Human-mouse chimeric monoclonal antibody against human LGI1 protein, products and uses thereof

Abstract

The present application belongs to the technical field of monoclonal antibody preparation, and particularly relates to a human-mouse chimeric monoclonal antibody against human G protein-coupled receptor family member 1 (LGI1 protein) as well as products and applications thereof. The variable regions of the heavy chain and the light chain of the human-mouse chimeric monoclonal antibody against human LGI1 protein according to the present application are derived from mice, and the constant regions are human antibody constant regions. On the basis of retaining the activity of the parent to the maximum extent, the human-mouse chimeric monoclonal antibody can replace the function of serum to a certain extent. Meanwhile, the human-mouse chimeric monoclonal antibody can solve the technical problems that the Fc region of the existing monoclonal antibody against human LGI1 protein cannot react with the antibody against human IgG, the sensitivity difference between batches of antibodies is large, and the antibody cannot be produced in batches. Further, the human-mouse chimeric monoclonal antibody against human LGI1 protein can detect LGI1 protein and LGI1 autoantibody.

Description

TECHNICAL FIELD

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

[0002] LGI1, leucine rich gliomain activated 1, is a kind of neuron secreted protein, mainly existing in hippocampus, amygdala, temporal lobe cortex and other marginal systems, and plays a crucial role in brain development, maintaining neuron excitability and synaptic transmission, and is closely related to nervous system diseases. LGI1 deletion can cause disordered stratification of neurons in the cerebral cortex and reduce the thickness of the external granular cell layer of the cerebellum during the embryonic period. More importantly, LGI1 has been confirmed to be related to central nervous system myelination and differentiation of oligodendrocytes. Previous studies have shown that LGI1 can play a regulatory role in glutamate synaptic plasticity and transmission of glutamate in the hippocampus. In addition, LGI1 autoantibodies can interfere with the interaction of inter-synaptic proteins and cause a series of clinical manifestations such as cognitive impairment, mental and behavioral abnormalities, sleep disorders, etc. Anti-LGI1 antibody has become one of the markers of autoimmune encephalitis.

[0003] In the detection system of autoantibodies, the reference product is 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 value and trace, so it is necessary to provide a positive reference product that can replace the positive serum of patients, and the chimeric antibody or humanized antibody can meet such a demand.

[0004] The existing commercial LGI1 antibody is a rabbit / mouse polyclonal antibody, and such an antibody: (1) there are inevitable differences between different batches, and it is impossible to ensure that each batch has the same sensitivity; (2) the sequence of the antibody is not disclosed, and it is impossible to form a low-cost, large-scale industrial batch production; (3) the Fc region of the antibody is of mouse / rabbit origin, and cannot react with the antibody against human IgG. Therefore, it is of great significance to develop a new monoclonal antibody against LGI1 protein. SUMMARY

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

[0006] The application 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 complementarity determining region CDR1, a light chain complementarity determining region CDR2 and a light chain complementarity determining region CDR3, and the amino acid sequences of the light chain complementarity determining region CDR1, the light chain complementarity determining region CDR2 and the light chain complementarity determining region CDR3 are respectively shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; the light chain constant region is a human light chain constant region;

[0007] the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the heavy chain variable region comprises a heavy chain complementarity determining region CDR1, a heavy chain complementarity determining region CDR2 and a heavy chain complementarity determining region CDR3, the amino acid sequence of the heavy chain complementarity determining region CDR1 is shown as SEQ ID NO: 8, the amino acid sequence of the heavy chain complementarity determining region CDR2 is shown as SEQ ID NO: 9 or SEQ ID NO: 13, and the amino acid sequence of the heavy chain complementarity determining region CDR3 is shown as SEQ ID NO: 10 or SEQ ID NO: 14; the heavy chain constant region is a human heavy chain constant region.

