Alpha-synuclein oligomer-specific antibodies and their use in parkinson's disease auxiliary diagnostic kits

Abstract

The application discloses an alpha-syn oligomer specific antibody and application thereof in Parkinson auxiliary diagnosis reagent kits, belongs to the technical field of immunoassay, and provides an antibody targeting alpha-syn oligomer and a chemiluminescence reagent kit for detecting alpha-syn oligomer. The amino acid sequences of CDRH1-CDRH3 of the antibody heavy chain are sequentially shown in SEQ ID NO:1-SEQ ID NO:3, the amino acid sequence of CDRL1 of the light chain is shown in SEQ ID NO:4, the amino acid sequence of CDRL2 of the light chain is LA, and the amino acid sequence of CDRL3 of the light chain is shown in SEQ ID NO:5. The antibody has high affinity and high sensitivity, the production cycle is obviously shortened, and is more suitable for being applied to the field of in-vitro diagnosis reagents as a core raw material.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology and relates to an α-syn oligomer-specific antibody and its application in a Parkinson's disease auxiliary diagnostic kit. Background Technology

[0002] Parkinson's disease (PD) is an age-related, progressive, and irreversible neurodegenerative disease that commonly affects middle-aged and elderly individuals. Clinically, it manifests as core motor symptoms such as bradykinesia, resting tremor, rigidity, and postural instability without loss of consciousness, accompanied by various non-motor symptoms including decreased sense of smell, sleep disturbances, autonomic dysfunction, cognitive impairment, and depression and anxiety.

[0003] Studies have found that the main pathological features of Parkinson's disease (PD) include the progressive death of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, and the presence of Lewy bodies, primarily composed of abnormal aggregations of α-synuclein (α-syn), within the remaining neurons. It is generally believed that α-synuclein oligomers (α-syn oligomers), intermediate products of the conversion of α-synuclein from soluble monomers to insoluble amyloid filaments, are the core pathological form leading to neurotoxicity and synaptic dysfunction. These oligomers can ultimately cause dysfunction of the basal ganglia motor circuit through multiple mechanisms, including disrupting cell membrane integrity, inducing mitochondrial dysfunction, interfering with protein degradation systems, and activating neuroinflammation. Evidence suggests that the formation of α-syn oligomers leads to axonal and neuronal damage, ultimately resulting in progressive neuronal loss and Parkinson's disease-related symptoms. In patients with α-synucleinopathy, the level of α-syn oligomers is significantly elevated in brain tissue, cerebrospinal fluid, and peripheral blood, and is closely related to the degree of loss of dopaminergic neurons in the substantia nigra, the severity of motor symptoms, and the rate of disease progression. However, it is usually not elevated in patients with common Alzheimer's disease (AD). Therefore, α-syn oligomers are considered a specific biomarker to distinguish AD from α-synucleinopathy. Summary of the Invention

[0004] This application provides a specific antibody against α-syn oligomers, specifically an α-syn oligomer-specific antibody, Anti-o-α-syn-rRmab-1. The monoclonal antibody prepared using single-B cell technology in this application exhibits higher sensitivity and a significantly shorter production cycle. The kit prepared using Anti-o-α-syn-rRmab-1 has high sensitivity and a low detection limit, providing a tool for the auxiliary diagnosis of diseases related to α-syn oligomers. Furthermore, this application of the antibody or kit in the auxiliary diagnosis of Parkinson's disease is further provided.

[0005] On the one hand, this application provides an antibody or antigen-binding fragment thereof that specifically binds to α-syn oligomers.

