A rabbit-derived monoclonal antibody against human phosphorylated tau217 and a preparation method and application thereof

Abstract

The present disclosure provides a rabbit-derived monoclonal antibody against human phosphorylated tau217 and a preparation method and application thereof, and belongs to the field of biological detection and bioengineering technology. The rabbit-derived monoclonal antibody against human phosphorylated tau217 comprises a light chain variable region and a heavy chain variable region, and the monoclonal antibody comprises HZK33-B0010, HZK33-B0011 and HZK33-B0029. The present disclosure develops three rabbit monoclonal antibodies that recognize p-tau217, which do not cross-react with non-phosphorylated tau protein or phosphorylated tau protein at other sites, can highly specifically bind to p-tau217, and thus develops a more specific related humoral marker detection kit.

Description

Technical Field

[0001] This disclosure pertains to the fields of biological detection and bioengineering, and particularly relates to a rabbit-derived monoclonal antibody against human phosphorylated tau217, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a prevalent degenerative disease of the central nervous system. Its important pathological features include β-amyloid (Aβ) plaque deposition, neurofibrillary tangles (NFTs) formed by phosphorylated tau protein, and neuronal damage and loss. AD patients typically experience memory loss and cognitive decline for 10 to 15 years.

[0003] Tau protein is a microtubule-associated protein encoded by the microtubule-associated protein tau (MAPT) gene, which contains transcripts with three or four microtubule-binding domains (3R and 4R). Under physiological conditions, tau protein plays a role in stabilizing microtubule structure and regulating intracellular transport. However, in the development of neurodegenerative diseases such as Alzheimer's disease (AD), tau protein aggregates to form non-fibroid free radicals (NFTs).

[0004] Related studies have shown that decades before the onset of Alzheimer's disease (AD), tau proteins in the brain undergo post-translational modifications such as hyperphosphorylation, acetylation, glycosylation, and truncation. In the cerebrospinal fluid of AD patients, various tau protein alterations can be detected, including phosphorylated tau protein at positions 181 (p-tau181), 205 (p-tau205), 217 (p-tau217), and 231 (p-tau231). Therefore, detecting pathological changes in tau proteins is considered an important means of early diagnosis of AD. Summary of the Invention

[0005] In view of the above, in order to at least partially solve at least one of the aforementioned technical problems, this disclosure provides a rabbit-derived monoclonal antibody against human phosphorylated tau217, its preparation method, and its application.

[0006] According to one embodiment of this disclosure, a rabbit-derived monoclonal antibody against human phosphorylated tau217 is provided. The rabbit-derived monoclonal antibody includes a light chain variable region and a heavy chain variable region. The rabbit-derived monoclonal antibody includes HZK33-B0010, HZK33-B0011, and HZK33-B0029. Specifically, the heavy chain variable region of HZK33-B0010 includes the CDR sequences shown in SEQ ID No. 13-SEQ ID No. 15, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 16-SEQ ID No. 18; the heavy chain variable region of HZK33-B0011 includes the CDR sequences shown in SEQ ID No. 19-SEQ ID No. 21, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 22-SEQ ID No. 24; the heavy chain variable region of HZK33-B0029 includes the CDR sequences shown in SEQ ID No. 25-SEQ ID No. 27, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 16-SEQ ID No. 18. The CDR sequences shown are No. 28 to SEQ ID No. 30.

[0007] According to another embodiment of this disclosure, a nucleic acid molecule is provided that encodes the aforementioned rabbit monoclonal antibody.

[0008] According to another aspect of the present disclosure, a recombinant expression vector is provided, comprising the aforementioned nucleic acid molecules.

[0009] According to another embodiment of this disclosure, a host cell is provided, comprising the above-described recombinant expression vector.

[0010] According to another aspect of this disclosure, a composition is provided comprising at least one of the above-described rabbit monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, and the above-described host cell.

[0011] According to another aspect of this disclosure, a composition is provided comprising the above-described rabbit monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, the above-described host cells, or the above-described composition, for use in preparing a kit for detecting human phosphorylated tau217 protein.

[0012] According to another embodiment of this disclosure, a method for preparing a rabbit-derived monoclonal antibody is provided, comprising the step of culturing the aforementioned host cells.

[0013] According to the embodiments of this disclosure, three rabbit-derived monoclonal antibodies that recognize p-tau217 have been developed. These antibodies do not cross-react with non-phosphorylated tau proteins or phosphorylated tau proteins at other sites (such as p-tau181, p-tau205, and p-tau231). Specifically, the three antibodies include HZK33-B0010, HZK33-B0011, and HZK33-B0029. Compared to mouse or rat antibodies, rabbit-derived monoclonal antibodies, due to their greater genetic diversity and simpler immunoglobulin structure, are easier to humanize for subsequent clinical applications to reduce potential immunogenicity. The p-tau217 monoclonal antibody prepared in this disclosure can bind to p-tau217 with high specificity, and can be used to develop more specific detection kits for related humoral markers. Attached Figure Description

[0014] Figure 1 The following is a flow cytometry result of the preparation of monoclonal antibodies for B cell sorting in this embodiment of the present disclosure, wherein A is a forward scattering light-area map, B is a 7-aminoactinomycin D screening map, C is an immunoglobulin G signal map, and D is a phycocyanin-area map;

[0015] Figure 2 This is a schematic diagram illustrating the principle of obtaining variable region fragments of antibody heavy and light chains by nested PCR amplification as disclosed in this invention.

[0016] Figure 3A This is the binding curve of HZK33-B0010 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention; Figure 3B The binding curve of HZK33-B0010 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention is shown. Figure 3C The binding curve of HZK33-B0011 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention is shown. Figure 3D The binding curve of HZK33-B0011 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention is shown. Figure 3E This is the binding curve of HZK33-B0029 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention; Figure 3F This is the binding curve of HZK33-B0029 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention;

[0017] Figure 4A This is an image showing the immunoblotting results of the HZK33-B0010 antibody binding to a human brain protein sample in an embodiment of the present invention. Figure 4B This is an image showing the immunoblotting results of the HZK33-B0011 antibody binding to a human brain protein sample in an embodiment of the present invention. Figure 4CThis is an image showing the immunoblotting results of the HZK33-B0029 antibody binding to a human brain protein sample in an embodiment of the present invention.

