Uch-l1 specific antibody and its application in auxiliary diagnosis kit of traumatic brain injury

Abstract

The application discloses a UCH-L1 specific antibody and application thereof in an auxiliary diagnosis kit for traumatic brain injury, and belongs to the technical field of immune analysis. The application provides an antibody targeting UCH-L1 and a chemiluminescence kit for detecting UCH-L1. The antibody provided by the application has high affinity, high sensitivity and a significantly shortened production cycle, and is more suitable for being applied to the field of in-vitro diagnosis reagents as a core raw material. The kit disclosed by the application can be used for the auxiliary diagnosis of diseases related to UCH-L1, such as traumatic brain injury.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology and relates to a UCH-L1 specific antibody and its application in a diagnostic kit for traumatic brain injury. Background Technology

[0002] Traumatic brain injury (TBI) is an acute neurological disorder caused by external mechanical force applied to the head. It can occur at any age and its severity ranges from mild concussion to severe diffuse axonal damage. Clinical manifestations include a complex set of symptoms, including immediate or delayed loss of consciousness, cognitive decline, sensorimotor abnormalities, and altered mood and behavior. According to the Glasgow Coma Scale (GCS), TBI is classified as mild (13-15 points), moderate (9-12 points), and severe (3-8 points). Mild TBI patients may experience transient altered consciousness, headache, and dizziness, most of which recover completely. Moderate TBI progresses to prolonged coma and definite neurological deficits. Severe TBI patients fall into a persistent coma with severe long-term cognitive and physical impairments, potentially leading to a vegetative state or death.

[0003] Studies have found that the main pathological mechanisms of total brain injury (TBI) include axonal breakage and microvascular rupture caused by primary mechanical injury, as well as secondary neuroinflammatory responses, oxidative stress, mitochondrial dysfunction, and apoptosis cascades. In this complex pathological process, ubiquitin-terminal hydrolase L1 (UCH-L1), a neuron-specific deubiquitinating enzyme, has become a key biomarker for assessing the severity of neuronal damage due to its release dynamics and functional status. UCH-L1 is highly enriched in neuronal cytoplasm. When TBI leads to neuronal structural damage and blood-brain barrier disruption, UCH-L1 is rapidly released into the extracellular space and enters the cerebrospinal fluid and blood circulation. Its release level is directly related to the degree of neuronal cell body and axonal damage. Studies have shown that within hours of TBI, serum and cerebrospinal fluid levels of UCH-L1 in patients significantly increase, and its peak concentration is highly positively correlated with the severity of injury, radiographic abnormalities (such as intracranial hemorrhage and cerebral edema), and poor functional prognosis (such as the Glasgow Outcome Scale). Of particular note is that the elevation window of UCH-L1 occurs earlier than traditional neuroimaging changes and can distinguish high-risk patients with intracranial lesions in mild TBI. Therefore, it has shown important value in acute injury assessment, prognosis prediction and treatment decision-making, and has been approved by the U.S. Food and Drug Administration as an auxiliary diagnostic biomarker for TBI. Summary of the Invention

[0004] This application provides a specific antibody against UCH-L1, specifically a UCH-L1 specific antibody, Anti-UCH-L1-rRmab-1. The monoclonal antibody prepared using single-B cell technology in this application has higher sensitivity and a significantly shorter production cycle. The kit prepared using Anti-UCH-L1-rRmab-1 has high sensitivity and a low detection limit, providing a tool for the auxiliary diagnosis of UCH-L1-related diseases. Furthermore, this application provides the use of this antibody or kit in the auxiliary diagnosis of traumatic brain injury.

[0005] On the one hand, this application provides an antibody or antigen-binding fragment thereof that specifically binds to UCH-L1.

[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 AA; 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.

[0008] 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-L3 comprising a light chain variable region CDR-L4 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.

[0009] 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 AA; 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 AA, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0010] 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.

[0011] On the one hand, this application provides a polynucleotide that encodes the aforementioned antibody or antigen-binding fragment.

[0012] On the one hand, this application provides a vector containing the polynucleotide of this application.