[0006] In some embodiments, the antibody or antigen-binding fragment comprises at least one, two, three, four, five, or six CDRs selected from the following: (a) a heavy chain variable region CDR-H1 comprising a heavy chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR-H2 comprising a heavy chain variable region CDR-H2 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H4 comprising a heavy chain variable region CDR-H5 ... (e) a light chain variable region CDR-L1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of LA; and (f) a light chain variable region CDR-L3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0007] In some embodiments, the antibody or antigen-binding fragment comprises at least one, at least two, or all three of the VH CDR sequences selected from the following: (a) a heavy chain variable region CDR-H1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR-H2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 2; and (c) a heavy chain variable region CDR-H3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding fragment comprises at least one, at least two, or all three of the VL CDR sequences selected from the following: (a) a light chain variable region CDR-L1 comprising a light chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 4; (b) a light chain variable region CDR-L2 comprising a light chain variable region CDR-L3 comprising a light chain variable region CDR-L3 comprising a light chain variable region CDR-L3 comprising a light chain variable region CDR-L4 comprising a light chain variable region CDR-L5 comprising a light chain variable region CDR-L3 comprising a light chain variable region CDR-L5 comprising a light chain variable region CDR-L1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 5.In some embodiments, the antibody or antigen-binding fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the following: (i) a heavy chain variable region CDR-H1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR-H2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR-H3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 3; and (b) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the following: The VL domain of the CDR sequence includes: (i) a light chain variable region CDR-L1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 4; (ii) a light chain variable region CDR-L2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of LA; and (iii) a light chain variable region CDR-L3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment of this application comprises: CDR-H1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR-H2 with an amino acid sequence as shown in SEQ ID NO: 2, CDR-H3 with an amino acid sequence as shown in SEQ ID NO: 3, CDR-L1 with an amino acid sequence as shown in SEQ ID NO: 4, CDR-L2 with an amino acid sequence of LA, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises at least one or two heavy chain variable regions selected from: (a) a heavy chain variable region VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6; and (b) a light chain variable region VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO: 7.

[0009] On the one hand, this application provides a polynucleotide that encodes the aforementioned antibody or antigen-binding fragment.

[0010] On the one hand, this application provides a vector containing the polynucleotide of this application.

[0011] In some embodiments, the vector includes a viral vector, an expression vector, or a recombinant expression vector. In some embodiments, the expression vector can be any suitable recombinant expression vector selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, ZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, the expression vector is pcDNA3.1.

[0012] On the one hand, this application provides a host cell that contains the vector of this application or whose genome integrates the polynucleotides described in this application.

[0013] In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a 293 cell.

[0014] On the one hand, this application provides a kit for detecting α-syn oligomers.

[0015] In some embodiments, the kit comprises the antibody or antigen-binding fragment of this application. In some embodiments, the kit is used for non-diagnostic immunoassay of α-syn oligomers. In some embodiments, the kit is a chemiluminescence immunoassay, electrochemiluminescence immunoassay, or ELISA. In some embodiments, the kit is a chemiluminescence immunoassay kit based on a double-antibody sandwich principle, comprising: magnetic beads coated with a first antibody and a second antibody labeled with a chemiluminescent agent. In some embodiments, the chemiluminescent agent is selected from at least one of acridinium ester, alkaline phosphatase (ALP), and horseradish peroxidase (HRP).

[0016] In some embodiments, the second antibody is the antibody or antigen-binding fragment described in this application. In some embodiments, the amino acid sequences of the heavy chains CDRH1-CDRH3 of the first antibody are as shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively; the amino acid sequence of the light chain CDRL1 is as shown in SEQ ID NO: 20; the amino acid sequence of the light chain CDRL2 is DA; and the amino acid sequence of the light chain CDRL3 is as shown in SEQ ID NO: 21. In some embodiments, the first antibody comprises a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO: 16.

[0017] On the one hand, this application provides the use of the antibody or antigen-binding fragment as described above in the preparation of reagents or kits for detecting α-syn oligomers.

[0018] On the one hand, this application provides the use of the antibody or antigen-binding fragment as described above and the kit as described above in assisting the treatment of Parkinson's disease.

[0019] On the one hand, this application provides a method for preparing an antibody or antigen-binding fragment as described above, including culturing host cells as described in this application; and recovering the antibody or antigen-binding fragment.

[0020] Beneficial effects: This application provides a new antibody targeting α-syn oligomers and a kit containing the aforementioned antibody. The antibody produced by this application using single B cell technology has high affinity, high sensitivity, and a significantly shortened production cycle, making it more suitable as a core raw material for use in the field of in vitro diagnostic reagents. Attached Figure Description

[0021] Figure 1 Image showing antigen-specific single B cells sorted by flow cytometry.

[0022] Figure 2 This is a correlation curve between the detection results and clinical values ​​of the anti-α-syn oligomeric monoclonal antibody used in this application for chemiluminescence detection of a gradient of values ​​in clinical samples. Detailed Implementation

[0023] The present disclosure will be described in detail below with reference to the embodiments. However, the implementation of the present disclosure is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present disclosure. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present disclosure.

[0024] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. If multiple definitions exist for any term herein, those defined in this section shall prevail.