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

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

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

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

[0012] (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids on the basis of the amino acid shown as (I) and having the same function;

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

[0014] The application 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 according to the technical solution described above;

[0016] 2) a recombinant vector comprising the nucleic acid molecule according to 1);

[0017] 3) an engineered cell comprising the nucleic acid molecule according to 1) or the recombinant vector according to 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 a light chain variable region comprises a nucleotide sequence as shown in SEQ ID NO: 1;

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

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

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

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

[0026] Preferably, the product is a product prepared based on an immunological detection technology.

[0027] The present application also provides a method for detecting LGI1 protein, which detects the human-mouse chimeric monoclonal antibody against human LGI1 protein according to the technical solution described above based on an immunological detection technology; the method is a method for non-diagnostic treatment purposes.

[0028] Advantages:

[0029] The present application provides a human-mouse chimeric monoclonal antibody against human LGI1 protein, the variable regions of the heavy chain and the light chain of the human-mouse chimeric monoclonal antibody against human LGI1 protein are from mice, and the constant regions are human antibody constant regions, which can replace the function of serum to the greatest extent while retaining the activity of the parent, and 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 batches of antibodies is large, and the antibodies cannot be mass-produced. Further, the human-mouse chimeric monoclonal antibody against human LGI1 protein can detect, such as visualize and locate, LGI1 protein and LGI1 autoantibody, judge the expression and location of LGI1 protein, and can use the monoclonal antibody of LGI1 protein as a ligand in affinity chromatography to realize the separation and purification of LGI1 protein. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0031] Figure 1 Figure 1 is the purification result of LGI1 protein in Example 1;

[0032] Figure 2 Figure 2 is the titer of the serum antibody of the immunized mouse identified by CBA method in Example 1;

[0033] Figure 3 Figure 3 is the CBA detection result of the three positive clones screened in Example 1;

[0034] Figure 4 Figure 4 is the ELISA detection result of the three positive clones screened in Example 1;

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

[0036] Figure 6 Figure 6 is the immunofluorescence staining result of human-mouse chimeric monoclonal antibody 48C10 and 48C10-1 with anti-LGI1 positive blood in Example 5. DETAILED DESCRIPTION

[0037] The application 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 complementarity determining region CDR1, a light chain complementarity determining region CDR2 and a light chain complementarity determining region CDR3, and the amino acid sequences of the light chain complementarity determining region CDR1, the light chain complementarity determining region CDR2 and the light chain complementarity determining region CDR3 are respectively shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the light chain constant region is a human light chain constant region.

[0038] The heavy chain comprises a heavy chain variable region and a heavy chain constant region; the heavy chain variable region comprises a heavy chain complementarity determining region CDR1, a heavy chain complementarity determining region CDR2 and a heavy chain complementarity determining region CDR3, the amino acid sequence of the heavy chain complementarity determining region CDR1 is shown as SEQ ID NO: 8, the amino acid sequence of the heavy chain complementarity determining region CDR2 is shown as SEQ ID NO: 9 or SEQ ID NO: 13, and the amino acid sequence of the heavy chain complementarity determining region CDR3 is shown as SEQ ID NO: 10 or SEQ ID NO: 14; and the heavy chain constant region is a human heavy chain constant region.

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

[0040] As an embodiment, the light chain variable region comprises an amino acid shown as SEQ ID NO: 2; and as another embodiment, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2, and specifically is: NIVMTQSPKCMSMSVGERV TLSCKASENVYTYVSWYQQKPEQSPKMLMYGASSRYTGVPDRFTGSGSATDFTLTISSVQAEDL ADYFCGQTYSYPYTFGSGTMLEIK; wherein the three light chain complementarity determining regions are in the bold part.

[0041] As an embodiment, the nucleotide sequence encoding the light chain variable region comprises the nucleotide sequence as 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'- AACATTGTGATGACCCAATCTCCCAAATGCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTCTATACTTATGTATCCTGGTATCAACAGAAACCAGAGCAGTCTCCAAAAATGCTGATGTACGGGGCATCCAGCCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATTTCTGTGGACAGACTTACAGCTATCCGTATACGTTCGGATCGGGGACCATGCTGGAAATAAAA-3'.