[0018] Figure 5A This is a comparison of the immunoblotting results of HZK33-B0010 antibody binding to human brain protein samples in an embodiment of the present invention; Figure 5B This is a comparison of the immunoblotting results of HZK33-B0011 antibody binding to human brain protein samples in an embodiment of the present invention; Figure 5C This is a comparison of the immunoblotting results of the HZK33-B0029 antibody binding to human brain protein samples in an embodiment of the present invention. Detailed Implementation

[0019] The embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the disclosure. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present disclosure for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0021] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0022] The term "homology" refers to the level of similarity or percentage identity between polynucleotide sequences in terms of percentage nucleotide positional similarity (i.e., sequence similarity or identity). As used here, homology also refers to the concept of similar functional properties between different polynucleotide molecules; for example, promoters with similar functions may have homologous cis elements. Polynucleotide molecules are homologous when they specifically hybridize under certain conditions to form a double-stranded molecule. Under these conditions (called stringent hybridization conditions), one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule sharing homology.

[0023] The term "promoter" refers to a polynucleotide molecule that, in its native state, is located upstream of or 5' of the translation start codon in the reading frame (or protein-coding region) and participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0024] The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribed polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are portions of a single, consecutive polynucleotide molecule, and more preferably, they are adjacent. For example, if a promoter regulates or mediates the transcription of a target gene within the cell, then the promoter is operably linked to the target gene.

[0025] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0026] The term "expression" refers to the transcription and / or translation of endogenous or exogenous genes in host cells.

[0027] The term "host cell" refers to a cell containing the polynucleotides of this disclosure, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome.

[0028] The term "monoclonal antibody," unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites.

[0029] The aforementioned CDR is a sequence defined according to the Kabat numbering system. Within the scope of this disclosure, the antibody may be in various forms, such as a full-length antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

[0030] In realizing the present invention, it was discovered that during the development of neurodegenerative diseases such as Alzheimer's disease (AD), tau protein aggregates to form neurofibrillary free radicals (NFTs), which leads to the transfer of soluble tau protein distributed along neuronal axons to the cell body, causing tau protein aggregation, synaptic dysfunction, and neuronal loss (tauopathy). Pathological tau protein exists in different forms, including monomers, oligomers, paired helical filaments (PFHs), and straight filaments, and their composition ratio is closely related to the type of neurodegenerative disease.

[0031] Various tau protein changes can be detected in the cerebrospinal fluid of AD patients. However, due to the invasive lumbar puncture procedure required for cerebrospinal fluid extraction, it is difficult to promote its large-scale application in the population. Related studies have shown that p-tau217 in plasma can also predict AD. Therefore, the development of anti-p-tau217 antibodies has great application potential.

[0032] Specifically, according to one aspect of this disclosure, a rabbit-derived monoclonal antibody against human phosphorylated tau217 is provided. The monoclonal antibody includes a light chain variable region and a heavy chain variable region. The monoclonal antibody includes HZK33-B0010, HZK33-B0011, and HZK33-B0029. Specifically, the heavy chain variable region of HZK33-B0010 includes the CDR sequences shown in SEQ ID No. 13-SEQ ID No. 15, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 16-SEQ ID No. 18. The heavy chain variable region of HZK33-B0011 includes the CDR sequences shown in SEQ ID No. 19-SEQ ID No. 21, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 22-SEQ ID No. 24. The heavy chain variable region of HZK33-B0029 includes the CDR sequences shown in SEQ ID No. 25-SEQ ID No. 27, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 19-SEQ ID No. 21. The CDR sequences shown are No. 28 to SEQ ID No. 30.

[0033] The heavy chain variable region of the HZK33-B0010 monoclonal antibody contains the following CDR sequence:

[0034] (i) GFSLSTYT (SEQ ID No. 13);

[0035] (ii) IKSGGST (SEQ ID No. 14);

[0036] (iii) GRGGFRTAP (SEQ ID No. 15).

[0037] The light chain variable region of the HZK33-B0010 monoclonal antibody contains the following CDR sequence:

[0038] (i) QSVYKNNY (SEQ ID No. 16);

[0039] (ii) SAS (SEQ ID No. 17);

[0040] (iii) QGAYDCSSADCIA (SEQ ID No. 18).

[0041] The heavy chain variable region of the HZK33-B0011 monoclonal antibody contains the following CDR sequence:

[0042] (i) GFSLNTYT (SEQ ID No. 19);

[0043] (ii) IKSGGYT (SEQ ID No. 20);

[0044] (iii) GRGGYRTAP (SEQ ID No. 21).

[0045] The light chain variable region of the HZK33-B0011 monoclonal antibody contains the following CDR sequence:

[0046] (i) QSVYNNNY (SEQ ID No. 22);

[0047] (ii) AAS (SEQ ID No. 23);

[0048] (iii) QGAYDCSSADCIP (SEQ ID No. 24).

[0049] The heavy chain variable region of the HZK33-B0029 monoclonal antibody contains the following CDR sequence:

[0050] (i) GFSLSTYT (SEQ ID No. 25);

[0051] (ii) IKSGGNT (SEQ ID No. 26);

[0052] (iii) GRGGYKTAP (SEQ ID No. 27).

[0053] The light chain variable region of the HZK33-B0029 monoclonal antibody contains the following CDR sequence:

[0054] (i) QSVYKNNY (SEQ ID No. 28);

[0055] (ii) SAS (SEQ ID No. 29);

[0056] (iii) QGAYDCSSADCTP (SEQ ID No. 30).

[0057] According to embodiments of this disclosure, three antibodies recognizing p-tau217 were developed using rabbit-derived monoclonal antibodies without cross-reactivity with non-phosphorylated tau proteins or phosphorylated tau proteins at other sites (such as p-tau181, p-tau205, and p-tau231). Specifically, the three antibodies include HZK33-B0010, HZK33-B0011, and HZK33-B0029. Compared to mouse or rat antibodies, rabbit-derived monoclonal antibodies, due to their greater genetic diversity and simpler immunoglobulin structure, are easier to humanize for subsequent clinical applications to reduce potential immunogenicity. The p-tau217 monoclonal antibody prepared in this disclosure can bind to p-tau217 with high specificity, and can be used to develop more specific detection kits for related humoral markers.

[0058] According to embodiments of this disclosure, the heavy chain variable region sequence of HZK33-B0010 is shown in SEQ ID No. 31, and the light chain variable region sequence is shown in SEQ ID No. 34; the heavy chain variable region sequence of HZK33-B0011 is shown in SEQ ID No. 32, and the light chain variable region sequence is shown in SEQ ID No. 35; the heavy chain variable region sequence of HZK33-B0029 is shown in SEQ ID No. 33, and the light chain variable region sequence is shown in SEQ ID No. 36.

