Human-mouse chimeric monoclonal antibody for resisting human LGI1 protein as well as product and application of human-mouse chimeric monoclonal antibody

Abstract

The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a human-mouse chimeric monoclonal antibody for resisting human G protein-coupled receptor family member 1 (LGI1 protein) as well as a product and application of the human-mouse chimeric monoclonal antibody. The variable regions of the heavy chain and the light chain of the human-mouse chimeric monoclonal antibody of the anti-human LGI1 protein come from mice, and the constant regions of the heavy chain and the light chain of the human-mouse chimeric monoclonal antibody are human antibody constant regions, so that the human-mouse chimeric monoclonal antibody can replace the function of serum to a certain extent on the basis of retaining parent activity to the greatest extent; meanwhile, the technical problems that the Fc region of the existing anti-human LGI1 protein monoclonal antibody cannot react with the anti-human IgG antibody, the sensitivity difference among multiple batches of antibodies is large, and batch production cannot be realized can be solved. Furthermore, the human-mouse chimeric monoclonal antibody of the anti-human LGI1 protein can be used for detecting the LGI1 protein and an LGI1 autoantibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a human-mouse chimeric monoclonal antibody against human LGI1 protein and a product and application thereof. Background Art

[0002] LGI1, or leucine rich gliomainactivated 1 (LGI1), is a protein secreted by neurons, mainly present in the limbic system such as the hippocampus, amygdala, and temporal cortex. It plays a vital role in brain development, maintaining neuronal excitability, and synaptic transmission, and is closely related to neurological diseases. LGI1 deficiency can cause disordered stratification of neurons in the cerebral cortex and reduce the thickness of the cerebellar granule cell layer in the embryonic period. More importantly, LGI1 has been shown to be associated with central nervous system myelination and oligodendrocyte differentiation. Previous studies have shown that LGI1 can play a regulatory role in glutamate synaptic plasticity and glutamate transmission in the hippocampus. In addition, LGI1 autoantibodies interfere with the interaction of synaptic proteins and cause a series of clinical manifestations, such as cognitive impairment, mental and behavioral abnormalities, and sleep disorders. Anti-LGI1 antibodies have become one of the markers of autoimmune encephalitis.

[0003] In the detection system of autoantibodies, reference products are an important part of the quality control of the detection system. Usually, the positive serum of patients can be used as a positive reference product, but the cost of obtaining positive serum is high and it is difficult to determine the value and trace the source. Therefore, it is necessary to provide a positive reference product that can replace the positive serum of patients. Chimeric antibodies or humanized antibodies can meet such needs.

[0004] Currently available commercial LGI1 antibodies are rabbit / mouse polyclonal antibodies, which: (1) inevitably have differences between different batches, and cannot guarantee the same sensitivity between batches; (2) the antibody sequence is not public, and cannot be formed into low-cost, large-scale industrial batch production; (3) its Fc region is mouse / rabbit sequence, which cannot react with anti-human IgG antibodies. Therefore, the development of new monoclonal antibodies against LGI1 protein is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a human-mouse chimeric monoclonal antibody against human LGI1 protein and its product and application. The human-mouse chimeric monoclonal antibody against human LGI1 protein has human properties and can be directly used as a substitute for positive patient serum, thereby solving the technical problems that the Fc region of the existing anti-human LGI1 protein monoclonal antibody cannot react with anti-human IgG antibodies, the sensitivity differences between multiple batches of antibodies are large, and batch production is impossible.

[0006] The present invention provides a human-mouse chimeric monoclonal antibody against human LGI1 protein, comprising a light chain and a heavy chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain variable region comprises a light chain complementary determining region CDR1, a light chain complementary determining region CDR2 and a light chain complementary determining region CDR3, and the amino acid sequences of the light chain complementary determining region CDR1, the light chain complementary determining region CDR2 and the light chain complementary determining region CDR3 are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; the light chain constant region is a human light chain constant region;

[0007] The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO:8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO:9 or SEQ ID NO:13, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO:10 or SEQ ID NO14; the heavy chain constant region is a human heavy chain constant region.

[0008] Preferably, the light chain variable region comprises the amino acids shown in SEQ ID NO: 2;

[0009] The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.

[0010] Preferably, the human-mouse chimeric monoclonal antibody against human LGI1 protein comprises an amino acid sequence shown in (I) or (II):

[0011] (I) the light chain variable region sequence shown in SEQ ID NO: 2, and the heavy chain variable region sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12;

[0012] (II) an amino acid sequence having the same function as that shown in (I) by substituting, deleting or adding one or more amino acids;

[0013] (III) an amino acid sequence having at least 50% homology with the amino acid sequence shown in (I) and having sequence identity or similarity with (I); said (III) does not include the amino acid sequence shown in (II).

[0014] The present invention also provides a biomaterial, comprising one or more of 1) to 3):

[0015] 1) A nucleic acid molecule expressing the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution;

[0016] 2) a recombinant vector, wherein the recombinant vector comprises the nucleic acid molecule described in 1);

[0017] 3) an engineered cell, the engineered cell comprising the nucleic acid molecule described in 1) or the recombinant vector described in 2);

[0018] Preferably, the recombinant vector comprises a light chain recombinant vector and / or a heavy chain recombinant vector;

[0019] The light chain recombinant vector comprises a nucleic acid molecule expressing a light chain variable region and a nucleic acid molecule expressing a light chain constant region;

[0020] The heavy chain recombinant vector comprises a nucleic acid molecule expressing a heavy chain variable region and a nucleic acid molecule expressing a heavy chain constant region.