[0013] 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.

[0014] 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.

[0015] In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a 293 cell.

[0016] On the one hand, this application provides a kit for detecting UCH-L1.

[0017] 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 UCH-L1. 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).

[0018] 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.

[0019] 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 UCH-L1.

[0020] On the one hand, this application provides the application of the antibody or antigen-binding fragment as described above and the kit as described above in assisting traumatic brain injury.

[0021] 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 (2) recovering the antibody or antigen-binding fragment.

[0022] Beneficial effects: This application provides a new antibody targeting UCH-L1 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 shorter production cycle, making it more suitable as a core raw material for use in the field of in vitro diagnostic reagents. Attached Figure Description

[0023] Figure 1 Image showing antigen-specific single B cells sorted by flow cytometry.

[0024] Figure 2 This is a correlation curve between the detection results and clinical values ​​of the anti-UCH-L1 monoclonal antibody used in this application for chemiluminescence detection of a fixed-value gradient clinical sample. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Example 1: Preparation of monoclonal antibody against ubiquitin C-terminal hydrolase L1 (UCH-L1)

[0029] 1. Antigen preparation

[0030] In this embodiment, the full-length region of ubiquitin C-terminal hydrolase L1 (UCH-L1) was selected and prepared into a recombinant protein with a purity >80% by fermentation expression in prokaryotic Escherichia coli, which is used for the development of specific monoclonal antibodies.

[0031] Human UCH-L1, full-length protein sequence:

[0032] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSV LKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA (SEQ ID NO:8)

[0033] When designing recombinant clones, GST and 6×His tags were added to the C-terminus of UCH-L1, respectively. The GST tag was designed for animal immunization, and the 6×His tag was designed for animal serum titer detection and antibody screening.

[0034] 2. Preparation of animal immune libraries

[0035] Human UCH-L1-GST recombinant 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. Serum titers were measured after the third immunization.

[0036] A small blood sample was collected from the marginal ear vein and, after clotting, centrifuged at 8000 g to prepare serum. UCH-L1-His recombinant 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.

[0037] 3. Antigen-specific single B cell sorting

[0038] 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.

[0039] The original material was sorted, and UCH-L1-His was selected and coupled with FITC dyes.

[0040] The anti-rabbit IgG-Fc specific secondary antibody is a self-developed antibody conjugated with PE dye.

[0041] During cell labeling, DAPI dye is added to distinguish between dead and live cells.

[0042] B-cell sorting scheme: Dead / Live- / IgG+ / Antigen+.

[0043] 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 6Cells 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.

[0044] 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 1 The 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.

[0045] 4. Preparation of cDNA from rabbit single B cells

[0046] 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.

[0047] 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 minutes.

[0048] 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.

[0049] 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.

[0050] 5. Amplification of antibody-encoding genes using rabbit single-cell B-cell PCR technology

[0051] A single B-cell cDNA library can be used to retrieve genes encoding naturally paired antibody heavy and light chains.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Amplification of the antibody heavy chain coding region gene; forward primer sequence is:

[0057] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0058] Antibody heavy chain coding region gene amplification, reverse primer sequence is:

[0059] tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 10).

[0060] The forward primer sequence for amplifying the antibody light chain coding region gene is as follows:

[0061] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0062] Antibody light chain coding region gene amplification, reverse primer sequence is:

[0063] tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 11).

[0064] Extraction of antibody light and heavy chain encoding genes: Add to the PCR amplification system according to the 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.

[0065] 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.

[0066] 6. Construction and expression of antibody heavy and light chain recombinant expression plasmids

[0067] 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.

[0068] For efficient recombination of vectors and coding genes, choose the seamless cloning kit, and purchase the C115# kit from the Vazyme website.

[0069] 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.

[0070] 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.

[0071] The bacterial detection PCR of the recombinant plasmid uses the upstream primer sequence of caagctggctagcgtttaaactt (SEQ ID NO:12).

[0072] The downstream primer sequence for antibody heavy chain bacterial detection PCR is: ctcatttacccggagagcg (SEQ ID NO:13).