[0059] The amino acid sequence of the B0010-H variable region (rabbit IgG1) (SEQ ID No. 31):

[0060] QSLEESGGRLVTPGTPLTLTCTVSGFSLSTYTVSWVRQAPGKGLEWIGLIKSGGSTYYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCGRGGFRTAPWGPGTLVTVSS.

[0061] B0011-H variable region amino acid sequence (rabbit IgG1) (SEQ ID No. 32):

[0062] QSLEESGGRLVTPGTPLTLTCTVSGFSLNTYTVSWVRQAPGKGLEWIGLIKSGGYTYYASWTKGRFTISKTSSTTVDLKITSPTTEDTATYFCGRGGYRTAPWGPGTLVTVSS.

[0063] The amino acid sequence of the variable region B0029-H (rabbit IgG1) (SEQ ID No. 33):

[0064] QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYTMTWVRQAPGKGLEWIGLIKSGGNTYYASWAKGRFTISKTSSTTVALKITSPTTEDTATYFCGRGGYKTAPWGPGTLVTVSS.

[0065] B0010-L variable region amino acid sequence (rabbit κ chain) (SEQ ID No. 34):

[0066] AAVMTQTASPVSAAVGGTVTINCQASQSVYKNNYLAWFQQKPGQPPKLLIYSASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSADCIAFGGGTEVVVR.

[0067] B0011-L variable region amino acid sequence (rabbit κ chain) (SEQ ID No. 35):

[0068] AIDMTQTASPVSAAVGGTVTINCQASQSVYNNNYLAWFQQKPGQPPKLLIYAASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSADCIPFGGGTEVVVR.

[0069] B0029-L variable region amino acid sequence (rabbit κ chain) (SEQ ID No. 36):

[0070] AVVLTQTASPVSAAVGGTVTINCQASQSVYKNNYLAWFQQKPGQPPNLLIYSASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSADCTPFGGGTEVVVR.

[0071] According to embodiments of this disclosure, the heavy chain sequence of HZK33-B0010 is shown in SEQ ID No. 1, and the light chain sequence is shown in SEQ ID No. 4; the heavy chain sequence of HZK33-B0011 is shown in SEQ ID No. 2, and the light chain sequence is shown in SEQ ID No. 5; the heavy chain sequence of HZK33-B0029 is shown in SEQ ID No. 3, and the light chain sequence is shown in SEQ ID No. 6.

[0072] B0010-H full-length amino acid sequence (rabbit IgG1) (SEQ ID No. 1):

[0073] MGWSLILLFLVAVATRVLSQSLEESGGRLVTPGTPLTLTCTVSGFSLSTYTVSWVRQAPGKGLEWIGLIKSGGSTYYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCGRGGFRTAPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0074] Full-length amino acid sequence of B0011-H (rabbit IgG1) (SEQ ID No.2):

[0075] MGWSLILLFLVAVATRVLSQSLEESGGRLVTPGTPLTLTCTVSGFSLNTYTVSWVRQAPGKGLEWIGLIKSGGYTYYASWTKGRFTISKTSSTTVDLKITSPTTEDTATYFCGRGGYRTAPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0076] Full-length amino acid sequence of B0029-H (rabbit IgG1) (SEQ ID No.3):

[0077] MGWSLILLFLVAVATRVLSQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYTMTWVRQAPGKGLEWIGLIKSGGNTYYASWAKGRFTISKTSSTTVALKITSPTTEDTATYFCGRGGYKTAPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*。

[0078] Full-length amino acid sequence of B0010-L (rabbit κ chain) (SEQ ID No.4):

[0079] MGWSCIILFLVATATGVHSAAVMTQTASPVSAAVGGTVTINCQASQSVYKNNYLAWFQQKPGQPPKLLIYSASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSADCIAFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。

[0080] Full-length amino acid sequence of B0011-L (rabbit κ chain) (SEQ ID No.5):

[0081] MGWSCIILFLVATATGVHSAIDMTQTASPVSAAVGGTVTINCQASQSVYNNNYLAWFQQKPGQPPKLLIYAASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSA DCIPFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0082] B0029-L full-length amino acid sequence (rabbit κ chain) (SEQ ID No. 6):

[0083] MGWSCIILFLVATATGVHSAVVLTQTASPVSAAVGGTVTINCQASQSVYKNNYLAWFQQKPGQPPNLLIYSASTLASGVSSRFSGSGSGTQFTLTISGVQCDDAATYYCQGAYDCSSA DCTPFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0084] According to an embodiment of another aspect of this disclosure, a nucleic acid molecule encoding any of the rabbit-derived monoclonal antibodies described above is provided. The nucleic acid molecule comprises the base sequence shown in any one of SEQ ID No. 7 to SEQ ID No. 12 and SEQ ID No. 37 to SEQ ID No. 42.

[0085] B0010-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID No. 7):

[0086]

[0087] B0011-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID No. 8):

[0088]

[0089] B0029-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID No. 9):

[0090]

[0091] Full-length nucleotide sequence of B0010-L (rabbit κ chain) (SEQ ID No.10):

[0092] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCAGCCGTGATGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCCCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTATAGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0093] Full-length nucleotide sequence of B0011-L (rabbit κ chain) (SEQ ID No.11):

[0094] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCCATCGATATGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAACAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATTTATGCTGCATCCACTCTGGCATCTGGGGTCTCATCCCGGTTTAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTATACCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0095] Full-length nucleotide sequence of B0029-L (rabbit κ chain) (SEQ ID No.12):

[0096] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCCGTCGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAACCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCCCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTACACCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0097] Nucleotide sequence of the variable region of B0010-H (rabbit IgG1) (SEQ ID No. 37):

[0098] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTACCTATACAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGATTGATTAAAAGTGGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTGGTTTCAGAACTGCCCCTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA。

[0099] Variable region nucleotide sequence of B0011-H (rabbit IgG1) (SEQ ID No.38):

[0100] CAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATACCTATACAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGATTGATTAAAAGTGGTGGTTACACATACTACGCGAGCTGGACGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTGGTTATAGAACTGCCCCTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA。

[0101] Variable region nucleotide sequence of B0029-H (rabbit IgG1) (SEQ ID No.39):

[0102] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTACCTATACAATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGATTGATTAAAAGTGGTGGTAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACGGTGGCTCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTGGTTATAAAACTGCCCCGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA。

[0103] Nucleotide sequence of the variable region of B0010-L (rabbit κ chain) (SEQ ID No. 40):

[0104] GCAGCCGTGATGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCCCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTATAGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA。

[0105] Nucleotide sequence of the variable region of B0011-L (rabbit κ chain) (SEQ ID No. 41):

[0106] GCCATCGATATGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAACAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATTTATGCTGCATCCACTCTGGC ATCTGGGGTCTCCCGGTTTAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTATACCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA.