[0021] Preferably, the nucleic acid molecule expressing the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 1;

[0022] The nucleic acid molecule expressing the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:11.

[0023] The present invention also provides an antibody derivative, which is a complex obtained by modifying the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution.

[0024] The present invention also provides the use of the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution, the biological material described in the above technical solution or the antibody derivative described in the above technical solution in the preparation of one or more products for detecting LGI1 protein, identifying LGI1 protein and detecting LGI1 autoantibodies.

[0025] The present invention also provides a product, which comprises one or more of the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution, the biological material described in the above technical solution, and the antibody derivative described in the above technical solution.

[0026] Preferably, the product is prepared based on immunoassay technology.

[0027] The present invention also provides a method for detecting LGI1 protein, which is based on immunological detection technology to detect the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution; the method is a method for non-diagnostic and therapeutic purposes.

[0028] Beneficial effects:

[0029] The present invention provides a human-mouse chimeric monoclonal antibody against human LGI1 protein, wherein the variable regions of the heavy chain and light chain of the human-mouse chimeric monoclonal antibody against human LGI1 protein are from mice, and the constant region is a human antibody constant region, which can replace the function of serum to a certain extent while retaining the parent activity to the greatest extent; at the same time, it can solve the technical problems that the Fc region of the existing anti-human LGI1 protein monoclonal antibody cannot react with the anti-human IgG antibody, the sensitivity difference between multiple batches of antibodies is large, and batch production is not possible. Furthermore, the human-mouse chimeric monoclonal antibody against human LGI1 protein can detect LGI1 protein and LGI1 autoantibodies, such as visualization and positioning, and judge the expression and positioning of LGI1 protein; the monoclonal antibody of LGI1 protein can also be used as a ligand in the affinity layer system to achieve the separation and purification of LGI1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0031] Figure 1 This is a diagram showing the purification results of the LGI1 protein in Example 1;

[0032] Figure 2 The titer of serum antibodies of immunized mice was identified by CBA method in Example 1;

[0033] Figure 3 The CBA test results of the three positive clones screened in Example 1;

[0034] Figure 4 The ELISA test results of the three positive clones screened in Example 1 are as follows;

[0035] Figure 5 The immunofluorescence staining results of the human-mouse chimeric monoclonal antibody 48C10 and the human-mouse chimeric monoclonal antibody 48C10-1 in Example 4;

[0036] Figure 6 The immunofluorescence staining results of human-mouse chimeric monoclonal antibodies 48C10 and 48C10-1 and anti-LGI1 positive blood in Example 5 are shown. DETAILED DESCRIPTION

[0037] The present invention provides a human-mouse chimeric monoclonal antibody against human LGI1 protein, comprising a light chain and a heavy chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain variable region comprises a light chain complementary determining region CDR1, a light chain complementary determining region CDR2 and a light chain complementary determining region CDR3, and the amino acid sequences of the light chain complementary determining region CDR1, the light chain complementary determining region CDR2 and the light chain complementary determining region CDR3 are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; the light chain constant region is a human light chain constant region;

[0038] The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO:8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO:9 or SEQ ID NO:13, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO:10 or SEQ ID NO14; the heavy chain constant region is a human heavy chain constant region.

[0039] In the present invention, the amino acid sequences of the three light chain complementary determining regions of the light chain variable region are as follows: light chain complementary determining region CDR1: KASENVYTYVS (SEQ ID NO: 3); light chain complementary determining region CDR2: GASSRYT (SEQ ID NO: 4); light chain complementary determining region CDR3: GQTYSYPYT (SEQ ID NO: 5).

[0040] As one embodiment, the light chain variable region includes the amino acids shown in SEQ ID NO:2; as another embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:2, specifically: NIVMTQSPKCMSMSVGERV TLSCKASENVYTYVSWYQQKPEQSPKMLMYGASSRYTGVPDRFTGSGSATDFTLTISSVQAEDL ADYFCGQTYSYPYTFGSGTMLEIK; wherein the bold parts are three light chain complementary determining regions.

[0041] As one embodiment, the nucleotide sequence encoding the light chain variable region includes the nucleotide sequence shown in SEQ ID NO:1; as another embodiment, the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:1, specifically: 5'-AACATTGTGATGACCCAATCTCCCAAATGCATGTCCATGTCAGTAGGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTCTATACTTATGTATCCTGGTATCAACAGAAACCAGAGCAGTCTCCAAAAATGCTGATGTACGGGGCATCCAGCCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATTTCTGTGGACAGACTTACAGCTATCCGTATACGTTCGGATCGGGACCATGCTGGAAATAAAA-3'.

[0042] As an embodiment, the light chain constant region of the present invention is a human kappa type light chain constant region. In a specific embodiment, the amino acid sequence of the human kappa type light chain constant region is shown in SEQ ID NO: 16, specifically RTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC*; the nucleotide sequence encoding the human kappa type light chain constant region shown in SEQ ID NO: 16 is shown in SEQ ID NO: 15, specifically: 5'-AGAACTGTGGCTGCACCATCTGTCTTCATCTT CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3'.

