Monoclonal antibody of anti-human Yo protein as well as application and detection method of monoclonal antibody

Abstract

The invention discloses an anti-human Yo protein monoclonal antibody as well as application and a detection method thereof, and belongs to the technical field of monoclonal antibody preparation. The monoclonal antibody provided by the invention can efficiently and specifically recognize a key functional region of Yo protein, meanwhile, a constant region (Fc region) of the monoclonal antibody has human-derived characteristics and can be accurately adapted to anti-human IgG secondary antibody-based mainstream detection systems such as immunofluorescence, ELISA (enzyme-linked immuno sorbent assay) and IHC (infectious histocompatibility complex), and a quality control product prepared on the basis of the monoclonal antibody gets rid of dependence on positive serum of a patient and has a good application prospect. Stable supply can be achieved through industrial production, quality indexes such as antibody purity and titer can be accurately regulated and controlled, the technical bottlenecks that a traditional serum quality control product is scarce in source, complex in component, non-uniform in quality and poor in stability are solved, and standardized quality control guarantee is provided for a Yo protein autoantibody detection reagent.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody preparation technology, specifically relating to a monoclonal antibody against human Yo protein, its application, and detection method. Background Technology

[0002] Cerebellar Degeneration-Related Protein 2 (CDR2), also known as Yo, PCA-1, or CDR62, is a 545-amino acid protein associated with paraneoplastic cerebellar degeneration (PCD). PCD is an immune-mediated syndrome, a heterogeneous group of rare paraneoplastic neurological disorders affecting the nervous system. PCD is characterized by subacute cerebellar ataxia and primarily occurs in patients with ovarian, uterine, fallopian tube, or breast cancer. In the 1970s, TROTTER et al. first detected antibodies that cross-reacted with cerebellar Purkinje cells in the serum of PCD patients. Later, it was confirmed that one of these antibodies reacted only with cerebellar Purkinje cells, and this antibody that specifically binds to Purkinje cells was named anti-Yo autoantibody. PCD with positive anti-Yo autoantibodies typically presents with symptoms of subacute cerebellar degeneration, primarily manifesting as cerebellar ataxia in the trunk and limbs, lasting from several weeks to months. Occasionally, myelopathy or peripheral neuropathy may also occur, and the prognosis is poor. The average age of onset is 60-70 years, with women making up the majority of patients. 80%-90% of patients also have ovarian, uterine, or breast cancer, and these patients will develop an immune response to both the Yo expression from cancer cells and the Yo expression from Purkinje neurons. When PCD patients present with anti-Yo autoantibodies, aggressive diagnosis and treatment of the underlying cancer are necessary.

[0003] In the fields of Yo protein expression identification, functional research, and clinical testing, there is an urgent need for antibodies that specifically recognize Yo protein with high stability and versatility. However, existing commercially available antibodies have significant technical shortcomings: most mainstream products are murine / rabbit polyclonal antibodies, which suffer from poor batch-to-batch consistency in specificity and affinity due to limitations in the preparation process, and their complex production process leads to high prices, seriously affecting the reproducibility of test results and the cost of use. In addition, although some murine / rabbit monoclonal antibodies have better specificity, their constant region (Fc region) is derived from foreign species, making them unsuitable for mainstream detection systems such as immunofluorescence, ELISA, and IHC based on anti-human IgG secondary antibodies, posing immunogenicity issues in clinical applications. At the same time, when preparing test reagents for detecting Yo protein autoantibodies, quality control products are required to ensure the effectiveness and accuracy of the test. However, existing quality control products mainly rely on collecting serum from positive patients, which suffers from problems such as scarce sources, ethical restrictions, complex composition, and large individual differences in antibody titers. This makes it difficult to standardize the quality control products, and they have poor stability, making industrial-scale production impossible and failing to meet the needs of industrialization, promotion, and long-term application of test reagents.

[0004] Therefore, developing a monoclonal antibody against human Yo protein aims to address the technical shortcomings of existing antibodies and the challenges in preparing quality control products. This involves optimizing antibody species compatibility, reducing immunogenicity, improving batch-to-batch consistency, and developing standardized, scalable quality control products based on this chimeric antibody to meet the practical needs of Yo protein expression identification, clinical testing, and reagent quality control. This has become a pressing technical problem in this field. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a monoclonal antibody against human Yo protein, its application and detection method, so as to solve the technical problems that existing products are difficult to standardize, have poor stability and cannot be industrialized and scaled up.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses a monoclonal antibody against human Yo protein, comprising a light chain and a heavy chain; The variable region of the light chain includes three light chain complementarity-determining regions, and the three light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 have amino acid sequences as shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; The variable region of the heavy chain includes three heavy chain complementarity-determining regions, CDR-H1, CDR-H2, and CDR-H3, which have amino acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0007] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:9; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:10. The constant region of the light chain is the human light chain constant region, and its amino acid sequence is shown in SEQ ID NO:11; the constant region of the heavy chain is the human heavy chain constant region, and its amino acid sequence is shown in SEQ ID NO:12.