[0107] B0029-L variable region nucleotide sequence (rabbit κ chain) (SEQ ID No. 42):

[0108] GCCGTCGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAACAACTACCTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAACCTCCTGATCTATTCTGCATCCACTCTGGC ATCTGGGGTCTCATCCCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGCTTATGATTGTAGTAGTGCTGATTGTACACCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA.

[0109] According to an embodiment of another aspect of this disclosure, a recombinant expression vector is provided, comprising a nucleic acid molecule with a base sequence as shown in any one of SEQ ID No. 7 to SEQ ID No. 12 and SEQ ID No. 37 to SEQ ID No. 42 above.

[0110] Specifically, this disclosure does not limit the types of recombinant expression vectors. For example, expression vectors can be constructed using isolated mammalian cells, and corresponding recombinant expression vectors can be obtained through adaptive design.

[0111] According to embodiments of this disclosure, a vector refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0112] According to another aspect of the present disclosure, a host cell comprising the above-described nucleotide sequence or the above-described expression vector is provided.

[0113] For example, genes encoding antibody heavy and light chains (whether the complete sequence of the variable region fused with the constant region, or the variable region fused with the vector's own constant region sequence) are inserted into the multiple cloning site of such vectors to form heavy chain expression plasmids and light chain expression plasmids, and then co-transfected into HEK293 cells for expression to obtain the corresponding host cells.

[0114] According to another aspect of this disclosure, a composition is provided comprising at least one of the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, and the above-described host cell.

[0115] According to embodiments of this disclosure, the composition further includes a pharmaceutically acceptable carrier.

[0116] According to embodiments of this disclosure, the above composition may include pharmaceutically acceptable excipients, diluents or carriers, etc.; it may also include essential components as a detection kit.

[0117] Specifically, pharmaceutically acceptable excipients refer to substances added to pharmaceutical formulations other than the active ingredient, used to improve the physical properties, stability, or bioavailability of the drug. They can be classified into various types according to their function, such as disintegrants, binders, and lubricants. They must meet conditions such as being non-toxic, non-irritating, and not chemically reacting with the active ingredient to ensure the safety and efficacy of the drug. Diluents are mainly used to adjust drug concentrations to achieve appropriate dosage specifications. Diluents need to be chemically stable, not affecting the activity of antibody or antigen-binding fragments, and have good flowability and compressibility to facilitate formulation molding.

[0118] Specifically, the essential components of a test kit may include solid-phase supports, such as ELISA plates, nitrocellulose membranes, and chips, for coating antigens or antibodies; calibrators and quality controls, including a series of standards containing known concentrations of p-tau217, for plotting standard curves and monitoring the effectiveness of the testing process; labeled secondary antibodies, such as horseradish peroxidase-labeled goat anti-rabbit IgG, for signal amplification and detection; substrate chromogenic solutions / chemiluminescent substrates, which react with labeled enzymes to generate detectable signals (color or light); and washing, diluents, and blocking solutions, for sample dilution, blocking of non-specific binding sites, and washing between steps.

[0119] In some specific embodiments of this disclosure, the monoclonal antibody composition may exist in various forms, such as liquid, lyophilized powder, and other formulations.

[0120] According to another aspect of this disclosure, a composition comprising the above-described rabbit monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, the above-described host cells, or the above-described composition, is provided for use in the preparation of a kit for detecting human phosphorylated tau217 protein.

[0121] Specifically, nucleic acid molecules may also include all regulatory sequences necessary to initiate the expression of nucleic acid molecules encoding antibody or antigen-binding fragments. These regulatory sequences, under compatible conditions, guide the expression of the coding sequence in suitable host cells. Regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence may also be a suitable transcription terminator sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operatively linked to the 3' end of a nucleic acid sequence encoding an antibody or antigen-binding fragment. Any terminator that functions in a selected host cell may be used in this disclosure. Any leader sequence that functions in a selected host cell may be used in this disclosure. The regulatory sequence may also be a signal peptide coding region encoding an amino acid sequence linked to the amino terminus of a protein (i.e., an antibody or antigen-binding fragment) that guides the encoded protein (i.e., the antibody or antigen-binding fragment) into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein (i.e., the antibody or antigen-binding fragment) into the secretory pathway of the host cell used may be used in this disclosure. Adding a regulatory sequence that can regulate protein (i.e., antibody or antigen-binding fragment) expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein (i.e., the antibody or antigen-binding fragment) should be operatively linked to the regulatory sequence.

[0122] According to embodiments of this disclosure, the kit is used for one or more of the following: treating or preventing neurodegenerative diseases, diagnosing or assisting in the diagnosis of neurodegenerative diseases, monitoring p-tau217 levels, differentiating neurodegenerative diseases from other forms of disease, and assessing the effectiveness of treatment and disease progression in neurodegenerative diseases.

[0123] According to another aspect of the embodiments of this disclosure, neurodegenerative diseases include Alzheimer's disease, Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia, and other forms of disease include Lewy body dementia and frontotemporal degeneration.

[0124] According to another embodiment of this disclosure, a method for preparing a monoclonal antibody is provided, including the step of culturing the host cells described above.

[0125] Specifically, an expression vector containing the heavy and light chain nucleotide sequences encoding the monoclonal antibody disclosed herein can be introduced into a suitable mammalian host cell (e.g., but not limited to, HEK293 cells, CHO cells, or SP2 / 0 cells) to obtain a recombinant host cell capable of expressing the antibody.

[0126] The technical solutions of this disclosure will be further explained below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, all reagents used are commercially available or can be prepared by known methods, and all test / experiment methods used are conventional methods in the art.

[0127] Example 1: Preparation of HZK33 monoclonal antibody

[0128] 1.1 Peptide Conjugation

[0129] The immunogenic peptide SinoA12330 was designed based on the target p-tau217, and its sequence is CRTPSLP(pT)PPTREPK (SEQ ID No. 43). Simultaneously, the negative screening peptide SinoA12331 was designed, and its sequence is CRTPSLPTPPTREPK (SEQ ID No. 44).

[0130] Peptide coupling reagents: heterobifunctional crosslinking agent (Sulfo-SMCC, Thermo), 30 Kd ultrafiltration tube (manufacturer: Sartorius), 0.45μm needle filter (manufacturer: Sartorius), 0.2μm needle filter (manufacturer: Sartorius), human serum albumin (HS-A, Yiqiao Shenzhou).