[0043] In the present invention, the amino acid sequences of the three heavy chain complementary determining regions of the heavy chain variable region are as follows: heavy chain complementary determining region CDR1: NYGLS (SEQ ID NO: 8); heavy chain complementary determining region CDR2: EVYPRTGNTYYNEKFKG (SEQID NO: 9) or EVYPRSGNTYYNEKFKG (SEQ ID NO: 13); heavy chain complementary determining region CDR3: SLYYDYGWYFDV (SEQID NO: 10) or SFYYDYGWYFDV (SEQ ID NO: 14); that is, the heavy chain complementary determining region CDR2 shown in SEQ ID NO: 13 is obtained by replacing the amino acid T at position 6 of the heavy chain complementary determining region CDR2 shown in SEQ ID NO: 9 with amino acid S; the heavy chain complementary determining region CDR3 shown in SEQ ID NO: 14 is obtained by replacing the amino acid L at position 2 of the heavy chain complementary determining region CDR3 shown in SEQ ID NO: 10 with amino acid F.

[0044] As one embodiment, the heavy chain variable region of the present invention includes an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:12; as another embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or SEQ ID NO:12: specifically: QVQLQQSGAELARPGASVKLSCTASGYTFTNYGLSWVKQRTGQGLEWIGEVYPRTGNTYYNEKFKGKATLTADKSSRTAYMELRSLTSEDSAVYFCARSLYYDYGWYFDVWGA GTTVTVSS (SEQID NO:7) and QVQLQQSGAELARPGASVKLSCTASGYTFTNYGLSWVKQRTG QGLEWIGEVYPRSGNTYYNEKFKGKATLTADKSSRTAYMELRSLTSEDSAVYFCARSFYYDYG WYFDVWGAGTTVTVSS (SEQ ID NO:12); wherein the bold parts are three heavy chain complementary determining regions.

[0045] As one embodiment, the nucleotide sequence encoding the heavy chain variable region of the present invention includes the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:11; as another embodiment, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO:6 or SEQ ID NO:11, specifically: 5'-CAGGTTCAACTGCAGCAGTCTGGAGCTGAGCTGGCGA GGCCTGGGGCTTCAGTGAAGCTGTCCTGCACGGCTTCTGGCTACACCTTCACAAACTATGGTTTAAGTTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGGTTTATCCTCGAACAGGTAATACTTACTACAATGAGAAATTCAAGGGCAAGGC CACACTGACTGCAGACAAATCCTCCAGAACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCTCTCTACTATGATTACGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA-3'(SEQ ID NO:6) and 5'-CAGGTTCAACTGCAGCAGTCTGGAGCTGAGCTG GCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCACGGCTTCTGGCTACACCTTCACAAACTATGGTTTAAGTTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGGTTTATCCTCGAAGTGGTAATACTTACTACAATGAGAAATTCAAGGGCAAG GCCACACTGACTGCAGACAAATCCTCCAGAACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCTTTCTACTATGATTACGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA-3'(SEQ ID NO:11).

[0046] As an embodiment, the heavy chain constant region of the present invention is a human IgG1 heavy chain constant region. In a specific embodiment, the amino acid sequence of the human IgG1 heavy chain constant region is as shown in SEQ ID NO: 18, specifically: ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*; The nucleotide sequence of the human IgG1 heavy chain constant region shown in NO:18 is shown in SEQ ID NO:17, specifically: 5'-GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA-3'。

[0047] As an embodiment, the human-mouse chimeric monoclonal antibody against human LGI1 protein comprises an amino acid sequence shown in (I) or (II):

[0048] (I) the light chain variable region sequence shown in SEQ ID NO: 2, and the heavy chain variable region sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12;

[0049] (II) an amino acid sequence having the same function as that shown in (I) by substituting, deleting or adding one or more amino acids;

[0050] (III) an amino acid sequence having at least 50% homology with the amino acid sequence shown in (I) and having sequence identity or similarity with (I); said (III) does not include the amino acid sequence shown in (II).

[0051] As an embodiment, the functional variants of the human-mouse chimeric monoclonal antibodies against human LGI1 protein described in the present invention are also included in the protection scope of the present invention, i.e., the amino acid sequence shown in (III); the "functional variant" refers to a protein having obvious or significant sequence identity or similarity compared to the parent antibody (i.e., the human-mouse chimeric monoclonal antibodies against human LGI1 protein of the present invention), and the functional variant retains the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the antibodies (parent antibodies) described herein, which retain the ability to recognize the target protein to a similar degree, to the same degree, or to a higher degree than the parent antibody. With reference to the parent antibody, the functional variant may, for example, have at least about 50%, 70%, 75%, 80%, 85%, 90%, 95% or higher homology with the parent antibody in terms of amino acid sequence.

[0052] As another embodiment, conservative amino acid substitutions are known in the art and include substitutions of one amino acid having specific physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may be substitutions of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a non-polar side chain with another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of an alkaline / positively charged polar amino acid with another alkaline / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.).

[0053] As another embodiment, a person skilled in the art can easily mutate the nucleotide sequence corresponding to the human-mouse chimeric monoclonal antibody against human LGI1 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 90% or more homology with the nucleotide sequence corresponding to the antibody or antigen-binding fragment thereof of the present invention, as long as they encode the above-mentioned antibody of the present invention, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, and are also included in the protection scope of the present invention.

[0054] The present invention also provides a biomaterial, comprising one or more of 1) to 3):

[0055] 1) A nucleic acid molecule expressing the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution;

[0056] 2) a recombinant vector, wherein the recombinant vector comprises the nucleic acid molecule described in 1);

[0057] 3) An engineered cell, wherein the engineered cell comprises the nucleic acid molecule described in 1) or the recombinant vector described in 2).

