5 ml of methylphenidate per mouse one week prior). Each mouse received approximately 5 × 10⁶ cells intraperitoneally. 7 Cells. Feed the mice for 8-12 days, carefully observing their condition. When the mouse abdomen resembles a frog's abdomen (indicating significant ascites), ascites and serum can be collected. After collecting the ascites, place it on ice, centrifuge promptly, collect the supernatant ascites antibodies, aliquot, and store at -80°C.
[0127] 3. Antibody purification
[0128] Mouse ascites antibodies were purified using the protein A affinity purification method, and this purification procedure was outsourced to Bio-Sensing Pharmaceutical Co., Ltd. The purified antibody was 3B3.
[0129] Example 3: Identification of 3B3 antibody subtypes
[0130] The 3B3 antibody purified in Example 2 was subjected to subtype identification.
[0131] 1. Experimental materials and equipment
[0132] Mouse monoclonal antibody subtype identification kit (Proteintech, catalog number: KMI-2), full-wavelength microplate reader, antibody to be identified—purified 3B3 antibody.
[0133] 2. Experimental Methods
[0134] 1) First, dilute the 20×PBST solution to 1×PBST solution, then use the 1×PBST solution to dilute the 100× goat anti-mouse IgM+IgG-HRP to 1× goat anti-mouse IgM+IgG-HRP; then use the 1×PBST solution to dilute the antibody to be tested to 0.5μg / mL.
[0135] 2) Equilibrate the kit at room temperature for 30 minutes, add 50 μL of purified 3B3 antibody to the sample wells of the kit, and then add 50 μL of 1× goat anti-mouse IgM+IgG-HRP to the sample wells. Mix gently on a mixer or by gently tapping the sides of the plate holder for 1 minute. Cover with the sealing film and incubate at room temperature for 1 hour.
[0136] 3) Discard the liquid in the wells, wash the plate three times with 1×PBST, pat dry on absorbent paper, and add 100 μL of the freshly prepared colorimetric solution (Note: the colorimetric solution formula is A:B = 1:100, i.e., mix 10 μL of A solution with 1 mL of B solution, and use immediately after mixing) to the wells. Incubate at room temperature in the dark for 10-20 min. Add 100 μL of stop solution to each well.
[0137] 4) Result Interpretation. The results are read using a microplate reader at OD450. The well with the highest OD value corresponds to the specific subtype.
[0138] 3. Experimental Results
[0139] The results of the OD value detection are shown in Table 1.
[0140] Table 1. Detection results of OD values for monoclonal antibody subtype identification.
[0141]
[0142] As shown in Table 1, the heavy chain type of the 3B3 antibody is IgG2b, and the light chain type is Kappa.
[0143] Example 4: Western blot analysis of the specificity of 3B3 antibody against phosphorylated full-length α-syn protein (P-α-syn) and wild-type full-length α-syn protein (W-α-syn).
[0144] 1. Experimental materials and equipment
[0145] 1) Antibody to be identified: 3B3 ascites antibody
[0146] 2) Protein samples: P-α-syn protein solution, W-α-syn protein solution
[0147] 3) Antibody: Goat anti-mouse IgG-HRP
[0148] 4) Other reagents and materials: 0.5M Tris-HCl (pH 6.8), 1.5M Tris-HCl (pH 8.8), 30% acrylamide, 10% SDS, 10% APS, TEMED, 4× sample buffer, protein marker, electrophoresis buffer, semi-dry electrophoresis buffer, TBST solution, skim milk powder, DDW, tap water, ECL colorimetric solution, developing and fixing solution, PVDF membrane, X-ray film, exposure film, etc.
[0149] 5) Instruments and equipment: pipettes, horizontal shakers, incubators, scissors, tweezers, rollers, vertical electrophoresis tubes, etc.
[0150] 2. Experimental Methods
[0151] 1) SDS-polyacrylamide gel electrophoresis
[0152] ①Preparation of separating gel: Prepare separating gel (10% separating gel). The composition of the separating gel is shown in Table 2. Slowly pour the separating gel between the glass plates, add a layer of distilled water 2-3 cm on top to isolate the air, and let it stand for more than 45 minutes until the separating gel solidifies.
[0153] Table 2 Composition of the separating gel
[0154] Concentration 10％ Resolution 18-75 kD 1.5 M Tris-HCl (pH 8.8) 3ml 30% acrylamide 4ml H2O 5ml 10% SDS 120 μl 10% APS 50 μl TEMED 10 μl
[0155] ② Preparation of the stacking gel: After the separating gel has fully polymerized (a clear interface exists between the gel surface and the aqueous phase above), absorb the supernatant with absorbent paper. Prepare the stacking gel according to the formula (see Table 3 for the composition of the stacking gel). Slowly pour the stacking gel between the glass plates, avoiding the formation of air bubbles. Then immediately and carefully insert the comb (be careful not to create air bubbles when inserting the comb). Let it stand for at least 30 minutes until the stacking gel has completely solidified. Remove the comb, and then remove the clamps.
[0156] Table 3 Composition of the stacking gel
[0157] 0.5 M Tris-HCl (pH 6.8) 1.25ml 30% acrylamide 0.75ml H2O 3.00ml 10% SDS 50 μl 10% APS 20 μl TEMED 10 μl
[0158] ③ Place the gel plate in the electrophoresis tank: Depending on the situation, inject electrophoresis buffer into the electrophoresis tank. Use a microsyringe to flush the sample wells with electrophoresis buffer to remove unpolymerized acrylamide.
[0159] ④ Sample preparation: Mix the sample to be separated with the sample preparation buffer (4×sample buffer) at a ratio of 3:1, boil for 3 minutes (place in the solution after the water has boiled), cool to room temperature, and centrifuge for 1 minute using a handheld centrifuge.