[0073] The downstream primer sequence for antibody light chain bacterial detection PCR is: acctcaacagtcacccctattg (SEQ ID NO:14).

[0074] Recombinant positive clones were sent for testing: Five PCR-positive clones from both the antibody heavy chain and light chain were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 7. Antigen specificity assessment of recombinant expression supernatant

[0079] Antigen-coated plate preparation: Screening for original human UCH-L1-His and UCH-L1-GST recombinant proteins.

[0080] Screening protocol for human UCH-L1 specific antibodies: If the supernatant of the antibody to be tested simultaneously binds to both UCH-L1-His and UCH-L1-GST proteins, it is preliminarily identified as a UCH-L1 specific monoclonal antibody.

[0081] 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.

[0082] 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).

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

[0084]

[0085] Table 1-2: Antigen-antibody affinity data for some antibodies (below)

[0086]

[0087] Indirect ELISA results: 1000 monoclonal antibodies were co-transfected with UCH-L1 specific antibodies for initial screening. Antibodies that reacted with UCH-L1-His and UCH-L1-GST were also used to preliminarily screen out 100 specific monoclonal antibodies.

[0088] 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.

[0089] 8. Recombinant antibody ELISA paired screening.

[0090] The specific recognition of ubiquitin C-terminal hydrolase L1 (UCH-L1) protein is achieved through pairing screening with UCH-L1-specific antibodies. Since the screening and identification of UCH-L1 protein-specific coated antibodies have already been completed, this embodiment selects the identified UCH-L1 coated antibodies and conducts sandwich ELISA experiments on 100 monoclonal antibodies initially screened by ELISA to identify specific labeled antibodies that can pair and bind to the full-length UCH-L1 protein.

[0091] The UCH-L1 protein is coated with antibody Rabbit-Anti-UCH-L1-mAb-A, and the heavy chain variable region sequence is as follows:

[0092] QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSNYGVSWVRQAPGKGLEWIGIIYATNNTYYANWAKGRFTISKTSTTVDLKITSPTTEDAATYFCAREGRQYYDMDLWGPGTLVTVSS (SEQ ID NO: 15);

[0093] (CDR-H1: GFSLSNYG (SEQ ID NO:17); CDR-H2: IYATNNT (SEQ ID NO:18); CDR-H3: AREGRQYYDMDL (SEQ ID NO:19))

[0094] The light chain variable region sequence is as follows:

[0095] AYDMTQTPASVSAAVGGTVTINCQASESIYSYLSWYQQKPGQPPKLLIYDASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQSYTGDEVDNIFGGGTEVVVK (SEQ ID NO: 16);

[0096] (CDR-L1: ESIYSY (SEQ ID NO:20); CDR-L2: DA; CDR-L3: QQSYTGDEVDNI (SEQ ID NO:21))

[0097] One hundred monoclonal antibodies were initially screened out. UCH-L1-His recombinant protein was detected by sandwich ELISA. Finally, the top 15 antibodies with the highest paired detection signal values ​​were selected and their detection in clinical samples was verified on a chemiluminescence platform (only the detection results of 20 pairs of paired antibodies are shown as an example).

[0098] Table 2: Detection data of paired antibodies against UCH-L1-His recombinant protein (partial)

[0099]

[0100] 9. Screening of recombinant antibodies using a chemiluminescence platform

[0101] 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.

[0102] Recombinant UCH-L3 and UCH-L1 proteins were expressed and biotinylated as selectogens. The sequence information of UCH-L1 is shown in the immunogen information, and the specific information of UCH-L3 is shown in Table 3.

[0103] Table 3: Original Information Table for Chemiluminescence Platform Screening of Recombinant Antibodies

[0104]

[0105] 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.

[0106] The coating antibody, labeled SA, and screener were prepared according to the above method. Each screener was then detected using a fully automated chemiluminescence analyzer according to the set program. The preferred monoclonal antibody that specifically recognizes UCH-L1 is the one that reacts only with the UCH-L1 protein and not with other reacting antibodies.