[0025] The technical solutions provided in this disclosure will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this disclosure.

[0026] Example 1: Preparation of a specific monoclonal antibody against human α-synuclein oligomers (α-syn oligomers)

[0027] 1. Antigen preparation

[0028] In this embodiment, α-syn recombinant protein monomers with a purity >80% were prepared by fermentation expression in prokaryotic Escherichia coli. Oligomeric proteins with a polymorphism of 10-100 were then prepared by shaking in an incubator for the development of specific monoclonal antibodies.

[0029] Full-length sequence of human α-syn protein:

[0030] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQID NO: 8)

[0031] When designing the cloning of recombinant protein monomers, a 6×His tag is added to the C-terminus of α-syn to facilitate protein purification.

[0032] 2. Preparation of animal immune libraries

[0033] Human α-syn oligomer protein was used to immunize five New Zealand rabbits. For the first round of immunization, 500 μg of immunogen was mixed with an equal amount of Freund's adjuvant to prepare an emulsion, which was then injected subcutaneously at multiple sites. Two weeks later, a second immunization was performed, with 250 μg of immunogen mixed with an equal amount of Freund's adjuvant to prepare an emulsion, which was then injected subcutaneously at multiple sites. The serum titer of the animals was measured after the third immunization.

[0034] A small blood sample was collected from the marginal ear vein and, after clotting, centrifuged at 8000 g to prepare serum. α-syn oligomer protein was plated, and serum titer was determined by ELISA. Rabbits with high serum titers were given a booster immunization via subcutaneous injection of 250 μg immunogen at multiple sites, followed by spleen collection. The rabbit spleen was physically ground and filtered through a porous mesh to prepare a single-cell suspension.

[0035] 3. Antigen-specific single B cell sorting

[0036] This embodiment is based on the specific recognition of surface markers of lymphocyte B cells by flow cytometry antibodies, and uses flow cytometry cell sorting to obtain specific single B cells from single-cell suspensions.

[0037] The original protein was sorted, and α-syn oligomer protein was selected and coupled with FITC dye.

[0038] The anti-rabbit IgG-Fc specific secondary antibody is a self-developed antibody conjugated with PE dye.

[0039] During cell labeling, DAPI dye is added to distinguish between dead and live cells.

[0040] B-cell sorting scheme: Dead / Live- / IgG+ / Antigen+.

[0041] Cell labeling procedure: Rabbit lymphocyte suspension, centrifuged at 300 g for 5 min, added 5 mL of buffer, mixed by inverting, centrifuged at 300 g for 5 min. Discard the supernatant, repeat once, take 30 μL of cell suspension for cell counting, and take 40 μL of cell suspension for blank control tubes and single-stain tubes to be labeled. The remaining cell suspension is used as sample tubes, centrifuged at 300 g for 5 min, and resuspended with a small amount of PBS. Blank tubes are left untreated. For single-stain tubes, add PBS to 100 μL, and add 2 μL of PE, 2 μL of FITC, and 2 μL of DAPI dye respectively. Sample tubes are labeled with PE dye at a ratio of 1.5 μL / 10n. 6 Cells, FITC dye 2 μg / 10 6 Cells were added in the calculated amount, and the corresponding antibody was added in the dark. The cells were incubated at 4°C for 30 min. After antibody incubation, 2 mL of buffer was added, gently mixed, and centrifuged at 300 g for 5 min. The cells were washed three times. The cells were resuspended in 1 mL of buffer, filtered, and ready for sorting.

[0042] After completing fluorescence compensation adjustment, the viable cell population, PE and FITC double-positive signal cell populations were sequentially delineated (sorting diagram as shown in the figure). Figure 1The flow cytometer was programmed to sort antigen-specific B cells into 96-well plates, with only one cell per well. After sorting, the plates needed to be immediately stored at low temperature; dry ice boxes were provided in this example for short-term storage. The wells contained cell lysis buffer, and the sorted 96-well PCR plates were directly used for single-B-cell PCR experiments.

[0043] 4. Preparation of cDNA from rabbit single B cells

[0044] The preparation of single-cell cDNA libraries was based on SMART 5'RACE technology, and all reagents used were the Vazyme N711 kit from Nanjing Novizan Biotechnology Co., Ltd., which is commercially available. The amplification systems involved in the experiments described in the examples can be found in the N711 kit instructions.