[0042] As an embodiment, the light chain constant region of the present application 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 CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3'.

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

[0044] As an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth 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 set forth in SEQ ID NO: 7 or SEQ ID NO: 12: specifically: QVQLQQSGAELARPGASVKLSCTASGYTFTNYGLSWVKQRTGQGLEWIGEVYPRTGNTYYNEKFKGKATLTADKSSRTAYMELRSLTSEDSAVYFCARSLYYDYGWYFDVWGA GTTVTVSS (SEQ ID NO: 7) and QVQLQQSGAELARPGASVKLSCTASGYTFTNYGLSWVKQRTG QGLEWIGEVYPRSGNTYYNEKFKGKATLTADKSSRTAYMELRSLTSEDSAVYFCARSFYYDYG WYFDVWGAGTTVTVSS (SEQ ID NO: 12); wherein the bolded part is three heavy chain complementarity determining regions.

[0045] As an embodiment, the nucleotide sequence encoding the heavy chain variable region comprises the amino acid sequence as 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, in particular: 5'-CAGGTTCAACTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCACGGCTTCTGGCTACACCTTCACAAACTATGGTTTAAGTTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGGTTTATCCTCGAACAGGTAATACTTACTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGAACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCTCTCTACTATGATTACGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA-3'(SEQ ID NO: 6) and 5'-CAGGTTCAACTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCACGGCTTCTGGCTACACCTTCACAAACTATGGTTTAAGTTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGGTTTATCCTCGAAGTGGTAATACTTACTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGAACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCTTTCTACTATGATTACGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA-3'(SEQ ID NO: 11).

[0046] As an embodiment, the heavy chain constant region of the present application is a human IgGl heavy chain constant region. In a specific embodiment, the amino acid sequence of the human IgGl heavy chain constant region is shown in SEQ ID NO: 18, specifically: ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*; the nucleotide sequence encoding the human IgGl heavy chain constant region shown in SEQ ID NO: 18 is shown in SEQ ID NO: 17, specifically: 5'-GCCTCCACCAAGGGCCC ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA-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) a light chain variable region sequence shown in SEQ ID NO: 2, and a heavy chain variable region sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12;

[0049] (II) an amino acid sequence which is substituted, deleted or added with one or more amino acids based on the amino acid shown in (I) and has the same function;

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

[0051] As an embodiment, the functional variant of the human-mouse chimeric monoclonal antibody against human LGI1 protein of the present application is also included in the protection scope of the present application, i.e. the amino acid sequence shown in (III); the "functional variant" refers to a protein which has obvious or significant sequence identity or similarity compared with the parent antibody (i.e. the human-mouse chimeric monoclonal antibody against human LGI1 protein of the present application), and the functional variant retains the biological activity of the parent antibody. The functional variant covers, for example, the following variants of the antibody (parent antibody) described herein, which retain the ability to recognize the target protein to a similar degree, to the same degree or to a higher degree compared with 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 in the amino acid sequence with the parent antibody.

[0052] As another embodiment, the conservative amino acid substitution is known in the art and includes the substitution of one amino acid with specific physical and / or chemical properties with another amino acid with the same or similar chemical or physical properties. For example, the conservative amino acid substitution can be the substitution of one acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g. Asp or Glu), the substitution of one 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.), the substitution of one basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g. Lys, His, Arg, etc.), the substitution of one 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, the nucleotide sequence corresponding to the human-mouse chimeric monoclonal antibody against human LGI1 protein described in the present application can be mutated by known methods, such as methods of directed evolution and point mutation, by those of ordinary skill in the art. Those nucleotides artificially modified, having 90% or more homology with the nucleotide sequence corresponding to the antibody or antigen-binding fragment thereof described in the present application, as long as they encode the above-mentioned antibody of the present application, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application, and are also included in the scope of protection of the present application.

[0054] The present application also provides a biological material 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 comprising the nucleic acid molecule of 1);

[0057] 3) an engineered cell comprising the nucleic acid molecule of 1) or the recombinant vector of 2).