[0058] As an embodiment, the backbone vector of the recombinant vector of the present invention includes a plasmid or a viral vector; as another embodiment, the backbone vector of the recombinant vector can be a plasmid vector; as another embodiment, the plasmid vector is but not limited to pcDNA3.1, pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT , pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K and one or more of pPIC3.5K; in a specific embodiment, the backbone vector of the recombinant vector is pcDNA3.1.

[0059] As an embodiment, the recombinant vector includes a light chain recombinant vector and / or a heavy chain recombinant vector.

[0060] As an embodiment, the nucleic acid molecule may comprise natural, non-natural or altered nucleotides. In some specific embodiments, the nucleic acid molecule does not comprise any insertion, deletion, inversion and / or replacement. However, in other specific embodiments, it may be suitable for the nucleic acid molecule to comprise one or more insertions, deletions, inversions and / or replacements, and therefore, the nucleic acids formed by these insertions, deletions, inversions and / or replacements are equally within the scope of the present invention.

[0061] As one embodiment, the light chain recombinant vector comprises a nucleic acid molecule expressing a light chain variable region and a nucleic acid molecule expressing a light chain constant region; as another embodiment, the light chain recombinant vector comprises the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:15.

[0062] As one embodiment, the heavy chain recombinant vector comprises a nucleic acid molecule expressing a heavy chain variable region and a nucleic acid molecule expressing a heavy chain constant region; as another embodiment, the heavy chain recombinant vector comprises the nucleotide sequence shown in SEQ ID NO:6 and SEQ ID NO:17, or comprises the nucleotide sequence shown in SEQ ID NO:11 and SEQ ID NO:17.

[0063] As one embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell; as another embodiment, the prokaryotic cell includes but is not limited to a bacterial cell, such as Escherichia coli; as another embodiment, the eukaryotic cell includes but is not limited to a yeast cell, an insect cell, an animal cell or a plant cell; as another embodiment, the yeast cell may be but is not limited to a Pichia pastoris or a Saccharomyces cerevisiae cell; the insect cell may be but is not limited to a CHO cell, a COS cell, a NSO cell, a 293T cell, a HT-1080 cell, a BHK (baby hamster kidney cell), a HEK (human embryonic kidney cell), an Expi293F or a PERC.6 (human retinal cell).

[0064] As one embodiment, the method of introducing the recombinant vector into the host cell includes physical methods, chemical methods or biological methods; as another embodiment, the physical method includes calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection or electroporation; as another embodiment, the chemical method includes a colloidal dispersion system or a lipid-based system; as another embodiment, the biological method includes the use of a DNA vector, a lentiviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector or an adeno-associated virus vector.

[0065] The present invention also provides an antibody derivative, which is a complex obtained by modifying the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution. As an embodiment, the modification includes conjugation modification with a detectable marker; as another embodiment, the detectable marker includes one or more of a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold or a colored latex.

[0066] The present invention also provides the use of the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution, the biological material described in the above technical solution and the antibody derivative described in the above technical solution in the preparation of products for detecting LGI1 protein, identifying LGI1 protein and detecting LGI1 autoantibodies.

[0067] The present invention also provides a product, which comprises one or more of the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution, the biological material described in the above technical solution, and the antibody derivative described in the above technical solution. As an embodiment, the product can be a kit; as another embodiment, the kit can be a kit prepared based on immunoassay technology, such as an immunofluorescence detection kit, a blotting detection kit, an ELISA detection kit, a flow sorting kit, or an IHC detection kit.

[0068] The present invention also provides a method for detecting LGI1 protein, which is based on immunological detection technology and the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the above technical solution for detection; the method is a method for non-diagnostic treatment purposes. As an embodiment, the immunological detection technology can be radioimmunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, chemiluminescent-labeled immunoassay, colloidal gold-labeled immunoassay or colored latex-labeled immunoassay. As an embodiment, the biological samples that can be tested by the above immunoassay or assay include but are not limited to plasma, whole blood, serum, tissue, cells and cell extracts, and tissue cell extracts.

[0069] As an embodiment, the enzyme-labeled immunoassay includes but is not limited to indirect immunofluorescence, direct ELISA or dot blot (linear blot). In a specific embodiment, the steps of the enzyme-labeled immunoassay can be: adsorbing human LGI1 protein or polypeptide (antigen) onto a solid phase support NC membrane, blocking the solid phase with a blocking protein (e.g., skim milk powder or bovine serum albumin), contacting and incubating a human-mouse chimeric monoclonal antibody (test antibody) against human LGI1 protein with the solid phase, removing unreacted antibodies, and adding a labeled second antibody that specifically reacts with the test antibody to the solid phase to determine the amount of the label on the solid phase.

[0070] Technical terms:

[0071] "Antibody" refers to any form of an antibody that exhibits the desired biological activity. Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized monoclonal antibodies, fully humanized monoclonal antibodies, single-chain antibodies, or portions thereof that are capable of binding to their antigens, such as Fab fragments or fragments produced from Fab expression libraries, etc.

[0072] "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes.

[0073] Based on the variable region amino acid sequence contained in a given antibody or fragment thereof of the present invention, those skilled in the art can routinely determine the CDR contained therein. For example, the CDR in the variable region amino acid sequence is defined using the Kabat scheme, the AbM scheme, the Chothia scheme or the Contact scheme.