[0045] Single B cell RNA reverse transcription: After sorting, thaw the 96-well plates and place them in a PCR instrument to run the program. After the program is completed, let them stand on ice for 2 min.

[0046] Single-stranded cDNA synthesis in B cells: After the reverse transcription reaction is complete, the single-stranded synthesis system can be added. After adding the system, gently mix the wells and place them in a PCR instrument to run the program. After the program is completed, incubate the samples on ice for 2 minutes.

[0047] Single-cell B-cell DNA double-strand synthesis: After the synthesis reaction of the cDNA single-strand product is completed, the double-strand synthesis system can be added. After adding the system, gently mix the well plate, centrifuge, and then place it in a PCR instrument to run the program. After the program is completed, incubate the well plate samples on ice.

[0048] 5. Amplification of antibody-encoding genes using rabbit single-cell B-cell PCR technology

[0049] A single B-cell cDNA library can be used to retrieve genes encoding naturally paired antibody heavy and light chains.

[0050] All reagents used for gene amplification were the Vazyme P515 kit from Nanjing Novizan Biotechnology Co., Ltd., which are commercially available. The amplification systems used in the experiments described in this example can be found in the P515 kit instructions.

[0051] The upstream primer contains a homologous arm that interlocks with the 3' end of the promoter CMV gene sequence, so the antibody-encoding gene can be directly used to construct the recombinant expression cassette after retrieval.

[0052] The downstream primer for the antibody heavy chain encoding gene is located in a constant region and contains a homologous arm that interacts with the BGH-polyA gene sequence.

[0053] The downstream primer for the light chain encoding gene is located in a constant region and contains a homologous arm complementary to the BGH-polyA gene sequence. Therefore, after the antibody encoding gene is retrieved, it can be directly used to construct a recombinant expression cassette.

[0054] The forward primer sequence for amplifying the antibody heavy chain coding region gene is as follows:

[0055] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0056] Antibody heavy chain coding region gene amplification, reverse primer sequence is:

[0057] tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 10).

[0058] The forward primer sequence for amplifying the antibody light chain coding region gene is as follows:

[0059] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0060] Antibody light chain coding region gene amplification, reverse primer sequence is:

[0061] tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 11).

[0062] Extraction of antibody light and heavy chain encoding genes: Add to the PCR amplification system according to the kit instructions, mix gently in the well plate, place in the PCR instrument and run the program. After the program is completed, place the well plate samples on ice.

[0063] In this embodiment, the pairing positivity rate of the amplification products encoding the antibody light and heavy chains in the same 96-well plate was over 80%, and the bands were clear as detected by agarose gel electrophoresis, indicating that both the single-cell flow cytometry sorting and the encoding gene amplification experiment were effective. The amplification products were used for the construction of recombinant expression plasmids.

[0064] 6. Construction and expression of antibody heavy and light chain recombinant expression plasmids

[0065] The recombinant expression vector, pcDNA3.1 (Invitrogen), was purchased from the ThermoFisher SCIENTIFIC website. Before recombinant construction, the expression vector was linearized by single digestion with HindIII restriction enzyme, which was purchased from the New England Biolabs website.

[0066] For efficient recombination of vectors and coding genes, choose the seamless cloning kit, and purchase the C115# kit from the Vazyme website.

[0067] Construction of recombinant expression plasmid: The amplified products encoding the antibody heavy and light chains were circularized with the pcDNA3.1 linearized vector using seamless cloning technology, and then transformed into E. coli DH5α competent cells. The plasmids were then plated on LB fixation medium plates and incubated overnight at 37°C with the plates inverted.

[0068] Selection of recombinant positive clones: For the initial screening of heavy and light chains of antibodies, 8 single colonies are picked from each chain, and the colony positivity rate is determined by PCR testing. If the positivity rate is low, single colonies can be picked for further testing.

[0069] The bacterial detection PCR of the recombinant plasmid uses the upstream primer sequence of caagctggctagcgtttaaactt (SEQ ID NO:12).

[0070] The downstream primer sequence for antibody heavy chain bacterial detection PCR is: ctcatttacccggagagcg (SEQ ID NO:13).

[0071] The downstream primer sequence for antibody light chain bacterial detection PCR is: acctcaacagtcacccctattg (SEQ ID NO:14).