[0058] As an embodiment, the backbone vector of the recombinant vector described in the present application comprises 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 one or more of, but not limited to, pcDNA3.1, pBAD, pQE-12, pGEX, pBluescript, pET-series expression vector, 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 pPIC3.5K; in a specific embodiment, the backbone vector of the recombinant vector is pcDNA3.1.

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

[0060] As an embodiment, the nucleic acid molecule can comprise natural, unnatural or altered nucleotides. In some specific embodiments, the nucleic acid molecule does not comprise any insertion, deletion, inversion and / or substitution. However, in some other specific embodiments, it can be appropriate for the nucleic acid molecule to comprise one or more insertions, deletions, inversions and / or substitutions, and thus the nucleic acid formed by these insertions, deletions, inversions and / or substitutions is also within the scope of the present application.

[0061] As an embodiment, the light chain recombination 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 recombination vector comprises the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 15.

[0062] As an embodiment, the heavy chain recombination 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 recombination vector comprises the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 17, or the nucleotide sequence shown in SEQ ID NO: 11 and SEQ ID NO: 17.

[0063] As an embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell; as another embodiment, the prokaryotic cell comprises but is not limited to a bacterial cell such as Escherichia coli; as another embodiment, the eukaryotic cell comprises but is not limited to a yeast cell, an insect cell, an animal cell or a plant cell; as another embodiment, the yeast cell can be but is not limited to a Pichia or Saccharomyces cell; the insect cell can be but is not limited to a CHO cell, a COS cell, an NSO cell, a 293T cell, an 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 an embodiment, the way of introducing the recombination vector into the host cell comprises a physical method, a chemical method or a biological method; as another embodiment, the physical method comprises calcium phosphate precipitation, lipofection, particle bombardment, microinjection or electroporation; as another embodiment, the chemical method comprises a colloidal dispersion system or a lipid-based system; as another embodiment, the biological method comprises a DNA vector, a lentivirus vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector or an adeno-associated virus vector transfer method.

[0065] The application also provides an antibody derivative, which is a complex obtained by modifying the human-mouse chimeric monoclonal antibody against human LGI1 protein as described above. As an embodiment, the modification comprises conjugation modification using a detectable label; as another embodiment, the detectable label comprises one or more of a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold or colored latex.

[0066] The application also provides the use of the human-mouse chimeric monoclonal antibody against human LGI1 protein as described above, the biomaterial as described above and the antibody derivative as described above in the preparation of a product for one or more of detecting LGI1 protein, identifying LGI1 protein and detecting LGI1 autoantibody.

[0067] The application also provides a product comprising one or more of the human-mouse chimeric monoclonal antibody against human LGI1 protein as described above, the biomaterial as described above and the antibody derivative as described above. As an embodiment, the product can be a kit; as another embodiment, the kit can be a kit prepared based on an immunodetection 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 application also provides a method for detecting LGI1 protein, which is based on an immunological detection technology and the human-mouse chimeric monoclonal antibody against human LGI1 protein as described above; 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 sample that can be tested by the above-mentioned immunodetection or assay includes but is not limited to plasma, whole blood, serum, tissue, cells and extracts of cells, extracts of tissue cells.

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

[0070] Technical terms:

[0071] "Antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, fully humanized antibodies, single-chain antibodies, or portions of antibodies that are capable of binding to their antigens such as Fab fragments or fragments produced from Fab expression libraries, and the like.

[0072] "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is a region in 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 an epitope.

[0073] Based on the variable region amino acid sequence comprised by a given antibody or fragment thereof according to the present application, the CDRs contained therein can be routinely determined by the skilled person. For example, the CDRs in the variable region amino acid sequence can be defined using the Kabat scheme, the AbM scheme, the Chothia scheme or the Contact scheme.

[0074] In case an antibody is defined in relation to specific CDR sequences as defined according to the present application, the scope of said antibody also encompasses antibodies whose variable region sequence comprises said specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries as defined according to the present application due to the application of different schemes (e.g. different assignment system rules or combinations).