[0074] When referring to antibodies defined by specific CDR sequences defined herein, the scope of the antibodies also covers antibodies whose variable region sequences comprise the specific CDR sequences but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0075] The CDR of the antibody of the present invention can be manually assessed to determine the boundaries according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0076] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0077] Example 1

[0078] Preparation of mouse monoclonal antibodies

[0079] Step 1 Preparation of recombinant LGI1 protein

[0080] The gene sequence of human LGI1 was found from the GenBank sequence database, with the sequence number AF055636.1. The gene sequence was synthesized by Jinweizhi Company into the pET-28a vector, with the insertion site between HindIII and Xho I, to obtain the pET-28a-LGI1 recombinant vector. The constructed and correctly sequenced recombinant plasmid was transferred into Escherichia coli Arctic Express (Agilent) expression competent cells; a single clone was picked and inoculated into LB medium, and the culture was shaken at 37°C until the OD 600 The value was 0.5 to 1, IPTG was added at a final concentration of 0.5 mM for induction, and the cells were expressed at 16°C overnight; the cells were collected, ultrasonically disrupted, and the supernatant was collected after centrifugation. The LGI1 protein expressed by pET-28a was purified by Ni-NTA (QIAGEN) to obtain the LGI1 recombinant protein, and SDS-PAGE was performed. The results showed that the LGI1 protein that can be used to immunize mice was successfully prepared in this example. The results are as follows Figure 1 (Marker was purchased from Thermo Company, catalog number 26616).

[0081] Step 2 Preparation of cell slides overexpressing LGI1 protein

[0082] By molecular biological methods, the LGI1 gene was inserted into pCDNA3.1 with the insertion sites of Nhe I and Not I to obtain a recombinant plasmid; the recombinant plasmid was transfected into 293T cells with a cell density of 30% to 40% using PEI transfection reagent (the transfection reagent was purchased from thermo company, and the transfection was performed according to the instructions) (a 6cm×6cm cell slide had been placed in advance in a 10cm cell culture dish), 48h after transfection, acetone was added and fixed at 4°C for 5min, washed twice with PBS, and after drying, the 6cm×6cm cell slide was cut into a size of 0.25cm×0.25cm for use, and the cell slide transfected with empty pCDNA3.1 was used as a control, and its preparation method was the same as that of the cell slide overexpressing the LGI1 protein.

[0083] Step 3: Mouse antigen immunization

[0084] The LGI1 recombinant protein prepared in step 1 was used as an antigen to immunize five female Balb / c mice aged 6-8 weeks, and four immunizations were performed in total. In the first immunization, the antigen was mixed with equal volumes of Freund's adjuvant, fully ground into a water-in-oil emulsion, and 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally; two weeks after the first immunization, the second immunization was carried out by mixing 40 μg antigen with incomplete Freund's adjuvant in a volume ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle; the third immunization was carried out two weeks after the second immunization, and the dosage, method, and route of antigen injection were the same as those of the second immunization. After an interval of 2 weeks, 100 μL (40 μg) of antigen without adjuvant was injected intraperitoneally for boosting immunization. Three days later, the tail blood was collected and its titer was detected by indirect immunofluorescence assay (CBA). The specific detection process was as follows: the collected tail blood was diluted 20 times and 100 times with PBS respectively and incubated on the cell slides overexpressing LGI1 protein obtained in step 2 as the primary antibody, incubated at room temperature for 1 hour, and washed 3 times with PBST, each time for 5 minutes; incubated with Alexa The cells were incubated with goat anti-mouse secondary antibody labeled with 594 (Jackson, product number 115-585-146) for 40 min, washed 3 times with PBST, 5 min each time, and photographed under a fluorescence microscope. The results are shown in the figure. Figure 2 As shown, Figure 2 The scale bar is 150 μm.

[0085] Depend on Figure 2 It can be concluded that the cell slides overexpressing LGI1 protein combined with antibodies in the tail blood of 5 mice to produce signals. Figure 2 Based on the test results in the comprehensive serum antibody titer, mouse No. 2 was selected for subsequent experiments.

[0086] Step 4 Cell fusion and hybridoma culture

[0087] The antigen was injected intraperitoneally to strengthen the immunization, and the animals were killed 3 days later to obtain spleen cells for cell fusion. 7SP2 / 0 cells and spleen cells in logarithmic growth phase and in good growth state were washed 3 times with serum-free culture medium (37°C), and the active cells were counted (not less than 90%); after mixing myeloma cells and spleen cells at a ratio of 1:10, the supernatant was discarded by centrifugation, and the excess supernatant was absorbed with sterile filter paper; 1mL of 40% PEG solution was added drop by drop to the cell mass within 60 seconds, and the centrifuge tube was gently rotated or flicked with fingers; 1mL of serum-free culture medium was added to the rotating centrifuge tube within 60 seconds; then 20mL of serum-free culture medium was slowly added within 5 minutes; centrifuged (800 rpm, 8 minutes), the supernatant was removed, and 10mL of complete culture medium was used to suspend and gently mix; a 96-well plate was taken, and 50 microliters of cell suspension was added to each well; the plate was placed in a 5% CO2 incubator and cultured at 37°C, and replaced with HAT selective culture medium (thermo, catalog number: 21060017) after 24 hours.