[0131] Peptide conjugation methods:

[0132] (1) Take 10 mL of recombinant human papillomavirus type 16 virus-like particle (HPV-16 VLP) solution and filter it using a 0.45 μm aqueous syringe filter to sterilize and remove any possible aggregates. Then, measure the absorbance of the filtrate at 280 nm wavelength (UV280) using a UV spectrophotometer. Calculate the total mass (mg) of the carrier protein in the solution based on the standard protein quantification curve of HPV-16 VLP.

[0133] (2) Weigh an appropriate amount of crosslinking agent Sulfo-SMCC and dissolve it in water to prepare an activator solution with a concentration of 4 mg / mL. Add the activator solution dropwise to the HPV-16 VLP solution. Incubate the reaction system at room temperature (25±2℃) in the dark for 2 hours.

[0134] (3) Filter the mixture after the above activation reaction again through a 0.45 μm filter membrane to remove any possible small amount of precipitate, and desalt and replace the buffer solution of the activated protein solution to remove unreacted free Sulfo-SMCC and its hydrolysis byproducts.

[0135] (4) Dissolve the synthesized peptide SinoA12330 in an appropriate amount of PBS to prepare a solution of the required concentration. Slowly add the peptide solution to an activated carrier protein solution of the same mass as the activated HPV-16 VLP (i.e., the protein mass calculated in step 1). Incubate the reaction system at room temperature (25±2℃) in the dark for 4 hours.

[0136] (5) After the coupling reaction is completed, the crude product solution of the target complex (HPV-16-SinoA12330) can be obtained. The concentration of the final product is calculated based on the amount of carrier protein initially added, and the concentration is calculated based on the amount of HPV-16 added: Concentration (mg / mL) = Amount of HPV-16 (mg) / Product volume (mL).

[0137] 1.2 Immunoassay and Serum Titer Detection

[0138] Immunological and serum titer assay reagents: complete Freund's adjuvant (Sigma), incomplete Freund's adjuvant (BD), coating solution (Sino-American), washing solution (Sino-American), blocking solution (Sino-American), secondary antibody diluent (Sino-American), sample diluent (Sino-American), chromogenic solution (Sino-American), stop solution (Sino-American), horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (IR).

[0139] Immunization and Serum Titer Detection Methods: Two healthy New Zealand white rabbits were selected for this experiment. For the initial immunization, 500 μg of antigen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously into the rabbits at multiple sites on the back. Subsequently, booster immunizations were performed every two weeks, using the same dose of antigen emulsified with an equal volume of incomplete Freund's adjuvant. After the fourth immunization, blood was collected from the marginal ear vein, and serum titer was determined using an indirect ELISA method to determine if a follow-up immunization was necessary. Once the serum titer reached the acceptable standard, peripheral blood was collected for B cell sorting.

[0140] 1.3 Antigen labeling and flow cytometry sorting of B cells

[0141] Antigen labeling and flow cytometry reagents for B cell sorting: Phycoerythrin (PE, Agilent), Allophycocyanin (APC, Agilent), Succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC, Thermo), N-ethylmaleimide (NEM, SIGMA), Protectant (SinoA12330), Phosphate-buffered saline (PBS, SinoA12330), Rabbit IgG antibody (Anti-rabbit IgG), Rabbit IgM antibody (Anti-rabbit IgM), Peptide SinoA12330-crosslinker-APC fluorescently labeled complex (SinoA12330-SMCC-APC), Peptide SinoA12331-crosslinker-PE fluorescently labeled complex (SinoA12331-SMCC-PE), 7-aminoactinomycin (D7-AAD).

[0142] PE marking method:

[0143] (1) Concentrate PE to approximately 3 mg / mL and determine its accurate concentration using ultraviolet spectrophotometry. Subsequently, add crosslinking agent SMCC at a certain molar ratio and react at room temperature in the dark. The N-hydroxysuccinimide (NHS) end of SMCC will react with the primary amino group on the surface of PE protein, thereby introducing maleimide (MAL) active groups onto PE.

[0144] (2) Mix the activated PE with an appropriate amount of peptide SinoA12331 dissolved in PBS and mix thoroughly. Place the mixture at 4°C and react overnight in the dark.

[0145] (3) After the reaction was completed, the conjugated product was subjected to multiple buffer changes (replaced with the required storage buffer PBS) using ultrafiltration centrifuge tubes to thoroughly remove unreacted peptides, free dyes, and small molecule byproducts. Finally, the concentration of the purified product was determined again by ultraviolet spectrophotometry, and the yield of the final PE-peptide conjugate was calculated.

[0146] APC tagging method:

[0147] (1) APC was concentrated to about 3 mg / mL. After the accurate concentration was determined by ultraviolet spectrophotometry, SMCC was added and the reaction was carried out at room temperature in the dark. The N-hydroxysuccinimide (NHS) end of SMCC was bound to the primary amino group on the surface of APC, thereby introducing maleimide (MAL) active groups onto APC.

[0148] (2) Mix the peptide SinoA12330 dissolved in PBS with the activated APC, mix thoroughly, and react overnight at 4°C in the dark.

[0149] (3) After the reaction was completed, the conjugated product was replaced with an ultrafiltration centrifuge tube with a suitable buffer such as PBS to remove unreacted peptides and small molecule impurities. Finally, the concentration of the purified product was determined by ultraviolet spectrophotometry, and the final yield of the APC-peptide conjugate was calculated.

[0150] Flow cytometry sorting of B cells:

[0151] (1) Add EDTA-anticoagulated rabbit whole blood to a centrifuge tube containing gradient separation medium (Ficoll) and centrifuge. After centrifugation, aspirate the white, cloudy cell layer (i.e., the white membrane layer rich in lymphocytes and monocytes) located between the separation medium interfaces to a new centrifuge tube. Add sufficient phosphate buffer to wash the cells, centrifuge, and discard the supernatant to obtain relatively pure peripheral blood mononuclear cells (PBMCs).

[0152] (2) Add IgG, IgM antibodies and fluorescently labeled proteins to the isolated PBMC cells and incubate them. After incubation, wash the cells with PBS 2-3 times to completely remove unbound free antibodies. Finally, resuspend the cells with an appropriate amount of PBS.

[0153] (3) The cells were detected by flow cytometry. The flow cytometry results are as follows: Figure 1 As shown.

[0154] Figure 1 The following is a flow cytometry result image of the monoclonal antibody prepared according to the embodiments of this disclosure, wherein A is a forward scattering light-area map, B is a 7-aminoactinomycin D screening map, C is an immunoglobulin G signal map, and D is an allophycocyanin-area map.