[0072] Recombinant positive clones were sent for testing: five clones of the heavy chain and five clones of the antibody light chain were selected from the PCR positive clones and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0073] Rabbit antibody gene sequence analysis: The V region of the antibody sequence was determined using the IMGT database, and the antibody sequence was analyzed for the CDR1 / CDR2 / CDR3 regions of the heavy chain and light chain. The correct sequence number of the PCR-positive clones was then derived and determined.

[0074] Small-scale expression of recombinant expression plasmids: Cloning of the correct sequence and small-scale extraction of antibody light and heavy chain plasmids from bacterial culture. The plasmids were then co-transfected into HEK293 mammalian cells. Ten days after transfection, the cell supernatant was collected by centrifugation. The supernatant was used for antigen specificity assessment, and cell supernatant purification was performed after the initial ELISA screening results were available.

[0075] In this embodiment, 100 plasmids were transfected in each round, meaning that 100 monoclonal antibodies could be obtained in each round of transfection. A total of 3 rounds of transfection experiments were performed.

[0076] 7. Evaluation of antigen specificity in recombinant expression supernatant

[0077] Antigen-coated plate preparation: The original selection was made from the oligomeric and high-purity monomeric forms of α-syn recombinant protein. The high-purity monomeric form of α-syn was prepared by ultrafiltration after purification of the fermentation supernatant of prokaryotic Escherichia coli using a nickel column.

[0078] Preliminary antibody screening protocol for the oligomeric form of α-syn (o-α-syn): Indirect ELISA is used to detect the supernatant of the antibody to be tested. If the OD value of the binding reaction with o-α-syn is >3, and the OD value of the binding reaction with high-purity α-syn monomer is <0.3, it is preliminarily identified as an o-α-syn specific antibody.

[0079] Indirect ELISA was used to detect cell supernatant. The cells were coated with self-produced rabbit secondary antibody Anti-Rabbit IgG mAb and HRP-labeled with goat anti-rabbit polyclonal antibody. The reactivity OD>1 indicated that the recombinant plasmid was normally expressed on 293 cells.

[0080] Indirect ELISA was used to detect cell supernatants, and the reactivity of the two antigen-coated plates was evaluated to obtain the initial screening results of the supernatants of the well plates (Tables 1-1 and 1-2 only show the detection data of supernatants of 50 cell lines).

[0081] Table 1-1: Antigen-antibody affinity data for some antibodies (top)

[0082]

[0083] Table 1-2: Antigen-antibody affinity data for some antibodies (below)

[0084]

[0085] Indirect ELISA results: 1000 monoclonal antibodies were initially screened. The OD value of the binding reaction with o-α-syn protein was >3, while the OD value of the binding reaction with high-purity α-syn monomer protein was <0.2. 100 monoclonal antibodies were selected.

[0086] The cell supernatant identified in the initial screening was purified by protein A to obtain a small amount of monoclonal antibody, with an average of 1-3 mg per strain.

[0087] 8. Recombinant antibody ELISA paired screening.

[0088] For the pairing diagnostic reagents for the oligomeric form of α-synuclein (o-α-syn), the screening and identification of α-syn protein-coated antibodies have been completed in the early stages. Therefore, in this embodiment, the identified α-syn-coated antibodies are selected, and sandwich ELISA experiments are carried out on 100 oligomeric-specific antibodies in the preliminary screening to screen out specific labeling antibodies that can be paired to detect o-α-syn.

[0089] The α-syn protein is coated with the antibody Rabbit-Anti-α-syn-mAb-A, and the heavy chain variable region sequence is as follows:

[0090] QCQSMEESGGRLVTPGTPLTLTCTVSGFSLRNYGVNWVRQAAGKGLEWIGIIGSGGSTYYANWAKGRFTISKTSTTVDLIMTSLTTEDTATYFCAGLGAFNLWGQGTLVTVSS (SEQ ID NO: 15);

[0091] (CDR-H1: GFSLRNYG (SEQ ID NO:17); CDR-H2: IGSGGST (SEQ ID NO:18); CDR-H3: AGLGAFNL (SEQ ID NO:19))

[0092] The light chain variable region sequence is as follows:

[0093] AQVLTQTASSVSAAVGGTVTISCQSSQSVYNKNWLAWFQQKPGQPPKRLIYDASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAASYYCLGGYKGTVYVFGGGTEVVVK (SEQ ID NO: 16);

[0094] (CDR-L1: QSVYNKNW (SEQ ID NO:20); CDR-L2: DA; CDR-L3: LGGYKGTVYV (SEQ ID NO:21))

[0095] One hundred monoclonal antibodies were initially screened out. The o-α-syn protein was detected by sandwich ELISA. Finally, the top 20 antibodies with the highest paired detection signal values ​​were selected and verified on a chemiluminescence platform to detect the antibodies in clinical samples (the detection results of 20 pairs of paired antibodies are shown as an example only).