[0088] Step 5 Screening of positive hybridomas

[0089] 7 to 10 days after fusion, the medium is replaced with HAT selective culture medium in a variable volume, and then half the volume is replaced every 2 to 3 days; after 2 to 3 weeks, hybrid cell colonies appear, and the cells are large, round and transparent; when the colonies proliferate and grow to 1 / 3 of the well, the CBA method is used for antibody detection; cells with positive signals are cloned and cultured using the limiting dilution method, and a second screening is performed. After repeating 3 times, the CBA method is used to identify positive monoclonal clones. The CBA method is used to screen the cell slides overexpressing LGI1 prepared in step 2. The specific detection process is the same as the detection process described in step 3. The incubated hybridoma cell supernatant does not need to be diluted during the detection. For the screening results of anti-LGI1 antigen positive hybridomas, see Figure 3 ,in Figure 3 The results are observed under a 10x microscope.

[0090] Depend on Figure 3 It can be concluded that after three rounds of screening, three positive clone cells were screened out and named as 21D12, 33H8 and 48C10 respectively.

[0091] ELISA was used to verify and select positive clones. The detection steps were selected in the usual way, specifically: the LGI1 recombinant protein purified in step 1 was coated with carbonate buffer at pH 9.6 at 4°C overnight, 100 ng / well. The supernatant of the three positive hybridomas screened by CBA was diluted at 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, and then the titer was detected by ELISA. At the same time, the supernatant of cells transfected with empty pCDNA3.1 was used as a negative control, and the blood of No. 2 mouse was used as a positive control. The dilution multiple was used as the horizontal axis, and the OD value of the detection was used as the horizontal axis. 450 The value is the vertical axis, and the result is as follows Figure 4shown.

[0092] Depend on Figure 4 It can be seen that LGI1 protein can bind to the antibodies in the supernatants of hybridomas 21D12, 33H8, and 48C10 but not react with the supernatants of cells transfected with empty pCDNA3.1. Therefore, the antibodies secreted by hybridomas 21D12, 33H8, and 48C10 can not only recognize LGI1 protein expressed in eukaryotic cells, but also recognize prokaryotic LGI1 protein coated in ELISA wells. 450 The 48C10 with the highest value was used for subsequent experiments.

[0093] Step 6 Identification of antibody typing

[0094] The identification of antibody typing was performed by indirect ELISA. The specific process was as follows: overnight coating of purified LGI1 protein (100 ng / well), 2% BSA blocking, incubation with the hybridoma 48C10 supernatant obtained in step 5 at 37°C for 1 hour, washing 3 times with PBST, and then incubation with different types of HRP-labeled rabbit anti-mouse (total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, Ig kappa chain, Ig lambda chain) secondary antibodies diluted 1:5000 at 37°C for 30 minutes, washing 3 times with PBST, and then TMB color development for 5 minutes. The indirect ELISA results showed that the heavy chain subtype of 48C10 was IgG1, and the light chain subtype was kappa.

[0095] Example 2

[0096] Identification of the sequence of mouse monoclonal antibody against LGI1

[0097] The positive hybridoma 48C10 was cultured to a certain scale (cell number>3×10 6 After that, the cells were lysed with Trizol, and the total RNA of the hybridoma cell lysate was extracted using the Quick-RNA MicroPrep Kit. The RNA was separated by agarose gel electrophoresis, and then 5'RACE was performed using the Clontech SMARTerRACE 5' / 3'kit to obtain cDNA, which was used as a template. Max DNA Polymerase was used for PCR amplification to extract the antibody V region cDNA, which was sequenced by a sequencing company, and the CDR region of the antibody amino acid sequence was marked using the Kabat method. The nucleotide sequence of the light chain variable region of the 48C10 antibody is shown in SEQ ID NO: 1, and the nucleotide sequence of the heavy chain variable region of the 48C10 antibody is shown in SEQ ID NO: 6.

[0098] Example 3

[0099] Construction of recombinant vector of human-mouse chimeric monoclonal antibody against LGI1

[0100] 1. Construction of recombinant vector

[0101] According to the nucleotide sequences of the light chain variable region VL and the heavy chain variable region VH of the 48C10 antibody obtained in Example 2, the nucleotide sequence encoding the light chain variable region VL of the 48C10 antibody (shown in SEQ ID NO: 1), the nucleotide sequence encoding the heavy chain variable region VH of the 48C10 antibody (SEQ ID NO: 6), the human kappa light chain constant region sequence SEQ ID NO: 15 and the human IgG1 heavy chain constant region sequence SEQ ID NO: 16 were amplified by molecular biology methods using Fast Pfu DNA Polymerase (Full Formula Gold, AP221-01), and the nucleotide sequence of the light chain variable region VL of the 48C10 antibody and the human kappa light chain constant region sequence SEQ ID NO: 15 were ligated to the vector pcDNA3.1 using a homologous recombination seamless cloning kit (Full Formula Gold, CU201), and the vector linearization site was Not I (the connection order is the variable region of the antibody first, then the constant region, the same below), named pcDNA3.1-48C10-VL; the nucleotide sequence of the heavy chain variable region VH of the 48C10 antibody SEQ ID NO:6 and the human IgG1 heavy chain constant region sequence SEQ ID NO:16 were connected to the vector pcDNA3.1, the vector linearization site was NotI, named pcDNA3.1-48C10-VH, the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing, the sequenced recombinant plasmid was collected for standby use, the PCR amplification system, amplification procedure and recombination system can be selected according to the instructions; the amplification primers of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:6 and SEQ ID NO:16 are as follows:

[0102] SEQ ID NO: 1-F: 5'-TGCAGATATCCAGCACAGTGGCGGCCGCAACATTGTGATGACCCAAT C-3' (SEQ ID NO: 19);

[0103] SEQ ID NO: 1-R: 5'-AGACAGATGGTGCAGCCACAGTTCTTTTTATTTCCAGCATGGTCC-3' (SEQ ID NO: 20);

[0104] SEQ ID NO: 15-F: 5'-ATCGGGGACCATGCTGGAAATAAAAAGAACTGTGGCTGCACCATC-3' (SEQ ID NO: 21);

[0105] SEQ ID NO: 15-R: 5'-AACGGGCCCTCTAGACTCGACGCCGGCGCTAACACTCTCCCCTGTT GA-3' (SEQ ID NO: 22);

[0106] SEQ ID NO: 6-F: 5'-TGCAGATATCCAGCACAGTGGCCAGGTTCAACTGCAGCAGTC-3' (SEQ ID NO: 23);

[0107] SEQ ID NO: 6-R: 5'-AGACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGT-3' (SEQ ID NO: 24);

[0108] SEQ ID NO: 16-F: 5'-AGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATC-3' (SEQ ID NO: 25);

[0109] SEQ ID NO: 16-R: 5'-AACGGGCCCTCTAGACTCGACGTCATTTACCCGGAGACAGGGA-3' (SEQ ID NO: 26);

[0110] 2. Construction of mutants

[0111] By conventional methods of molecular biology, pcDNA3.1-48C10-VH was used as a template, and Fast MultiSite Mutagenes is System (full gold, FM201-01) was used to mutate some amino acids in the VH region of pcDNA3.1-48C10-VH. The sixth amino acid T in the CDR2 region of the heavy chain variable region VH of the 48C10 antibody was mutated to S, and the corresponding nucleotide sequence was mutated from ACA to AGT. The second amino acid L in the CDR3 region of the heavy chain variable region of the 48C10 antibody was mutated to F, and the corresponding nucleotide sequence was mutated from CTC to TTC. The nucleotide sequence of the mutated heavy chain variable region VH is shown in SEQ ID NO.11, and the mutated recombinant vector is marked as pcDNA3.1-48C10-1-VH. The PCR amplification system, amplification procedure and recombination reaction system can be selected according to the instructions. The connected recombinant plasmid is sent to Sangon Biotechnology for sequencing, and the sequenced recombinant plasmid is prepared for use. The mutation primer sequence is as follows:

[0112] SEQ ID NO:11-F1:5'-TATCCTCGAAGTGGTAATACTTACTACAATGA-3' (SEQ ID NO:27);

[0113] SEQ ID NO: 11-R1: 5'-TCATAGTAGAAAGATCTTGCACAGAAATAGA-3' (SEQ ID NO: 28);

[0114] SEQ ID NO: 11-F2: 5'-TGCAAGATCTTTCTACTATGATTACGGCTGG-3' (SEQ ID NO: 29);

[0115] SEQ ID NO: 11-R2: 5'-AAGTATTACCACTTCGAGGATAAACCTCTCCA-3' (SEQ ID NO: 30).

[0116] Example 4

[0117] Preparation of human-mouse chimeric antibody against LGI1

[0118] The following two groups of recombinant vectors were transferred into Expi293F cells (thermo) using the transfection reagent PEI (Aupumai): Group 1 was co-transfected with the recombinant plasmid pcDNA3.1-48C10-VL and the recombinant plasmid pcDNA3.1-48C10-VH, and the purified antibody was named human-mouse chimeric antibody 48C10; Group 2 was co-transfected with the recombinant plasmid pcDNA3.1-48C10-VL and the recombinant plasmid pcDNA3.1-48C10-1-VH, and the purified antibody was named human-mouse chimeric antibody 48C10. The obtained antibody was named human-mouse chimeric antibody 48C10-1; Expi293F cells were cultured using OPM medium (Cat. No.: P82019), and plasmid transfection and subsequent cell culture processes were carried out according to the instructions of the OPM-CD293 transient transfection system. On the third day after transfection, the culture supernatant was collected by centrifugation as the primary antibody and incubated on cells overexpressing LGI1. The secondary antibody was incubated with FITC-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No.: 109-095-170). The results are shown in Figure 2. Figure 5 As shown, Figure 5 The scale of the figures with rulers is 150 μm, and the figures without rulers are obtained by observation and photography under a 20x microscope; A is the staining result of the human-mouse chimeric antibody 48C10; B is the staining result of the human-mouse chimeric antibody 48C10-1; C is the staining result of the culture supernatant of incubated Expi293F cells.

[0119] Depend on Figure 5It can be seen that the mouse monoclonal antibody 48C10 can be successfully combined with the anti-human IgG secondary antibody after modification; the mutant 48C10-1 of the human-mouse chimeric monoclonal antibody 48C10 can successfully recognize the cell slides overexpressing LGI1 and is recognized by the anti-human IgG antibody. In this example, two anti-LGI1 human-mouse chimeric monoclonal antibodies 48C10 and their mutants 48C10-1 were successfully prepared. The human-mouse chimeric monoclonal antibody 48C10 and its mutant 48C10-1 were purified using protein A filler, and the protein concentration measured by the BCA method was 0.2 mg / mL (Solerbo BCA protein concentration determination kit).