[0008] Preferably, the monoclonal antibody against human Yo protein comprises having any one of the amino acid sequences shown in (I), (II), and (III): (I) The light chain variable region sequence shown in SEQ ID NO:1 and / or the heavy chain variable region sequence shown in SEQ ID NO:2; (II) and (I) show amino acid sequences that have at least 90% homology and the same function; (III) is an amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II).

[0009] Preferably, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody, or a murine antibody.

[0010] In a second aspect, the present invention discloses a recombinant expression vector comprising a nucleotide sequence of a monoclonal antibody expressing the above-described anti-human Yo protein.

[0011] A third aspect of the present invention discloses a host cell comprising the aforementioned vector.

[0012] In a fourth aspect, the present invention discloses the use of the above-mentioned monoclonal antibody against human Yo protein, recombinant expression vector or host cell in the preparation of one or more products for detecting Yo protein, identifying Yo protein and detecting Yo protein autoantibodies.

[0013] A fifth aspect of the present invention discloses a product for detecting Yo protein, comprising: The detection reagent or kit contains at least one of the above-mentioned monoclonal antibody against human Yo protein, recombinant expression vector, and host cell.

[0014] Preferably, the kit includes an immunofluorescence detection kit, a blot detection kit, an ELISA detection kit, a flow cytometry kit, or an IHC detection kit.

[0015] A sixth aspect of the present invention discloses a method for detecting Yo protein or polypeptide, comprising contacting a biological sample with the above-mentioned monoclonal antibody against human Yo protein, and detecting Yo protein or polypeptide in the sample. The biological samples include plasma, whole blood, serum, tissues, cells, or tissue and cell lysates containing Yo protein / peptide; The detection methods include enzyme-linked immunosorbent assay (ELISA), cell-based immunofluorescence assay, tissue-based immunofluorescence assay, dot blot assay, chemiluminescent immunoassay, radioimmunoassay, colloidal gold immunochromatography, and other non-enzyme-linked antibody binding methods.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a monoclonal antibody that specifically recognizes Yo protein, serving as a key raw material for the specific capture of Yo protein. It exhibits no cross-reactivity with other unrelated proteins, demonstrating superior specificity and improved accuracy. Compared to existing technologies, the monoclonal antibody provided by this invention efficiently and specifically recognizes key functional regions of the Yo protein. Furthermore, its constant region (Fc region) possesses human characteristics, allowing for precise adaptation to mainstream detection systems based on anti-human IgG secondary antibodies, such as immunofluorescence, ELISA, and IHC. Quality control products prepared based on this monoclonal antibody eliminate reliance on patient positive serum, enabling stable supply through industrial production. The antibody purity, titer, and other quality indicators can be precisely controlled, overcoming the technical bottlenecks of traditional serum-based quality control products, including scarcity of sources, complex composition, inconsistent quality, and poor stability. This provides standardized quality control assurance for Yo protein autoantibody detection reagents. The monoclonal antibody provided by this invention exhibits efficient expression in mammalian cell expression systems, a stable production process, and a simple preparation method, facilitating large-scale production. The resulting antibodies are of high purity and are suitable for long-term standardized research and industrial-scale preparation, meeting the long-term stable application needs of scientific research and clinical testing. Attached Figure Description

[0017] Figure 1 This is an SDS-PAGE electrophoresis image of the purified Yo-6his recombinant protein from Example 1.

[0018] Figure 2-1 This is a diagram showing the verification results of Yo-overexpressing cells on a smear. Figure 2-2 The results of specific antibody screening in immunized mouse serum are shown in the figure. Figure 3-1 This image shows the results of screening and verification of the supernatant of the positive hybridoma cell line in Example 1 using immunofluorescence. Figure 3-2 The results of screening the supernatant of the positive hybridoma cell line in Example 1 by ELISA; Figure 3-3 The results were validated using immunofluorescence.

[0019] Figure 4 This is an SDS-PAGE image of the purified 1H8C6E7 human-mouse chimeric monoclonal antibody from Example 2.