[0160] The P-α-syn protein samples and W-α-syn protein samples were diluted with 4× sample buffer and DDW to a final loading amount of 1 μg per lane.
[0161] ⑤ Sample loading: The sample protein loading and loading amount for each lane are shown in Table 4.
[0162] Table 4. Sample proteins and sample volumes for each lane.
[0163] Lane 1 2 3 Protein load Marker P-α-syn W-α-syn Load amount 4 μL 10 μL 10 μL
[0164] ⑥ Electrophoresis: Adjust the voltage to 50V and perform electrophoresis at a constant voltage. Once the protein enters the separating gel, the marker begins to separate. Increase the voltage to 150V until the bromophenol blue migrates to the vicinity of the bottom of the gel, about 0.5cm from the bottom. Then, turn off the power and stop electrophoresis.
[0165] ⑦ Remove the gel: Remove the glass plate from the electrophoresis apparatus, place it on a paper towel, and pry open the glass plate. Cut off a corner of the lower right part of the gel to mark its orientation, then place it in electrophoresis buffer and wash it on a shaker for 5 minutes.
[0166] 2) Semi-dry point transfer
[0167] ① Soak the prepared PVDF membrane in methanol for a few seconds until it becomes translucent, then remove it and place it in the electroporation buffer. Soak the prepared filter paper (6 sheets per gel) in the electroporation buffer. Use a roller to remove air bubbles.
[0168] ② Place the following items on the anode plate of the transfer tank in the following order: 3 sheets of filter paper soaked in buffer solution, PVDF membrane, gel, 3 sheets of filter paper soaked in buffer solution, then use a roller to remove air bubbles, and cover with the negative electrode cap.
[0169] ③ Connect the positive and negative terminals (red to red, black to black), turn on the power switch, adjust the current (100mA / PVDF membrane), and run the circuit at a constant current for 40 minutes. After the circuit is complete, remove the membrane.
[0170] 3) Immunoblotting
[0171] ① Sealing: Place the PVDF membrane in a freshly prepared 5% skim milk powder sealing solution and shake at room temperature for 1 hour.
[0172] ② Antibody binding: Dilute the 3B3 ascites antibody to 1 / 10000 with TBST solution containing 1% skim milk powder, place the blocked PVDF membrane in the antibody solution, and incubate overnight at 4°C.
[0173] ③ Wash the membrane with TBST solution on a horizontal shaker for 10 minutes each time, for a total of 3 times.
[0174] ④ Binding of secondary antibody: Dilute horseradish peroxidase-labeled secondary antibody at a ratio of 1:5000 with TBST solution containing 1% skim milk powder, place the membrane in the antibody solution, and shake slowly at room temperature for 1 hour.
[0175] ⑤ Wash away unbound secondary antibody: Wash 3 times with 100ml of rinsing buffer (TBST) and shake for 10 minutes each time.
[0176] 4) ECL color development (avoid light throughout the process)
[0177] ① Mix the two ECL chromogenic substrates, solution A and solution B, in equal volumes at a ratio of 1:1 (generally 0.5 ml / membrane for each).
[0178] ② After filtering off the excess liquid on the membrane, place the membrane on the exposure membrane, evenly cover the membrane surface with the mixture, and incubate for about 1 minute.
[0179] ③ Filter off any excess luminescent liquid from the membrane and place it in a darkroom. Cover the membrane with an X-ray film, close the darkroom, and expose for 0.5–10 minutes.
[0180] ④ Developing and fixing. After the X-ray film has been properly fixed, rinse it several times with tap water, air dry it, and store it.
[0181] 3. Experimental Results
[0182] Experimental results are as follows Figure 5 As shown. By Figure 5 It can be seen that the 3B3 antibody is positive for P-α-syn protein but negative for W-α-syn protein. P-syn protein tends to aggregate easily, especially into trimers. The 3B3 antibody has the ability to specifically recognize P-α-syn protein.
[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions (CDGs) of amino acid sequences SEQ ID NO. 1, positions 26-33, 51-58, and 97-103, respectively; and the light chain variable region comprises three CDGs of amino acid sequences SEQ ID NO. 2, positions 27-32, 50-52, and 89-97, respectively; wherein the antibody binds to human α-synuclein; and wherein the α-synuclein is serine phosphorylated α-synuclein at position 129.
2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.
2.
3. The antibody according to claim 2, characterized in that, Both the heavy chain variable region and the light chain variable region contain a framework region, which is derived from mice.
4. A biomaterial, wherein the biomaterial is any one of A1) to A5): A1) A nucleic acid molecule encoding any one of the antibodies described in claims 1 to 3; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1); A5) Cell lines containing the nucleic acid molecules described in A1).
5. The biomaterial according to claim 4, characterized in that, The nucleic acid molecule is a heavy chain variable region DNA molecule encoding SEQ ID NO.1 and / or a light chain variable region DNA molecule encoding SEQ ID NO.
2.
6. The use of the antibody of any one of claims 1 to 3 or the biomaterial of claim 4 in the preparation of a medicament for the prevention, relief and / or treatment of neurodegenerative disorders related to α-synuclein pathology; wherein the neurodegenerative disorder is Parkinson's disease, Lewy body dementia, Alzheimer's disease, multiple system atrophy, psychosis or Creutzfeldt-Jakob disease.
7. A medicament for the prevention, relief, and / or treatment of neurodegenerative disorders associated with α-synuclein pathology, characterized in that, The drug comprises any one of the antibodies of claims 1 to 3 or the biological material of claim 4.
8. The use of the antibody according to any one of claims 1 to 3 in the detection of α-synuclein, wherein the detection is a non-disease diagnostic test.
9. The use of the antibody according to any one of claims 1 to 3 in the preparation of a product for detecting α-synuclein.