[0155] like Figure 1 As shown, lymphocytes were delineated based on cell diameter (FSC) and intracellular granule complexity (SSC); cells not stained with the DNA dye 7-amino-actinomycin D (7-AAD) were delineated; IgG antibody-positive cells were delineated; and cells specifically binding to the target peptide were delineated. Different cell populations were rapidly distinguished using fluorescent labeling optical characteristics. Figure 1 The sorting strategy can gradually screen out target B cells from mixed cells, namely antigen-specific B cells that can bind to p-tau217.

[0156] 1.4 Expansion of rabbit-derived B-cell positive clones

[0157] Rabbit B cell positive clone amplification reagents: 3T3-mouse CD40 ligand feeder cells (3T3-msCD40L feeder cells, CellRON), mitomycin C (MCE), modified Durbeco medium (IMDM medium, GIBCO), phytohemagglutinin-M (PHA-M, Roche), fetal bovine serum (GIBCO), interleukin-2, interleukin-21 (IL2, IL21 cytokines, Sinocare).

[0158] Cell culture methods:

[0159] One day before B cell sorting, prepare mitomycin C-treated feeder cells and seed them into 96-well cell culture plates to allow them to adhere. On the day of sorting, inject single B cells that meet the screening criteria into wells containing feeder cells. After sorting, add culture medium containing mitogens and cytokines and incubate in a CO2 incubator for about one week. Take a portion of the culture supernatant for ELISA detection.

[0160] The reagents used in ELISA are shown in Table 1:

[0161] Table 1

[0162]

[0163] ELISA detection method:

[0164] (1) Dilute the project peptide and cross-peptide to working concentrations (0.5 μg / mL and 5 μg / mL, respectively) using coating buffer. Add 100 μL of the diluted antigen solution to each well of the ELISA plate, seal and coat overnight at 4°C.

[0165] (2) Discard the coating solution inside the plate and pat it dry on absorbent paper. Add 300 μL of blocking solution to each well, seal and incubate at room temperature (25°C) for 1 hour.

[0166] (3) Add 300 μL of washing solution to each well, let stand for about 30 seconds and then discard the liquid. Repeat twice and drain on absorbent paper.

[0167] (4) Use sample diluent to dilute the cell culture supernatant by a ratio of 5, and vortex thoroughly to mix well, and set aside.

[0168] (5) Add 100 μL of diluted sample supernatant to each well of the sealed and washed ELISA plate. Place the ELISA plate on a horizontal shaker and mix at low speed. Incubate at room temperature for 2 hours.

[0169] (6) Wash the plate 3 times with 300 μL / well washing solution, and pat dry the plate after the last wash.

[0170] (7) Dilute the horseradish peroxidase-labeled Goat Anti-Rabbit IgG Fc / HRP to the working concentration, mix well, add at 100 μL / well, and incubate at room temperature for 2 h.

[0171] (8) Same as step (3), but increase the number of washes to three to thoroughly remove unbound components.

[0172] (9) Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 3 min.

[0173] (10) Add 50 μL of stop solution to each well and immediately measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0174] ELISA test results:

[0175]

[0176] 1.5 Recombinant Antibody Expression

[0177] Recombinant antibody expression reagents: cell lysis buffer (Lysis Buffer, E-Tech), PCR kit (E-Tech), reverse transcription kit (SRT-200T, E-Tech), RNase inhibitor (Thermo), dithiothreitol (DTT, Thermo), deoxyribonucleoside triphosphate mixture (dNTPs, Thermo), one-step rapid cloning kit (Shanghai Yisheng).

[0178] Recombinant antibody expression method: After lysing qualified B cells from culture supernatant, cDNA was obtained by reverse transcription using the Sinocare Reverse Transcription Kit. The antibody heavy and light chain variable region sequences were then obtained through single B cell amplification technology (the amplification principle is as follows). Figure 2 As shown in the figure, after constructing the variable region fragments of the heavy and light chains into expression vectors, the correctly sequenced heavy and light chain expression plasmids were obtained, which were then transduced into HEK 293 for transient expression and secreted into complete IgG antibodies.

[0179] Figure 2 This is a schematic diagram illustrating the principle of obtaining variable region fragments of antibody heavy and light chains by nested PCR amplification as disclosed in this invention.

[0180] 1.6 Antibody Purification

[0181] The reagents used for antibody purification are shown in Table 2:

[0182] Table 2

[0183]

[0184] Antibody purification methods:

[0185] (1) Sample preparation. The collected cell supernatant was centrifuged using a benchtop centrifuge at 4000g for 30 min. The cell supernatant was collected after centrifugation and filtered through a 0.45μM filter membrane.

[0186] (2) Select a protein A column of appropriate specifications according to the expression level and connect it to the purification system; wash with ultrapure water for 3 column volumes to replace the 25% ethanol stored in the column, and then equilibrate with binding buffer (AC Binding + 1 / 5 stock buffer) for at least 3 column volumes until the UV absorption baseline is stable.

[0187] (3) Load the pretreated sample at an appropriate flow rate to ensure that the antibody and protein A filler are fully combined.

[0188] (4) After loading the sample, use the rinsing buffer to rinse the chromatography column for 5-10 column volumes at the same flow rate until the UV absorption signal returns to the baseline level to remove non-specifically bound proteins.

[0189] (5) Elute with elution buffer (AC Elution) and collect the elution peaks according to the real-time UV monitoring spectrum. Immediately add 2 M tris-HCl buffer (pH 8.0) to the collected eluent to neutralize it and prevent the antibody from being inactivated under acidic conditions.

[0190] (6) Add 2M Tris and neutralize the eluted antibody at pH 8.0.

[0191] (7) Use binding buffer (AC Binding) to equilibrate 3 column volumes to neutral.

[0192] (8) In-situ cleaning (CIP) of 5 column volumes or more.

[0193] (9) Use AC Binding to flush with alkali until the pH of the effluent returns to neutral.

[0194] (10) Use 25% ethanol to balance the two column volumes, and seal and store the fully balanced chromatography column.

[0195] The above steps allow antibodies to be captured from the cell culture supernatant. After elution and neutralization, high-purity antibody solutions B0010, B0011, and B0029 are obtained.

[0196] Example 2: Identification of Monoclonal Antibodies

[0197] 2.1 ELISA detection and identification

[0198] The ELISA test reagents are the same as those in Table 1 of Example 1.