[0020] Figure 5 The image shows the titer detection results of the 1H8C6E7 chimeric antibody in Example 3.

[0021] Figure 6 This is a diagram showing the results of the dot blot assay used in Example 3 to verify the specificity of the 1H8C6E7 chimeric antibody.

[0022] Figure 7 This is a graph showing the specificity results of the 1H8C6E7 chimeric antibody used as a positive control in Example 4.

[0023] Figure 8 This is a diagram showing the staining results of tissue sections used to detect Yo protein in Example 4. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0026] The present invention will now be described in further detail with reference to the accompanying drawings: The term “antibody” is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to the Yo antigen or fragments thereof.

[0027] The term "monoclonal antibody (MAb)" refers to a population of antibodies of a single species, consisting of a single light chain gene product and a single heavy chain gene product. The complementarity-determining region (CDR) of monoclonal antibodies is the same in all molecules of the antibody population, and it can bind to the antigen-binding site of a specific epitope of the antigen.

[0028] The term "chimeric antibody" refers to an antibody in which the sequences of the H-chain variable region and the L-chain variable region (VH and VL) are derived from a non-human animal species, and the constant region sequences (CH and CL) are derived from humans. The sequences of the variable regions are preferably derived from animal species that allow for the easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0029] The term "humanized monoclonal antibody" refers to the grafting of the CDR (code sequence) of a non-human animal antibody onto the framework of a human antibody; it is also known as a CDR-grafted antibody. The CDR sequence of the non-human animal species antibody is preferably derived from animal species that allow for the easy preparation of hybridomas, such as mice, rats, and rabbits.

[0030] Furthermore, according to the present invention, the antibody comprises a light chain variable region as shown in SEQ ID NO:1 and a heavy chain variable region as shown in SEQ ID NO:2.

[0031] The amino acid sequence of the light chain variable region, SEQ ID NO:1, is: MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSTSLLHSSGKHRLYWFLQRPGQSPQLLIYYMSNLDSGVPDRFSGSGSGTDFTLRISRVEAEDFGVYYCMQSLEYPLTFGAGTKLELK; The amino acid sequence of the heavy chain variable region, SEQ ID NO:2, is: MGWSWIFFFFLSGTAGVHCQVQLKQSGAELVRPGASVKLSCKASGYIFTDYYINWVKRRPGQGLEWIARFSPGSGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSGVYFCATHGMDYWGQGTSVTVSS.

[0032] In this article, the term "CDR" stands for "complementarity-determining region," which refers to the highly variable region of the heavy and light chains of an immunoglobulin, specifically the region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0033] Furthermore, according to the present invention, the amino acid sequences of the light chain variable regions LCDR1-LCDR3 are as shown in SEQ ID NO:3-5, wherein the amino acid sequence of LCDR1 is SEQ ID NO:3 (RSSTSLLHSSGKHRLY); the amino acid sequence of LCDR2 is SEQ ID NO:4 (YMSNLDS); and the amino acid sequence of LCDR3 is SEQ ID NO:5 (MQSLEYPLT); the amino acid sequences of the heavy chain variable regions HCDR1-HCDR3 are as shown in SEQ ID NO:6-8, wherein the amino acid sequence of HCDR1 is SEQ ID NO:6 (DYYIN); the amino acid sequence of HCDR2 is SEQ ID NO:7 (RFSPGSGNTYYNEKFKG); and the amino acid sequence of HCDR3 is SEQ ID NO:8 (HGMDY).

[0034] The term "constant region" (C region) refers to a region in the structure of an antibody molecule (immunoglobulin) whose amino acid sequence is relatively conserved and does not directly participate in antigen-specific binding. Together with the highly variable variable region (V region), it constitutes the basic structure of an antibody, primarily responsible for its effector function, structural stability, and typing, and is a crucial support for antibody functional diversity. The species origin of the constant region can be cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans. The heavy chain constant region can be selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0035] Furthermore, according to the present invention, the constant region includes the human heavy chain constant region IgG1 and the light chain constant region κ type. The amino acid sequence of the human light chain constant region κ type is shown in SEQ ID NO:11, and the amino acid sequence of the human heavy chain constant region IgG1 is shown in SEQ ID NO:12; specifically: The amino acid sequence of SEQ ID NO:11 is as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The amino acid sequence of SEQ ID NO:12 is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0036] The terms "gene," "DNA," or "nucleic acid molecule" include not only double-stranded DNA but also corresponding single-stranded DNA, the sense strand and antisense strand that constitute the double-stranded DNA. It is not specifically limited by its length. Unless otherwise stated, the term "gene (DNA)" in this specification includes double-stranded DNA (including human genomic DNA), single-stranded DNA (positive strand) (including cDNA), single-stranded DNA (complementary strand) having a sequence complementary to said positive strand, and fragments thereof.