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

[0076] For further illustrating the present application, the technical solutions provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0077] Example 1

[0078] Preparation of murine monoclonal antibodies

[0079] Step 1 Preparation of recombinant protein LGI1 protein

[0080] The gene sequence of human LGI1 was searched from GenBank sequence database, the sequence number was AF055636.1, the gene sequence was synthesized by Jinweizhi Company into pET-28a vector, the insertion site was between HindIII and Xho I, and the pET-28a-LGI1 recombinant vector was obtained. The constructed and sequenced recombinant plasmid was transformed into E. coli Arctic Express (Agilent) expression competent cells; a single colony was picked and inoculated in LB medium, and the bacteria were shaken at 37℃ until the OD 600 value was 0.5-1, IPTG was induced at a final concentration of 0.5 mM, and the bacteria were shaken at 16℃ overnight for expression; the bacterial cells were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The LGI1 protein expressed by pET-28a was purified by Ni-NTA (QIAGEN), and the LGI1 recombinant protein was obtained and detected by SDS-PAGE. The results showed that the LGI1 protein for immunizing mice was successfully prepared in the embodiment, and the results are shown in Figure 1 (Marker was purchased from thermo company, item number 26616).

[0081] Step 2 Preparation of cell climbing sheet overexpressing LGI1 protein

[0082] The LGI1 gene was inserted into pCDNA3.1 by molecular biology methods, the insertion site was Nhe I and Not I, and the recombinant plasmid was obtained; the recombinant plasmid was transfected into 293T cells (6cm×6cm cell climbing sheet was placed in 10cm cell culture dish in advance) with a cell density of 30%-40% using PEI transfection reagent (transfection reagent was purchased from thermo company, and the transfection was performed according to the instructions), and 48h after transfection, acetone was added for fixation at 4℃ for 5min, PBS was washed twice, and the 6cm×6cm cell climbing sheet was cut into 0.25cm×0.25cm size after drying, and was ready for use. The cell climbing sheet transfected with empty pCDNA3.1 was used as a control, and the preparation method was the same as that of the cell climbing sheet overexpressing LGI1 protein.

[0083] Step 3 Mouse antigen immunization

[0084] Five 6-8 week old female Balb / c mice were immunized with the LGI1 recombinant protein prepared in step 1 as the antigen, for a total of four immunizations. For the first immunization, the antigen and Freund's adjuvant were mixed in equal volumes and thoroughly ground into a water-in-oil emulsion. 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally. For the second immunization, two weeks after the first immunization, 40 μg antigen was mixed with incomplete Freund's adjuvant at a 1:1 volume ratio, and 100 μL (40 μg antigen) of the mixed antigen was injected intramuscularly into the right hind leg. The third immunization was performed two weeks after the second immunization, with the same dosage, method, and route of antigen injection as the second immunization. Two weeks later, 100 μL (40 μg) of unadjuvanted antigen was injected intraperitoneally to boost the immunization. Three days later, rat tail blood was collected and its titer was detected using indirect immunofluorescence assay (CBA). The specific detection procedure was as follows: The collected rat tail blood was diluted 20-fold and 100-fold with PBS as primary antibody and incubated on cell slides overexpressing LGI1 protein obtained in step 2. The cells were incubated at room temperature for 1 hour, washed three times with PBST for 5 minutes each time, and then incubated with Alexa... 594-labeled goat anti-mouse secondary antibody (Jackson, product number 115-585-146) was applied for 40 min, followed by washing with PBST three times for 5 min each time. The images were then taken using a fluorescence microscope. The results are as follows: Figure 2 As shown, where Figure 2 The scale bar is 150 μm.

[0085] Depend on Figure 2 It can be concluded that cell slices overexpressing LGI1 protein bound to antibodies in the tail blood of 5 mice, generating signals. Figure 2 Based on the combined results of the detection and serum antibody titers, mouse number 2 was selected for subsequent experiments.