[0120] Example 5

[0121] Application of anti-LGI1 human-mouse chimeric antibody

[0122] The cell slides overexpressing LGI1 prepared in Example 1 were incubated with sera from three patients with positive anti-LGI1 antibodies (diluted 1:10 with PBS, donated by Ruiao Medical Testing Company) and two anti-LGI1 human-mouse chimeric antibodies 48C10 and 48C10-1 purified by protein A obtained in Example 4 of the present application (diluted 1:200 with PBS), and the secondary antibody was incubated with FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-170). The staining results are as follows: Figure 6 As shown, Figure 6 The scale of the figures with ruler is 150μm, and the figures without ruler are obtained by observation under a 20x microscope.

[0123] Depend on Figure 6 It can be concluded that the two anti-LGI1 human-mouse chimeric antibodies can not only recognize the overexpressed LGI1 antigen and bind to the anti-human IgG antibody, but also produce a signal morphology similar to that of the anti-LGI1 antibody positive serum. Therefore, the modified human-mouse chimeric antibody 48C10 and the mutant antibody 48C10-1 of the human-mouse chimeric antibody 48C10 have the following three advantages: First, the enterprise can carry out large-scale production by itself, which solves the problem that the current mouse / rabbit monoclonal / polyclonal antibodies have batch differences and cannot form low-cost large-scale industrial production; second, it can replace the function of serum to a certain extent, which is helpful for the development of quality-controlled neuroimmune autoantibody detection kits; third, it provides a new set of LGI1 human-mouse chimeric monoclonal antibodies for identifying the expression and localization of LGI1 protein and purifying LGI1 protein.

[0124] At the same time, given that the signal morphology produced by the combination of anti-LGI1 human-mouse chimeric antibodies 48C10 and 48C10-1 with LGI1-overexpressing cell slides is similar to the signal morphology produced by positive blood, a large number of fluorescent images can be obtained through multiple experiments to enrich the image library of anti-LGI1 antibody-positive images, provide a large number of different styles of signals for the development of automatic immunofluorescence image processing systems, and improve the accuracy of automated interpretation.

[0125] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A human-mouse chimeric monoclonal antibody against human LGI1 protein, characterized in that: comprising a light chain and a heavy chain; the light chain comprising a light chain variable region and a light chain constant region; the light chain variable region comprising a light chain complementary determining region CDR1, a light chain complementary determining region CDR2 and a light chain complementary determining region CDR3, the amino acid sequences of the light chain complementary determining region CDR1, the light chain complementary determining region CDR2 and the light chain complementary determining region CDR3 are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; the light chain constant region is a human light chain constant region; The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO:8, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO:9 or SEQ ID NO:13, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO:10 or SEQ ID NO14; the heavy chain constant region is a human heavy chain constant region.

2. The human-mouse chimeric monoclonal antibody against human LGI1 protein according to claim 1, characterized in that: The light chain variable region comprises the amino acids shown in SEQ ID NO: 2; The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:

12.

3. The human-mouse chimeric monoclonal antibody against human LGI1 protein according to claim 1, characterized in that: The human-mouse chimeric monoclonal antibody against human LGI1 protein comprises an amino acid sequence shown in (I) or (II): (I) the light chain variable region sequence shown in SEQ ID NO: 2, and the heavy chain variable region sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12; (II) an amino acid sequence having the same function as that shown in (I) by substituting, deleting or adding one or more amino acids; (III) an amino acid sequence having at least 50% homology with the amino acid sequence shown in (I) and having sequence identity or similarity with (I); said (III) does not include the amino acid sequence shown in (II).

4. A biomaterial, characterized in that: Including one or more of 1) to 3): 1) A nucleic acid molecule expressing the human-mouse chimeric monoclonal antibody against human LGI1 protein according to any one of claims 1 to 3; 2) a recombinant vector, wherein the recombinant vector comprises the nucleic acid molecule described in 1); 3) an engineered cell, the engineered cell comprising the nucleic acid molecule described in 1) or the recombinant vector described in 2); Preferably, the recombinant vector comprises a light chain recombinant vector and / or a heavy chain recombinant vector; The light chain recombinant vector comprises a nucleic acid molecule expressing a light chain variable region and a nucleic acid molecule expressing a light chain constant region; The heavy chain recombinant vector comprises a nucleic acid molecule expressing a heavy chain variable region and a nucleic acid molecule expressing a heavy chain constant region.

5. The biomaterial according to claim 4, characterized in that The nucleic acid molecule expressing the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 1; The nucleic acid molecule expressing the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:

11.

6. An antibody derivative, characterized in that: The antibody derivative is a complex obtained by modifying the human-mouse chimeric monoclonal antibody against human LGI1 protein according to any one of claims 1 to 3.

7. Use of the human-mouse chimeric monoclonal antibody against human LGI1 protein according to any one of claims 1 to 3, the biological material according to claim 4 or 5, or the antibody derivative according to claim 6 in the preparation of a product for detecting one or more of LGI1 protein, identifying LGI1 protein, and detecting LGI1 autoantibodies.

8. A product, characterized in that The product comprises one or more of the human-mouse chimeric monoclonal antibody against human LGI1 protein according to any one of claims 1 to 3, the biological material according to claim 4 or 5, and the antibody derivative according to claim 6.

9. The product according to claim 8, characterized in that The product is prepared based on immunoassay technology.

10. A method for detecting LGI1 protein, characterized in that: The method is based on immunological detection technology and the human-mouse chimeric monoclonal antibody against human LGI1 protein according to any one of claims 1 to 3 for detection; the method is a method for non-diagnostic and therapeutic purposes.

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