[0037] According to the present invention, examples of nucleic acid molecules include the nucleotide sequence of the variable region VL of the light chain of a monoclonal antibody encoding the above-described Yo protein, as shown in SEQ ID NO:9: ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCCATTGGGGATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATTTCCTGCAGGTCTAGTACGAGTCTCCTGCACAGTAGTGGCAAGCATAGGTTGTATTGGTTCCTACAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATTATATGTCCAACCTTGATTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATTTTGGTGTTTATTATTGTATGCAAAGTCTAGAATATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA; The nucleotide sequence encoding the variable region VH of the above-mentioned Yo monoclonal antibody heavy chain is shown in SEQ ID NO:10: ATGGGATGGAGCTGGATCTTTTTCTTCTTCCTGTCAGGAACTGCAGGTGTCCACTGTCAGGTCCAGCTGAAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAACTGTCCTGCAAGGCTTCTGGCTACATTTTCACTGACTACTATATAAACTGGGTGAAGCGGAGGCCTGGACAGGGACTTGAGTGGATTGCAAGGTTTTCTCCTGGAAGTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAAAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGGTGTCTATTTCTGTGCAACCCATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; The nucleotide sequence encoding the constant region of human Ig Kappa light chain is shown in SEQ ID NO:13: AGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG; The nucleotide sequence encoding the constant region of the human IgG1 heavy chain is shown in SEQ ID NO:14: GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA。

[0038] The nucleic acid molecules of the present invention can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example by combinatorial chemical synthesis and recombinant methods.

[0039] The term "vector molecule" refers to a class of artificially modified nucleic acid molecules that can carry exogenous nucleic acid molecules (such as target genes, antibody coding sequences, etc.) into host cells (such as bacteria, yeast, mammalian cells) and achieve the replication, expression, or integration of exogenous nucleic acid molecules within the host cells. The vector in this invention is not limited and can be an expression vector, viral vector, etc. The expression vector is a eukaryotic expression vector, for example, one or more plasmid vectors selected from the pcDNA3.1, pFUSE, and pOptiVEC series; furthermore, according to this invention, the backbone vector of the recombinant expression vector is pcDNA3.1.

[0040] The term "host cell" refers to a cell capable of accepting exogenous nucleic acid molecules (such as antibody-encoded genes) or recombinant vectors, providing the enzymes, raw materials (nucleotides, amino acids), and environmental conditions required for the replication, transcription, and translation of exogenous genes, ultimately achieving the expression of exogenous proteins. Further, according to the present invention, the recombinant cells include prokaryotic cells or eukaryotic cells; the prokaryotic cells include, but are not limited to, bacterial cells, such as *Escherichia coli*; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, animal cells, or plant cells; the yeast cells may be, but are not limited to, *Pichia pastoris* or *Saccharomyces cerevisiae* cells; the animal cells may be, but are not limited to, CHO cells, COS cells, NSO cells, 293T cells, HT-1080 cells, BHK (juvenile hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, or PERC.6 (human retinal cells).

[0041] The term "peptide" refers to a protein, protein fragment, or fragment of a polypeptide sequence or analogue. Protein fragments and analogues are considered to be species of the genus *Polypeptide*. Further, according to the present invention, examples of polypeptides include the light chain immunoglobulin molecule represented by SEQ ID NO:1, the heavy chain immunoglobulin molecule represented by SEQ ID NO:2, and CDRs represented by SEQ ID NO:3, 4, 5, 6, 7, and 8, antibody molecules formed from combinations (or vice versa) comprising heavy chain immunoglobulin molecules and light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules. Wherein, *Yo* protein refers to a polypeptide, fragment, or analogue that retains the binding capacity of *Yo* protein.

[0042] The term "amino acid" refers to the 20 common amino acids and their abbreviations used in this invention, as is the case in conventional usage.