[0199] ELISA detection and identification method:

[0200] (1) Dilute the phosphorylated peptides to 0.1 μg / mL and 1 μg / mL respectively using coating buffer, and dilute the non-phosphorylated peptides to 1 μg / mL. Add 100 μL of the corresponding concentration of antigen solution to each well of the ELISA plate, seal and coat overnight at 4°C.

[0201] (2) Discard the coating solution in the plate and pat dry on absorbent paper. Add 300 μL of blocking solution to each well, seal and incubate at room temperature for 1 hour to cover the non-specific binding sites in the well.

[0202] (3) Discard the sealing solution, add 300 μL of washing solution to each well, wash the plate twice, and pat dry the plate on the last wash.

[0203] (4) Dilute antibodies HZK33-B0010, HZK33-B0011 and HZK33-B0029 (primary antibody) to 0.1 μg / mL, add 100 μL of each to the corresponding well plate, mix well and incubate at room temperature for 2 h.

[0204] (5) Discard the primary antibody and wash the plate three times with washing solution as in step 3. Pat the plate dry on the last wash to completely remove the unbound primary antibody.

[0205] (6) Dilute the horseradish peroxidase-labeled goat anti-rabbit IgG (Goat Anti-Rabbit IgGFc / HRP) secondary antibody to the recommended working concentration using antibody dilution buffer. Add 100 μL of the diluted secondary antibody to each well and incubate at room temperature for 1 hour.

[0206] (7) Discard the secondary antibody and wash the plate three times with washing solution as in step 3 to completely remove the unbound secondary antibody.

[0207] (8) Mix color development solution A and color development solution B at a ratio of 1:1, add 200 μL of the mixture to each well, and incubate at room temperature in the dark for 20 minutes.

[0208] (9) Add 50 μL of stop solution to each well, mix gently, and immediately use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0209] ELISA test results:

[0210]

[0211] 2.2 Detection of surface plasmon resonance (SPR) of HZK33-B0010, HZK33-B0011, and HZK33-B0029 antibodies with SinoA12330 and SinoA12331 peptides

[0212] Antibody conjugation steps:

[0213] (1) Cleaning: After replacing the CM5 chip and the running buffer, clean the chip twice with a mixture of 50 mM NaOH and 0.5% SDS, and then clean it once with 50 mM NaOH. The flow rate for both is 30 μL / min.

[0214] (2) Activation: Mix equal volumes of 0.4 M aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M aqueous solution of N-hydroxysuccinimide (NHS), and inject the mixture at a flow rate of 10 μL / min for 100 seconds to activate the Fc1 and Fc2 channels.

[0215] (3) Coupled proteins: The antibodies HZK33-B0010, HZK33-B0011 and HZK33-B0029 were diluted to a final concentration of 5 μg / mL with 10 mM sodium acetate at pH 4.0 and then coupled to the corresponding Fc2 / Fc3 / Fc4 channels and injected at a flow rate of 10 μL / min. The Fc1 channel was not fixed with antibodies and was used as a reference channel to remove non-specific signals in subsequent analysis.

[0216] (4) Closure: After coupling, the Fc1, Fc2, Fc3 and Fc4 channels are sealed with ethanolamine solution at 10 μL / min for 600 s.

[0217] Run buffer and sample preparation:

[0218] (1) Preparation of running buffer: Take 20× PBS concentrate and dilute it 20-fold with ultrapure water to obtain a 1× working concentration. Then, the diluted PBS buffer is vacuum filtered or pressure filtered through a 0.22 μm filter membrane to remove any possible microorganisms and particulate matter. After filtration, the solution is briefly sonicated to completely remove dissolved air bubbles, ensuring a stable liquid path and baseline during SPR detection. Finally, add the nonionic surfactant Tween-20 at a volume ratio of 0.05% (v / v) and vortex thoroughly to obtain the required running buffer PBST. This buffer should be allowed to stand until all air bubbles have disappeared before use.

[0219] (2) Sample preparation: The peptide sample to be tested was serially diluted using freshly prepared PBST running buffer. During dilution, the sample was first diluted to a predetermined highest starting concentration, and then, starting from this, it was continuously diluted 2-fold to prepare a total of 11 concentration gradients of sample solutions. In addition, a separate tube of sample containing only pure PBST running buffer without peptides was prepared as a 0 concentration control, which was used to correct the baseline and subtract the background signal in the subsequent SPR detection.

[0220] SPR kinetic testing (KD value determination) method:

[0221] A series of diluted peptide samples (HZK33-B0010, HZK33-B0011, or HZK33-B0029) were flowed through the surface of the antibody-immobilized chip at a flow rate of 30 μL / min for 120 seconds. Molecular binding signals were recorded in real time during this process. Analyte injection was then stopped, and the flow was switched to run buffer-only delivery for 180 seconds. The rate of dissociation of the bound analyte from the ligand was observed and recorded during this phase. After each binding-dissociation cycle, 10 mM glycine hydrochloride solution (Gly-HCl) (pH 3.0) was injected for 30 seconds to thoroughly remove any residual analyte from the chip surface, regenerating the chip to its initial state and ensuring the accuracy of the next concentration gradient detection.

[0222] Results Analysis: The results of the plasmon resonance affinity assays between HZK33-B0010, HZK33-B0011, and HZK33-B0029 antibodies and SinoA12330 and SinoA12331 peptides are attached. Figures 3A-3F As shown.

[0223] Figure 3A The binding curve of HZK33-B0010 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention is shown. Figure 3B The binding curve of HZK33-B0010 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention is shown. Figure 3C This is the binding curve of HZK33-B0011 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention; Figure 3D The binding curve of HZK33-B0011 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention is shown. Figure 3E This is the binding curve of HZK33-B0029 antibody and HZK33-1 (SinoA12330) in an embodiment of the present invention; Figure 3F The binding curve of HZK33-B0029 antibody and HZK33-2 (SinoA12331) in an embodiment of the present invention is shown.

[0224] according to Figures 3A-3F It can be seen that the three antibody clones, HZK33-B0010, HZK33-B0011, and HZK33-B0029, specifically bind to SinoA12330 but not SinoA12331, indicating that the antibodies HZK33-B0010, HZK33-B0011, and HZK33-B0029 can effectively distinguish p-tau217 from the unphosphorylated form of tau protein.

[0225] 2.3 Identification by Western blot assay

[0226] Immunoblotting assay

[0227] (1) Take a human brain tissue sample, add pre-cooled protein lysis buffer (containing protease inhibitor) for homogenization, and lyse on ice for 30 minutes. Then centrifuge at 4℃ and 12000rpm for 20 minutes, and collect the supernatant, which is the total protein sample. Determine the protein concentration using the BCA method, etc., and mix the sample with 5× loading buffer and boil at 100℃ for 5 minutes to denature the protein.