[0086] Step 4: Cell fusion and hybridoma culture

[0087] Immunization was boosted by intraperitoneal injection of antigen. Three days later, animals were sacrificed, and spleen cells were harvested for cell fusion. 5–6 × 10⁶ cells were used. 7The SP2 / 0 cells and the spleen cells in logarithmic growth phase and in good growth state are washed 3 times (37°C) with serum-free culture solution, and the viable cells (not less than 90%) are counted; the myeloma cells and the spleen cells are mixed at a ratio of 1:10, the supernatant is discarded after centrifugation, and the excess supernatant is absorbed with a sterilized filter paper; 1 mL of 40% PEG solution is added dropwise into the cell mass, and the addition is completed within 60 seconds, and at the same time, the centrifuge tube is slightly rotated or tapped with fingers; 1 mL of serum-free culture solution is added while rotating the centrifuge tube, and the addition is completed within 60 seconds; then, 20 mL of serum-free culture solution is slowly added within 5 minutes; centrifugation (800 rpm, 8 minutes) is performed, the supernatant is removed, 10 mL of complete culture solution is used for suspension, and the mixture is gently mixed; 96-well plates are taken, and 50 microliters of the cell suspension is added to each well; the plates are placed in a 5% CO2 incubator at 37°C, and after 24 hours, the HAT selective culture solution (thermo, item number: 21060017) is replaced.

[0088] Screening of the positive hybridoma of step 5

[0089] The HAT selective culture solution is used for replacement at a variable amount 7-10 days after the fusion, and the half amount is replaced every 2-3 days thereafter; the hybrid cell colonies appear 2-3 weeks later, the cells are large, round and transparent; when the colonies grow to 1 / 3 of the well, the antibody is detected by using the CBA method; the cells with positive signals are cultured by using the limited dilution method for cloning, and a second screening is performed, and after being repeated for 3 times, the positive monoclonal is identified by using the CBA method. The overexpressed LGI1 cell slides prepared in step 2 are used for screening by using the CBA method, and the detection process is the same as that described in step 3, and the hybridoma cell supernatant does not need to be diluted during the detection. The screening and determination results of the anti-LGI1 antigen positive hybridoma are shown in Table 1. Figure 3 , wherein Figure 3 is the result observed under 10 times magnification.

[0090] From Figure 3 It can be concluded that 3 positive clone cells are screened after 3 screenings, and are named as 21D12, 33H8 and 48C10 respectively.

[0091] The positive clones are verified by using ELISA, and the detection steps can be selected conventionally, and specifically, the LGI1 recombinant protein purified in step 1 is coated with carbonate buffer solution with a pH value of 9.6 at 4°C overnight, 100 ng / well, the supernatant of the 3 positive hybridomas screened by using the CBA method is used, and is diluted at a ratio of 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, and is used for ELISA detection titer, at the same time, the supernatant of the cells transfected with empty pCDNA3.1 is used as a negative control, and the blood of No. 2 mouse is used as a positive control. The dilution multiple is used as the abscissa, the detected OD 450 value is used as the ordinate, and a graph is drawn, and the result is shown in Table 2. Figure 4Figure 1 shows the results of the ELISA test.

[0092] Figure 2 shows the results of the ELISA test. Figure 4 It can be seen that the LGI1 protein can be combined with the antibodies in the supernatant of hybridoma 21D12, 33H8, 48C10, and does not react with the supernatant of cells transfected with empty pCDNA3.1. Therefore, the antibodies secreted by hybridoma 21D12, 33H8, 48C10 can not only recognize the LGI1 protein expressed in eukaryotic cells, but also recognize the prokaryotic LGI1 protein coated in the ELISA plate. The 48C10 with the highest OD value was selected for subsequent experiments. 450 Figure 3 shows the results of the ELISA test.