[0043] Furthermore, according to the present invention, the method for detecting or determining the amount of protein or polypeptide can be any known immunological detection technique, including immunoagglutination assays, immunoprecipitation assays, enzyme-linked immunosorbent assays, fluorescence immunoassay, immunoelectron microscopy, immunoblotting, and immunomagnetic bead assays. Immunological detection techniques are methods for detecting or determining the amount of antibody or antigen using labeled antigens or antibodies. Biological samples that can be tested by the above-described immunological detection or determination include, but are not limited to, plasma, whole blood, dried whole blood, serum, tissues, cells, or extracts of tissues and cells.

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art, based on their understanding of the spirit of the invention, can make various modifications or substitutions, and these equivalent forms also fall within the scope defined by the appended claims.

[0045] Example 1: Preparation of recombinant human Yo protein and mouse anti-human Yo monoclonal antibody 1. Preparation of recombinant human Yo protein Based on the human Yo cDNA sequence NM_001802.2 in GenBank, Genewiz synthesized a coding gene with a 6×His tag at its C-terminus and constructed it into the pET 32a expression vector. pET 32a-Yo Recombinant vector. The constructed and correctly sequenced plasmid was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium for induction of expression. The cells were collected, sonicated, centrifuged, and the cell supernatant and precipitate were collected. 8M urea was added to the cell precipitate to denature inclusion bodies. After centrifugation, the supernatant was collected, and the mixture was purified by affinity chromatography on a Ni column. After dialysis refolding and concentration, a high concentration of the target protein was obtained. Figure 1 ), labeled as Yo-6his as an antigen for future use.

[0046] 2. Preparation of mouse anti-human Yo monoclonal antibody Immunization of mice: Five 6-8 week old female Balb / c mice were subcutaneously immunized at multiple sites using Yo-6his protein as the immunogen. The immunogen was emulsified with Freund's adjuvant at a 1:1 (v / v) ratio, and the injection volume per mouse was 100 μL (of which the antigen content was 40 μg / mouse). The primary immunization used complete Freund's adjuvant, followed by a secondary immunization 14 days later. The secondary immunization used incomplete Freund's adjuvant, followed by a third immunization 14 days later, for a total of three antigen immunizations. After the final immunization, a pulse immunization was administered. Three days later, tail vein blood was collected from the mice, and serum was separated.

[0047] Immunotherapy efficacy assay: a. Genewiz Corporation was commissioned to construct the Yo cDNA gene with a 6×His tag at the C-terminus, synthesized in step 1, into the pcDNA3.1 vector to obtain... pcDNA3.1-Yo Recombinant vector, large-scale plasmid preparation for later use; use PEI transfection reagent to... pCDNA3.1-Yo The recombinant vector and empty pCDNA3.1 were transfected into 293T adherent cells and cultured. After 48 hours, the cells were fixed with acetone to prepare Yo-overexpressing cell slides and pCDNA3.1 control slides. The prepared Yo-overexpressing cell slides were then subjected to quality control using a commercially available Yo antibody. The results are as follows: Figure 2-1 As shown in the left image, a clear and dense red fluorescence is visible, indicating that the target protein (Yo) was successfully overexpressed in the cells, with a strong fluorescence signal and clear localization. The right image shows only very weak background fluorescence, with almost no specific signal, indicating that the empty vector control cells hardly express the target protein, ruling out interference from the vector itself or non-specific binding. The results demonstrate that the prepared Yo-overexpressing cells successfully climbed the slide.

[0048] b. Immunofluorescence experiments were performed using collected mouse venous blood. Blood from 5 immunized mice was diluted 1:50, and blood from 1 normal, non-immunized mouse was diluted 1:50. These were incubated on Yo-overexpressing cell smears and pCDNA3.1 control cell smears for 1 hour at room temperature. After washing three times with PBST, 1:200 diluted Alexa Fluor 594-labeled goat anti-mouse IgG (Jackson) was added, and the mixture was incubated for 30 minutes at room temperature. After washing three times with PBST, the cells were observed under a microscope. Figure 2-2 As shown, only the serum of mouse 5 exhibited strong and specific binding to Yo-overexpressing cells, indicating that this mouse successfully produced antibodies against the Yo protein. The remaining mice (1-4) either did not produce effective antibodies or had extremely low antibody levels. Therefore, mouse 5, with the strongest serum specificity, was selected for cell fusion.

[0049] Cell fusion: Immunized mice (size 5) were dissected, spleens were isolated and ground to obtain a spleen cell suspension. Mouse myeloma cells SP2 / 0 in logarithmic growth phase and in good growth condition were fused with the obtained spleen cells at a ratio of 1:10. After centrifugation, the supernatant was discarded; PEG was added as a fusion agent, and after incubation at room temperature for a short time, serum-free DMEM medium was immediately added to terminate the fusion reaction. The fused cell suspension was seeded into 96-well plates pre-coated with macrophages and cultured in HAT medium at 37°C in a 5% CO2 incubator.