[0228] (2) Prepare a 16% SDS-PAGE gel. Add the prepared protein sample to the well according to the predetermined loading amount. Add electrophoresis buffer to the electrophoresis tank, set the voltage to 120V, and electrophore for about 1 hour until the bromophenol blue indicator reaches the bottom of the gel.

[0229] (3) After electrophoresis, remove the gel and cut a 0.22 μm polyvinylidene fluoride (PVDF) membrane and filter paper according to the gel size. The PVDF membrane needs to be activated by soaking it in methanol for 5-10 seconds. Then, place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp in sequence, ensuring that there are no air bubbles between the layers. Place the transfer clamp into the transfer tank and add pre-cooled transfer buffer. Transfer the membrane at a constant current of 240 mA for 30 minutes under ice bath conditions.

[0230] (5) Block the membrane for 1 hour at room temperature using blocking buffer.

[0231] (6) Incubate the membrane with the appropriate diluted primary antibody at 4°C overnight; incubate the membrane with the conjugated secondary antibody dilution in the blocking buffer for 1 hour at room temperature.

[0232] (7) Mix the peroxide solution and enhancer in the ECL chemiluminescence substrate in equal volumes (500 μL each) to prepare a reaction solution. Evenly drop the reaction solution onto the surface of the PVDF membrane, ensuring the liquid covers all areas. Then, perform exposure detection in a chemiluminescence imager or dark chamber. Adjust the exposure time according to the signal intensity to obtain a clear band image, as shown in the figure. Figures 4A-4C As shown.

[0233] Figure 4AThis is an image showing the immunoblotting results of the HZK33-B0010 antibody binding to a human brain protein sample in an embodiment of the present invention. Figure 4B This is an image showing the immunoblotting results of the HZK33-B0011 antibody binding to a human brain protein sample in an embodiment of the present invention. Figure 4C This is an image showing the immunoblotting results of the HZK33-B0029 antibody binding to a human brain protein sample in an embodiment of the present invention.

[0234] according to Figures 4A-4C It can be seen that the rabbit B cell supernatant and the purified antibody clones HZK33-B0010, HZK33-B0011 and HZK33-B0029 can effectively distinguish p-tau217.

[0235] Example 3: Alzheimer's Disease Sample Detection

[0236] Brain tissue samples from patients with Alzheimer's disease (AD) and those from patients without Alzheimer's disease (non-AD) were collected and subjected to the same immunoblotting assay as in Example 2. The results are as follows. Figures 5A-5C As shown.

[0237] Figure 5A This is a comparison of the immunoblotting results of HZK33-B0010 antibody binding to human brain protein samples in an embodiment of the present invention; Figure 5B This is a comparison of the immunoblotting results of HZK33-B0011 antibody binding to human brain protein samples in an embodiment of the present invention; Figure 5C This is a comparison of the immunoblotting results of the HZK33-B0029 antibody binding to human brain protein samples in an embodiment of the present invention.

[0238] according to Figures 5A-5C As can be seen, the three antibodies detected clear bands at the same location (approximately 50-60 kDa) in most samples, and the band patterns were highly consistent. This demonstrates that the three antibodies can successfully detect p-tau217 in complex human brain tissue samples. Furthermore, the bands were strong in AD samples and weak or absent in control samples (non-AD), suggesting that the antibodies can be used to differentiate disease states.

[0239] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A rabbit-derived monoclonal antibody against human phosphorylated tau217, characterized in that, The monoclonal antibody includes a light chain variable region and a heavy chain variable region, and the monoclonal antibody includes HZK33-B0010, HZK33-B0011, and HZK33-B0029; wherein... The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK33-B0010 are shown in SEQ ID No. 13 to SEQ ID No. 15, and the light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in SEQ ID No. 16 to SEQ ID No. 18, respectively. The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK33-B0011 are shown in SEQ ID No. 19 to SEQ ID No. 21, and the light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in SEQ ID No. 22 to SEQ ID No. 24, respectively. The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK33-B0029 are shown in SEQ ID No. 25 to SEQ ID No. 27, and the light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in SEQ ID No. 28 to SEQ ID No. 30, respectively.

2. The rabbit-derived monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region sequence of HZK33-B0010 is shown in SEQ ID No. 31, and the light chain variable region sequence is shown in SEQ ID No. 34; The heavy chain variable region sequence of HZK33-B0011 is shown in SEQ ID No. 32, and the light chain variable region sequence is shown in SEQ ID No. 35; The heavy chain variable region sequence of HZK33-B0029 is shown in SEQ ID No. 33, and the light chain variable region sequence is shown in SEQ ID No.

36.

3. The rabbit-derived monoclonal antibody according to claim 1, characterized in that, The heavy chain sequence of HZK33-B0010 is shown in SEQ ID No. 1, and the light chain sequence is shown in SEQ ID No. 4; The heavy chain sequence of HZK33-B0011 is shown in SEQ ID No. 2, and the light chain sequence is shown in SEQ ID No. 5; The heavy chain sequence of HZK33-B0029 is shown in SEQ ID No. 3, and the light chain sequence is shown in SEQ ID No.

6.

4. A nucleic acid molecule, characterized in that, The code is a rabbit-derived monoclonal antibody as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule includes the base sequence shown in any one of SEQ ID No. 7 to SEQ ID No. 12 and SEQ ID No. 37 to SEQ ID No.

42.

6. A recombinant expression vector, characterized in that, Includes the nucleic acid molecules described in claim 4 or 5.

7. A host cell, characterized in that, Includes the recombinant expression vector as described in claim 6.

8. A composition, characterized in that, The composition comprises at least one of the rabbit monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the recombinant expression vector according to claim 6, and the host cell according to claim 7.

9. The composition according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers.

10. The use of a rabbit monoclonal antibody according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4 or 5, a recombinant expression vector according to claim 6, a host cell according to claim 7, or a composition according to claim 8 or 9 in the preparation of a kit for detecting human phosphorylated tau217 protein.

11. The application according to claim 10, characterized in that, The kit is used for one or more of the following: diagnosing or assisting in the diagnosis of neurodegenerative diseases, monitoring human phosphorylated tau217 levels, differentiating neurodegenerative diseases from other forms of disease, and assessing the treatment efficacy and disease progression of neurodegenerative diseases, wherein the other forms of disease are Lewy body dementia and frontotemporal degeneration.

12. The application according to claim 11, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

13. A method for preparing a rabbit-derived monoclonal antibody, comprising the step of culturing the host cells as described in claim 7.

Images