[0096] Table 2: Detection data of o-α-syn recombinant protein paired antibodies (partial)

[0097]

[0098] 9. Screening of recombinant antibodies using a chemiluminescence platform

[0099] The antibodies selected from the sandwich ELISA assay were coated onto magnetic beads. The specific procedure was as follows: 2 mg of magnetic beads were washed twice with activation buffer, then a certain amount of EDC was added and the mixture was vortexed and the supernatant was discarded by magnetic aspiration. The precipitate was added to 1000 μL of coupling buffer, followed by 40 μg of antibody, and vortexed for 2 h. Then, 100 μL of blocking buffer was added, and the mixture was vortexed for 3 h. Finally, 1000 μL of TTBST was added to wash the magnetic beads, followed by 1000 μL of preservation buffer.

[0100] Recombinant α-synuclein monomers were expressed and prepared into oligomers. The oligomers were then ultrafiltered to obtain oligomers with a purity >95%. α-Syn protein, β-Syn protein, γ-Syn protein, and α-syn oligomers were used as screening agents, and specific information on the screening agents is shown in Table 3.

[0101] Table 3: Original Information Table for Chemiluminescence Platform Screening of Recombinant Antibodies

[0102]

[0103] The streptavidin (SA) was labeled with acridinium ester, and the specific procedure was as follows: 100-(100 / C) SA Add 100 μL of coupling buffer to a 0.5 mL brown EP tube, and add (100 / C) SA Add 1 μL of SA to a 0.5 mL brown EP tube to achieve a final SA labeling concentration of 1 mg / mL. Add 5 mM acridine ester to the 0.5 mL brown EP tube, mix thoroughly using a vortex mixer, and then react vertically for 2 h at room temperature (20-25℃) to purify and remove free acridine ester.

[0104] The coated antibody, labeled SA, and screening antigen were prepared according to the above method. Each screening antigen was then detected using a fully automated chemiluminescence analyzer according to the set program. Antibodies that preferentially recognize α-syn oligomer proteins, do not recognize α-Syn monomeric proteins, and show no cross-reactivity with β-Syn and γ-Syn proteins are preferred monoclonal antibodies that specifically recognize α-syn oligomers.

[0105] Of the 20 monoclonal antibodies screened by sandwich ELISA, 5 were selected specifically for recognizing o-α-syn by chemiluminescence platform detection. Table 4 shows the detection results of two pairs of paired antibodies as an example.

[0106] Table 4: Partial Detection Data of o-α-syn Specific Antibody Chemiluminescence Platform

[0107]

[0108] The selected specific antibody, Anti-o-α-syn-rRmab-1, has the following heavy chain variable region sequence:

[0109] QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTNGVLWVRQAPGKGLEWIGIINSSGSTYYASWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARHAGTSNFNIWGPGTLVTVSS (SEQ ID NO: 6);

[0110] The light chain variable region sequence is as follows:

[0111] AIVMTQTPSSKSVPVGDTVTINCQASESVYSNNRLAWFQQKPGQPPKLLIYLASTLDSGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSKIDDIAFGGGTEVVVK (SEQ ID NO: 7).

[0112] Table 5: Amino acid sequence of o-α-syn antibody

[0113]

[0114] Example 2: Application of anti-α-synuclein oligomer (i.e., o-α-syn) monoclonal antibody in chemiluminescence detection of clinical samples

[0115] The selected o-α-syn specific antibody Anti-o-α-syn-rRmab-1 was applied to a chemiluminescence detection experiment to detect gradient samples prepared by polymer protein amplified using α-syn monomers via SAA seed, and the correlation between the detection results and the target value was compared.

[0116] High-value samples were prepared by adding α-syn monomers to plasma samples amplified using SAA seeding to form polymeric proteins. These high-value samples were then mixed with low-value plasma samples at different ratios to prepare clinical samples with different concentration gradients. The sample preparation process and dilution ratios are shown in Table 6.