[0050] Hybridoma cell screening: Observe the growth of hybridoma cells. After 10-14 days, when the cell culture supernatant turns yellow, a suitable amount of cell supernatant is aspirated for immunofluorescence antibody detection. Equal volumes of cell supernatant from each row of 12 wells in a 96-well plate are mixed, and Tween-20 is added to a final concentration of 0.2% before pooling. Based on the immunofluorescence results, cells from the row of 12 wells corresponding to the mixed samples with strong positive signals (1H and 17G) are selected for further culture and screening of parent clones. After 7-10 days of continued culture, the cell supernatant from each well of the mixed sample with strong positive signals is individually immunofluorescently detected for antibody detection. Finally, 1H8 and 17G7 wells are selected as positive. Figure 3-1 As shown, after the first and second screenings, the 1H8 hybridoma cell line exhibited the strongest antibody-binding activity, making it the optimal choice for subsequent monoclonal antibody preparation. The selected positive wells were re-verified by changing the medium and performing limiting dilutions to culture approximately one cell per well. After 7-10 days of culture, two clones with strong positive signals were selected. Hybridoma cells 1H8 and 17G7 were further screened using ELISA, yielding positive monoclonal clones 1H8C6, 1H8D9, 17G7G2, and 17G7D12. ELISA was then used to screen the third batch of positive hybridoma cells, ultimately selecting positive monoclonal clones 1H8C6E7 and 17G7D12D9. Figure 3-2 Then, immunofluorescence was used for verification. Figure 3-3 After multiple rounds of subcloning and verification, combined with immunofluorescence and ELISA results, two hybridoma cell lines that stably secrete anti-Yo antibodies were finally obtained: 1H8C6E7 and 17G7D12D9. (The 1H8C6E7 hybridoma cell line showed the most stable and strongest antibody binding activity and was the preferred line for subsequent experiments. The 17G7D12D9 cell line also had good antibody activity and could be used as a candidate line.)

[0051] Antibody typing identification: ELISA was performed on the cell supernatant of the two monoclonal antibody strains using commercially available antibodies (IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Ig Kappachain / Ig lambda chain). The results are shown in Table 1. Both cell lines secreted IgG1 / kappa type monoclonal antibodies. The 1H8C6E7 cell line was selected for subsequent construction of human-mouse chimeric antibodies.

[0052] Table 1

[0053] 3. Hybridoma cell variable region gene sequencing Hybridoma cells were collected after reaching the logarithmic growth phase (clone number 1H8C6E7). Total RNA was extracted from the hybridoma cells, and their light and heavy chain variable region genes were amplified using RACE technology. The antibody variable region genes obtained by PCR were ligated into a T vector, transformed, and positive clones were selected for sequencing to obtain the nucleotide and amino acid sequences of their light chain variable region (VL) and heavy chain variable region (VH). The complementarity-determining regions (CDRs) were defined according to Kabat rules. The specific antibody amino acid sequences are shown in SEQ ID NO: 1-8.

[0054] Example 2: Preparation of recombinant human-mouse chimeric monoclonal antibody against human Yo protein 1. Construction of recombinant vectors Referring to sequences in the Kabat database, molecular biology methods were employed using the pEASY®-UniSeamless Cloning and Assembly Kit from TransGold to ligate the nucleotide sequence (SEQ ID NO. 9) of the VL region of the light chain of the murine monoclonal antibody 1H8C6E7 encoding the Yo protein, along with the nucleotide sequence (SEQ ID NO. 13) of the constant region of the human kappa light chain, into the vector pcDNA3.1, labeled as... pcDNA3.1-1H8C6E7-VL The nucleotide sequence SEQ ID NO.10 of the variable region VH of the heavy chain of the murine monoclonal antibody 1H8C6E7 encoding the Yo protein and the nucleotide sequence SEQ ID NO.14 of the constant region of the heavy chain of human IgG4 were ligated into the vector pcDNA3.1 and labeled as follows. pcDNA3.1-1H8C6E7-VH The ligated recombinant plasmid is sent to Sangon Biotech for sequencing, and the sequenced recombinant plasmid is then prepared for use.