[0093] Step 6 Identification of antibody typing

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

[0095] Example 2

[0096] Identification of anti-LGI1 murine monoclonal antibody sequence

[0097] After the positive hybridoma 48C10 was cultured to a certain scale (cell number > 3 x 10 6 After the positive hybridoma 48C10 was cultured to a certain scale (cell number > 3 x 10 Max DNA Polymerase for PCR amplification, and the antibody V region cDNA was extracted and sent to a sequencing company for sequencing, and the CDR region of the antibody amino acid sequence was labeled 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 anti-LGIl human-mouse chimeric monoclonal antibody

[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 (Tiangen, AP221-01). The nucleotide sequence of the light chain variable region VL of the 48C10 antibody was connected to the human kappa light chain constant region sequence SEQ ID NO: 15 using homologous recombination seamless cloning kit (Tiangen, CU201) to connect the light chain variable region VL of the 48C10 antibody to the vector pcDNA3.1 with the linearization site of 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 was connected to the human IgG1 heavy chain constant region sequence SEQ ID NO: 16 to the vector pcDNA3.1 with the linearization site of Not I, named pcDNA3.1-48C10-VH, and the connected recombinant plasmid was sent to GenScript for sequencing. The sequenced recombinant plasmid was prepared for use, and the PCR amplification system, amplification program and recombination system were routinely 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 using the conventional method of molecular biology, the partial amino acids of the VH region of pcDNA3.1-48C10-VH were mutated by using Fast MultiSite Mutagenesis System (QIAGEN, FM201-01) with pcDNA3.1-48C10-VH as the template. The 6th 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 2nd amino acid L in the CDR3 region of the heavy chain variable region 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. The mutated recombinant vector is labeled as pcDNA3.1-48C10-1-VH. The PCR amplification system, amplification program and recombination reaction system can be selected according to the instructions, and the connected recombinant plasmid is sent to Shengong Biotechnology for sequencing. 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 anti-LGIl human-mouse chimeric antibody

[0118] The following two groups of recombinant vectors were transfected into Expi293F cells (thermo) using transfection reagent PEI (Opmi): group 1 was co-transfected with recombinant plasmid pcDNA3.1-48C10-VL and recombinant plasmid pcDNA3.1-48C10-VH, and the purified antibody was named human-mouse chimeric antibody 48C10; group 2 was co-transfected with recombinant plasmid pcDNA3.1-48C10-VL and recombinant plasmid pcDNA3.1-48C10-1-VH, and the purified antibody was named human-mouse chimeric antibody 48C10-1; Opmi medium (item number: P82019) was used for Expi293F cell culture, and the plasmid transfection and subsequent cell culture process were carried out according to the instructions of OPM-CD293 transient transfection system. On the third day after transfection, the culture supernatant was collected by centrifugation as the primary antibody, incubated on the LGIl overexpressing cell slide, and the secondary antibody was incubated with FITC-labeled goat anti-human IgG secondary antibody (Jackson, item number: 109-095-170). The results are shown in Figure 5 FIG. 1, wherein Figure 5 The scale bar is 150 pm, and the scale-free graph is observed under 20x lens; wherein A is the staining result of human-mouse chimeric antibody 48C10; B is the staining result of human-mouse chimeric antibody 48C10-1; C is the staining result of incubating Expi293F cell culture supernatant.

[0119] FIG. 2 shows the results of the binding of human-mouse chimeric antibody 48C10 to the extracellular domain of LGIl protein, wherein Figure 5It was found that the modified murine monoclonal antibody 48C10 could successfully bind to anti-human IgG secondary antibody; the mutant 48C10-1 of the human-mouse chimeric monoclonal antibody 48C10 could successfully recognize cell smears overexpressing LGI1 and be recognized by anti-human IgG antibody. In this example, two anti-LGI1 human-mouse chimeric monoclonal antibodies, 48C10 and its mutant 48C10-1, were successfully prepared. The human-mouse chimeric monoclonal antibody 48C10 and its mutant 48C10-1 were purified using protein A packing material, and the protein concentration was determined to be 0.2 mg / mL by the BCA method (Solepro BCA protein concentration assay kit).