[0117] Table 6: Preparation process and concentration of o-α-syn in clinical samples

[0118]

[0119] The selected α-syn oligomer-specific antibody, Anti-o-α-syn-rRmab-1, was used as the coating antibody. The specific coating method is as follows: 2 mg of magnetic beads were washed twice with activation buffer, then a certain amount of EDC was added and the mixture was vortexed and mixed. The supernatant was discarded by magnetic aspiration. 1000 μL of coupling buffer was added to the precipitate, followed by 40 μg of antibody. The mixture was vortexed and mixed for 2 h, then 100 μL of blocking buffer was added, and the mixture was vortexed and mixed for 3 h. Finally, 1000 μL of TBST was added to wash the magnetic beads, followed by 1000 μL of preservation buffer.

[0120] The selected α-syn oligomer-specific antibody Anti-o-α-syn-rRmab-1 was used as the labeling antibody for acridine ester labeling, and 100-(100 / C) was used as the labeling antibody. Ab Add 100 μL of coupling buffer to a 0.5 mL brown EP tube, and add (100 / C) Ab Add 1 μL of antibody solution to a 0.5 mL brown EP tube to achieve a final antibody labeling concentration of 1 mg / mL. Add 5 mM acridine ester to the 0.5 mL brown EP tube, mix thoroughly using a vortex mixer, and then react vertically for 2 h at room temperature (20-25℃) to purify and remove free acridine ester.

[0121] The coated antibody and labeled antibody were prepared according to the above method. The concentration of α-syn oligomers in samples P01-P08 and sample diluents was detected using a fully automated chemiluminescence analyzer. The results are shown in Table 7.

[0122] Table 7: Chemiluminescence Detection Results of Clinical Samples

[0123]

[0124] A scatter plot of the α-syn oligomer concentration of each sample was drawn, with the theoretical target value of α-syn oligomer concentration from sample preparation as the horizontal axis and the α-syn oligomer signal value detected by chemiluminescence as the vertical axis. Figure 2 The correlation coefficient between the α-syn oligomer concentration values ​​of the two detection methods was calculated. The correlation coefficient between the two detection methods was R = 0.9999. 2 =0.9999, meaning that when the preferred α-syn oligomer-specific antibody Anti-α-syn oligomer-rRmab-1 is used in chemiluminescence detection experiments, the detection results show a strong correlation with the theoretical protein concentration in the concentration range of 0-100 ng / mL. This preferred antibody can be used for downstream kit preparation.

[0125] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to α-syn oligomers, characterized in that, The amino acid sequences of the heavy chains CDRH1-CDRH3 are shown in SEQ ID NO:1-SEQ ID NO:3, respectively; the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:4; the amino acid sequence of the light chain CDRL2 is LA; and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO:

5.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It includes: the heavy chain variable region VH as shown in SEQ ID NO: 6 and the light chain variable region VL as shown in SEQ ID NO:

7.

3. A kit for detecting α-syn oligomers, characterized in that, The kit includes magnetic beads coated with a first antibody and a second antibody labeled with a chemiluminescent agent; Wherein, the second antibody in the antibody pair consisting of the first antibody and the second antibody is the antibody or its antigen-binding fragment as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The amino acid sequences of the heavy chains CDRH1-CDRH3 of the first antibody are shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively; the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO: 20; the amino acid sequence of the light chain CDRL2 is DA; and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO:

21.

5. The reagent kit according to claim 4, characterized in that, The first antibody comprises: a heavy chain variable region VH as shown in SEQ ID NO: 15 and a light chain variable region VL as shown in SEQ ID NO:

16.

6. The reagent kit according to claim 3, characterized in that, The chemiluminescent agent is selected from at least one of acridine ester, alkaline phosphatase, and horseradish peroxidase.

7. The use of the antibody or its antigen-binding fragment as described in claim 1 in the preparation of a kit for detecting α-syn oligomers.

8. The use of the antibody or its antigen-binding fragment as described in claim 1 in the preparation of a kit for the auxiliary diagnosis of Parkinson's disease.

9. A biomaterial, characterized in that, Choose from any of the following: a. A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in claim 1; b. A carrier containing the polynucleotide described in a; c. A host cell containing the vector described in b or whose genome integrates the polynucleotide described in a.

10. A kit for detecting α-syn oligomers, characterized in that, It comprises the antibody or its antigen-binding fragment as described in claim 1.

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