[0055] 2. Purification and expression of recombinant antibodies The validated light and heavy chain expression plasmids were co-transfected at a 1:1 ratio into Expi293F cells in logarithmic growth phase for transient expression. After transfection, cells were cultured for 3 days, and the cell culture supernatant was collected by centrifugation. The cell supernatant was purified using a Protein A affinity chromatography column, eluting the target antibody with 0.1M glycine-HCl buffer (pH 3.0) and neutralizing with 1M Tris-HCl buffer (pH 8.8). The purification results are shown below. Figure 4 As shown, the molecular weight is correct, the target band is single, and the purity is good. The antibody concentration was determined to be 0.218 mg / mL using the BCA method.

[0056] Example 3 Performance Validation of Recombinant Human-Mouse Chimeric Monoclonal Antibody Against Human Yo Protein 1. Recombinant antibody titer detection The titer was detected using an ELISA method. The specific procedure was as follows: A 96-well plate was coated with Yo-6his protein at a coating volume of 100 ng / well. Purified 1H8C6E7 recombinant antibody was serially diluted and added to each well at a volume of 100 μL / well. Positive and normal mouse sera were used as positive and negative controls, respectively. The plate was then incubated with a 1:5000 dilution of HRP-labeled goat anti-human IgG secondary antibody. After TMB color development for 5 min, the OD450 value was measured. A positive result was defined as an OD value ≥ 2.1 times that of the negative control. Results ( Figure 5 The results showed that the titer of the 1H8C6E7 antibody could reach 1:121,500.

[0057] 2. Specificity verification of recombinant antibodies Nova-1, Nova-2, HuB, HuC, HuD, CV2, Ma2, CDR2L, and Amphiphysin recombinant proteins were prepared according to Example 1 and combined with the Yo protein prepared in Example 1. Each recombinant protein was immobilized on a right-angled trapezoidal NC membrane (antigen distribution as shown in Example 1). Figure 6 (As shown in the schematic diagram), the recombinant antibody 1H8C6E7 purified in Example 3 and positive mouse serum were incubated separately, diluted 1:50, and incubated at room temperature for 1 h, followed by washing twice with PBST; AP-labeled goat anti-human / goat anti-mouse IgG antibody was diluted 2000 times (Jackson Biotech) and added to each well, incubated at room temperature for 30 minutes, and washed twice with 1% NaCl; after removing the washing solution from the wells, substrate dilution buffer BCIP / NBT was added, and the strips were allowed to stand at room temperature in the dark for 4 minutes for color development; the color development was stopped by rinsing the test strips with distilled water, and the results were read after the test strips dried. The results ( Figure 6 The results show that, compared with positive mouse serum, the recombinant antibody prepared in this invention reacts only with Yo(CDR2) protein and does not react with other proteins, especially not with Yo(CDR2L) protein, thus exhibiting better specificity.

[0058] Example 4: Application of recombinant human-mouse chimeric monoclonal antibody against human Yo protein 1. Application as a positive control material Referring to Example 1, Yo-overexpressing cell slides and control slides were prepared according to the method of this invention. These were incubated with the anti-Yo protein human-mouse chimeric antibody 1H8C6E7 obtained in Example 3 and serum from anti-Yo antibody-positive patients, respectively, and incubated with FITC-labeled goat anti-human IgG secondary antibody. The results were observed and photographed under a fluorescence microscope (e.g., ...). Figure 7As shown in the figure, the anti-Yo protein human-mouse chimeric antibody can specifically recognize the overexpressed antigen of Yo, and the anti-Yo protein human-mouse chimeric antibody produces a typical fluorescent signal on Yo-overexpressing cells that is highly similar to that of positive serum, while no signal is found on control cells transfected with empty vector. This confirms that it can be used as a positive quality control for the detection of anti-Yo autoantibodies. This not only provides a stable quality control for clinical testing, but also helps to build a large-scale standardized positive image library, laying a data foundation for the development of an artificial intelligence-based automatic interpretation system for immunofluorescence images.

[0059] 2. Application as a kit for detecting Yo protein a. Components: Human-mouse chimeric monoclonal antibody against Yo protein, 4% paraformaldehyde fixative, 0.3% Triton X-100 permeabilization buffer, 5% BSA blocking buffer, antibody dilution buffer, wash buffer, Alexa Fluor 594-labeled anti-human IgG secondary antibody, and anti-fluorescence quencher. The human-mouse chimeric monoclonal antibody against Yo protein is the core component of this kit.