[0120] Example 5

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

[0122] The LGI1 overexpressing cell smears prepared in Example 1 were incubated with serum from three patients positive for anti-LGI1 antibodies (diluted 1:10 with PBS, serum provided by Ruiao Medical Laboratory Co., Ltd.) and two anti-LGI1 human-mouse chimeric antibodies, 48C10 and 48C10-1, purified with protein A and obtained in Example 4 of this application (diluted 1:200 with PBS). 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, where Figure 6 The graphs with scales are 150 μm, while the graphs without scales were taken 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 anti-human IgG antibodies, but also produce signal morphologies similar to those of anti-LGI1 antibody-positive serum. Therefore, the modified human-mouse chimeric antibody 48C10 and its mutant antibody 48C10-1 have the following three advantages: First, companies can carry out large-scale production independently, solving the problem of batch-to-batch variability and the inability to achieve low-cost, large-scale industrial production of current mouse / rabbit monoclonal / polyclonal antibodies; second, they can, to some extent, replace the function of serum, which is helpful in developing quality-controlled neuroimmunological autoantibody detection kits; and third, they provide 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, in view that the signal pattern produced by the anti-LGI1 human-mouse chimeric antibody 48C10 and 48C10-1 and the LGI1 cell overexpression crawling slice binding is similar to the signal pattern produced by the positive blood, a large number of fluorescence pictures can be obtained through multiple experiments, a picture library of the anti-LGI1 antibody positive is enriched, a large number of different signal patterns are provided for the development of the immunofluorescence image automatic processing system, and the accuracy of the automatic interpretation is improved.

[0125] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A human-mouse chimeric monoclonal antibody against human LGI1 protein, characterized in that, It includes a light chain and a heavy chain; the light chain includes a light chain variable region and a light chain constant region; the light chain variable region includes a light chain complementarity-determining region CDR1, a light chain complementarity-determining region CDR2, and a light chain complementarity-determining region CDR3, the amino acid sequences of which 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-derived 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 complementarity-determining region (CDR1), a heavy chain complementarity-determining region (CDR2), and a heavy chain complementarity-determining region (CDR3), the amino acid sequences of which are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, or the amino acid sequences of which are shown in SEQ ID NO:8, SEQ ID NO:13, and SEQ ID NO:14, respectively; the heavy chain constant region is a human-derived 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 includes an amino acid as shown in SEQ ID NO:2; the heavy chain variable region includes an amino acid sequence as 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 having any one of (I) to (III): (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 that has the same function as the amino acids shown in (I) by substituting, deleting or adding one or more amino acids; (III) is an amino acid sequence that has at least 50% homology with the amino acid shown in (I) and has sequence identity or similarity with (I); (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 as described in any one of claims 1 to 3; 2) A recombinant vector comprising the nucleic acid molecule described in 1); 3) Engineered cells, wherein the engineered cells comprise the nucleic acid molecule described in 1) or the recombinant vector described in 2).

5. The biomaterial according to claim 4, characterized in that, The recombinant vector includes a light chain recombinant vector and / or a heavy chain recombinant vector; The light chain recombinant vector contains nucleic acid molecules expressing the variable region of the light chain and nucleic acid molecules expressing the constant region of the light chain; the heavy chain recombinant vector contains nucleic acid molecules expressing the variable region of the heavy chain and nucleic acid molecules expressing the constant region of the heavy chain.

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

11.

7. The use of the human-mouse chimeric monoclonal antibody against human LGI1 protein as described in any one of claims 1 to 3 or the biomaterial as described in any one of claims 4 to 6 in the preparation of products for detecting and / or identifying LGI1 protein.

8. A product characterized in that, The product includes the human-mouse chimeric monoclonal antibody against human LGI1 protein as described in any one of claims 1 to 3 and / or the biomaterial as described in any one of claims 4 to 6; the product is prepared based on immunoassay technology.

9. A method for detecting LGI1 protein, characterized in that, The method is based on immunological detection technology and the detection of human-mouse chimeric monoclonal antibody against human LGI1 protein as described in any one of claims 1 to 3; the method is not for diagnostic or therapeutic purposes.

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