[0107] Monoclonal antibodies were screened using a sandwich ELISA assay, and two antibodies specifically recognizing UCH-L1 were selected using a chemiluminescence platform. Table 4 shows the detection results of the two pairs of paired antibodies as an example.

[0108] Table 4: Partial Detection Data of UCH-L1 Specific Antibody Chemiluminescence Platform

[0109]

[0110] The selected specific antibody, Anti-UCH-L1-rRmab-1, has the following heavy chain variable region sequence:

[0111] QCQSVEESGGRLVTPGTPLTLTCTVSGFSLNRYGVSWVRQAPGKGLEWIGAINSTGSTYYASWAKGRFTISKTSTTVTLKMTSPTTEDTANYFCGRGGPGYRQ-DIWGPGTLVTVSS (SEQ ID NO: 6);

[0112] The light chain variable region sequence is as follows:

[0113] AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLGWYQQKLGQPPKQLIYAASTLPSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYTGNMHTFGGGTEVVVK (SEQ ID NO: 7).

[0114] Table 5: Amino acid sequence of UCH-L1 antibody

[0115]

[0116] Example 2: Application of anti-ubiquitin C-terminal hydrolase L1 (UCH-L1) monoclonal antibody in chemiluminescence detection of clinical samples

[0117] The selected UCH-L1 specific antibody Anti-UCH-L1-rRmab-1 was applied to chemiluminescence detection experiments to detect clinical samples with a set value gradient, and the correlation between the detection results and the clinical set values ​​was compared.

[0118] The concentration of UCH-L1 in the graded clinical samples was determined using the Simoa single-molecule immunoassay. The specific concentrations are shown in Table 6.

[0119] Table 6: UCH-L1 Concentration in Clinical Samples

[0120]

[0121] Rabbit-Anti-UCH-L1-mAb-A was fixed as the coating antibody. The specific coating method was as follows: 2 mg of magnetic beads were washed twice with activation buffer, then a certain amount of EDC was added and vortexed to mix. 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 to mix for 2 h, then 100 μL of blocking buffer was added and the mixture was vortexed to block for 3 h. Finally, 1000 μL of TBST was added to wash the magnetic beads, followed by 1000 μL of preservation buffer.

[0122] The selected UCH-L1 specific antibody Anti-UCH-L1-rRmab-1 was used as the labeling antibody for acridine ester labeling, and 100-(100 / C) was used. 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.

[0123] The coated antibody and labeled antibody were prepared according to the above method. The concentration of UCH-L1 in samples P01-P14 and sample diluent was detected using a fully automated chemiluminescence analyzer. The results are shown in Table 7.

[0124] Table 7: Chemiluminescence Detection Results of Clinical Samples

[0125]

[0126] A scatter plot of UCH-L1 concentration for each sample was drawn, with the concentration detected by Simoa single-molecule immunoassay on the x-axis and the concentration detected by chemiluminescence on the y-axis. Figure 2 The correlation coefficient between the two detection methods for UCH-L1 concentrations was calculated. The correlation coefficient between the two detection methods was R = 0.9947. 2 =0.9894, meaning that when the preferred UCH-L1 specific antibody Anti-UCH-L1-rRmab-1 is used in combination with a matching antibody for chemiluminescence detection experiments, the detection results show a strong correlation with the simoa single-molecule immunoassay. This preferred antibody can be used for downstream kit preparation.

[0127] 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 UCH-L1, 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 AA; 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 UCH-L1, 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, the amino acid sequences of the heavy chain CDRH1-CDRH3 of the first antibody are as shown in SEQ ID NO: 17-SEQ ID NO: 19, 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.

4. The reagent kit according to claim 3, 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.

5. 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.

6. The use of the antibody or its antigen-binding fragment as described in claim 1 in the preparation of a kit for detecting UCH-L1.

7. 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 traumatic brain injury.

8. 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.

9. A kit for detecting UCH-L1, characterized in that, It comprises the antibody or its antigen-binding fragment as described in claim 1.

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