[0060] b. Principle: This kit uses indirect immunofluorescence. First, a human-mouse chimeric monoclonal antibody against human Yo protein specifically binds to the Yo antigen in the tissue or cell to be tested. Then, an Alexa Fluor 594-labeled anti-human IgG secondary antibody binds to the specific antibody, forming an antigen-specific antibody-labeled fluorescent antibody complex, thereby enabling the detection of Yo protein in cells or tissues.

[0061] c. How to use: Frozen section rewarming: Remove frozen sections of rat brain tissue from the freezer and rewarm for 30 minutes to prevent detachment.

[0062] Fix the sections: Fix the sections with 4% paraformaldehyde (PFA) at room temperature for 30 minutes, then wash with PBS 3 times for 5 minutes each time.

[0063] Permeation treatment: Treat with 0.3% Triton X-100 at room temperature for 30 minutes.

[0064] Blocking: Block around the section with 5% BSA for 1 hour.

[0065] Primary antibody incubation: Dilute the human-mouse chimeric monoclonal antibody against human Yo protein 1:10, add it to the slide, and incubate at room temperature for 1 hour.

[0066] Secondary antibody incubation: After washing the sections with PBS, add fluorescently labeled anti-human IgG secondary antibody and incubate at 37°C for 1 hour, taking care to avoid light.

[0067] Image review and results analysis: Results observed under a fluorescence microscope. Based on... Figure 8It can be seen that Yo protein is expressed in Purkinje cells of rat cerebellum.

[0068] d. Application: This kit can specifically detect the expression distribution of the target protein Yo in cell and tissue samples, and perform qualitative analysis of Yo protein expression levels based on fluorescence signal intensity. It can clearly present the expression characteristics and subcellular localization of Yo protein within cells, providing reliable technical support for researchers to further explore the mechanisms of Yo protein-mediated cell function regulation and the pathogenesis of related diseases.

[0069] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A monoclonal antibody against human Yo protein, characterized in that, Including light chains and heavy chains; The variable region of the light chain includes three light chain complementarity-determining regions, and the three light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 have amino acid sequences as shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; The variable region of the heavy chain includes three heavy chain complementarity-determining regions, CDR-H1, CDR-H2, and CDR-H3, which have amino acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

2. A monoclonal antibody against human Yo protein according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:9; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:

10. The constant region of the light chain is the human light chain constant region, and its amino acid sequence is shown in SEQ ID NO:11; the constant region of the heavy chain is the human heavy chain constant region, and its amino acid sequence is shown in SEQ ID NO:

12.

3. The monoclonal antibody against human Yo protein according to claim 1, characterized in that, The monoclonal antibody against human Yo protein comprises having any one of the amino acid sequences shown in (I), (II), and (III): (I) The light chain variable region sequence shown in SEQ ID NO:1 and / or the heavy chain variable region sequence shown in SEQ ID NO:2; (II) and (I) show amino acid sequences that have at least 90% homology and the same function; (III) is an amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II).

4. The monoclonal antibody against human Yo protein according to claim 1, characterized in that, The antibodies include chimeric antibodies, humanized antibodies, or murine antibodies.

5. A recombinant expression vector, characterized in that, The nucleotide sequence comprising a monoclonal antibody expressing an anti-human Yo protein as described in any one of claims 1-4.

6. A host cell, characterized in that, The host cell includes the vector as described in claim 5.

7. The use of any one of the monoclonal antibodies against human Yo protein according to any one of claims 1-4, the recombinant expression vector according to claim 5, or the host cell according to claim 6 in the preparation of products for detecting Yo protein, identifying Yo protein, and detecting Yo protein autoantibodies.

8. A product for detecting Yo protein, characterized in that, include: The detection reagent or kit contains at least one of the monoclonal antibodies against human Yo protein according to any one of claims 1-4, the recombinant expression vector according to claim 5, and the host cell according to claim 6.

9. A product for detecting Yo protein according to claim 8, characterized in that, The kits include immunofluorescence detection kits, blot detection kits, ELISA detection kits, flow cytometry kits, or IHC detection kits.

10. A method for detecting Yo protein or polypeptide, characterized in that, This includes contacting a biological sample with a monoclonal antibody against human Yo protein according to any one of claims 1-4, and detecting Yo protein or polypeptide in the sample; The biological samples include plasma, whole blood, serum, tissues, cells, or tissue and cell lysates containing Yo protein / peptide; The detection methods include enzyme-linked immunosorbent assay (ELISA), cell-based immunofluorescence assay, tissue-based immunofluorescence assay, dot blot assay, chemiluminescent immunoassay, radioimmunoassay, colloidal gold immunochromatography, and other non-enzyme-linked antibody binding methods.

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