Human-mouse chimeric monoclonal antibody of NF186 protein, product of human-mouse chimeric monoclonal antibody and method for detecting NF186 protein

Abstract

The invention discloses a human-mouse chimeric monoclonal antibody of NF186 protein, a product of the human-mouse chimeric monoclonal antibody and a method for detecting the NF186 protein, and belongs to the technical field of monoclonal antibodies. The murine monoclonal antibody sequence of the NF186 protein is firstly obtained by means of molecular biology, then the murine monoclonal antibody sequence of the NF186 protein is modified to prepare the human-mouse chimeric monoclonal antibody of the NF186 protein, the human-mouse chimeric antibody can be expressed and purified on a large scale by using a recombinant protein expression and purification technology, a large number of antibodies can be obtained at a time at a low cost, and the human-mouse chimeric monoclonal antibody can be used for preparing the human-mouse chimeric monoclonal antibody of the NF186 protein. And the intra-batch difference and the inter-batch difference are controllable and can be kept consistent. The invention can effectively solve the technical problems that the current mouse / rabbit single / polyclonal antibody has batch-to-batch difference, the low-cost large-scale industrial production cannot be formed, and the Fc region cannot react with the anti-human IgG antibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and specifically relates to a human-mouse chimeric monoclonal antibody of NF186 protein and its product, and a method for detecting NF186 protein. Background Art

[0002] Neurofascin (NF) is a cell surface protein involved in the formation of nerve bundles discovered by Ranthjen et al. in chickens in 1987. It is a cell adhesion molecule that plays a vital role in the formation and stability of Ranvier nodes. Five different types of NF polypeptides have been reported, including NF140, NF155, NF166, NF180 and NF186. Studies have shown that on the axons of mature neurons, NF186 maintains the stability of neuronal structure by interacting with the cytoskeletal component ankyrin G. In the peripheral nervous system, NF186 interacts with glial proteins in the matrix and Schwann cell microvilli to promote the attachment of axons to Schwann cell microvilli. That is, the main role of NF186 is to form a nodal complex as a molecular barrier to limit the diffusion and migration of proteins and molecules in the nodal lateral region to the nodal region, thereby promoting the composition of the myelinated axonal domain and the saltatory conduction of signals along the axon. Therefore, insufficient expression of NF186 will directly affect the insulation of the myelin nodular region, which is manifested in patients with multiple sclerosis as disordered distribution of ion channels in the corresponding axonal membranes of various myelin regions, and decreased Na + The channel structure is destroyed and cannot aggregate. The Kv1.2 channels originally located in the proximal nodal region cross the nodal region to reach the nodal region, causing the failure of rapid nerve conduction in myelinated fibers.

[0003] At present, neurofascin-specific antibodies have been found in some human autoimmune demyelinating diseases, such as NF155 and NF186-specific autoantibodies found in the blood or cerebrospinal fluid of patients with multiple sclerosis and acute and chronic inflammatory demyelinating neuropathy. The Chinese invention patent "Human NF186 antigen, human NF186 antibody detection kit and its preparation method and application" with publication number CN109734791A discloses the preparation of a kit for detecting NF186 autoantibodies. The "Chinese Expert Consensus on the Diagnosis and Treatment of Chronic Inflammatory Demyelinating Polyradiculoneuropathy 2022 Edition" points out that different items can be selected according to the clinical characteristics of the patient to distinguish chronic inflammatory demyelinating polyradiculoneuropathy from other diseases. These items include the detection of NF186 protein antibodies (Chinese Medical Association Neurology Branch, etc. Chinese Expert Consensus on the Diagnosis and Treatment of Chronic Inflammatory Demyelinating Polyradiculoneuropathy 2022 [J]. Chinese Journal of Neurology, 2023, 56 (2): 125-132.).

[0004] In in vitro diagnostic reagents, reference products are an important part of the quality control of the detection system. Manufacturers use internal reference products for quality control in the product development and production stages. Common internal reference products include antibodies and antigens. There are two main sources of raw materials for internal reference products of antibodies: positive serum and humanized monoclonal antibodies. Due to the high cost of obtaining positive serum, it is difficult to determine the value and trace the source. Therefore, it is necessary to provide a positive control sample or positive reference product that can replace the patient's positive serum. Chimeric antibodies or humanized antibodies can meet such needs. However, the commercialized NF186 antibodies currently available are rabbit / mouse monoclonal / polyclonal antibodies. Since mouse / rabbit polyclonal antibodies are directly purified from the serum of immunized mice / rabbits, there are certain differences in immunized mice / rabbits at different times, and it is difficult to ensure that the types of antibodies between different batches are completely consistent; and the packaging specifications of commercial mouse / rabbit monoclonal / polyclonal antibodies are small, expensive, and the long-term purchase cost is too high. Specifically, this type of antibody has the following problems: (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 monoclonal antibody is not public, and low-cost, large-scale industrial mass production cannot be achieved; (3) Its Fc region is a mouse / rabbit sequence and cannot react with anti-human IgG antibodies. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a human-mouse chimeric monoclonal antibody of NF186 protein and its product and a method for detecting NF186 protein, so as to solve the technical problems that the current commercial antibodies have batch differences, cannot form low-cost large-scale industrial production, and their Fc regions cannot react with anti-human IgG antibodies. At the same time, a new NF186 monoclonal antibody can be provided for identifying the expression and localization of NF186 antigen.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a human-mouse chimeric monoclonal antibody of NF186 protein, comprising a light chain and a heavy chain, wherein:

[0008] The variable region of the light chain includes three light chain complementary determining regions, and the amino acid sequences of the three light chain complementary determining regions are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively;

[0009] The variable region of the heavy chain includes three heavy chain complementary determining regions, and the amino acid sequences of the three heavy chain complementary determining regions are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0010] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.6.

[0011] Further preferably, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody against the NF186 protein is as shown in SEQ ID NO.2; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody against the NF186 protein is as shown in SEQ ID NO.7.

[0012] Preferably, the human-mouse chimeric monoclonal antibody comprises a light chain having a complementarity determining region of an amino acid sequence that is at least 80% identical to the sequence of the variable region of the light chain, and the human-mouse chimeric monoclonal antibody comprises a heavy chain having a complementarity determining region of an amino acid sequence that is at least 80% identical to the sequence of the variable region of the heavy chain; and the complementary domains of the light chain and the complementary domains of the heavy chain maintain NF186 binding ability.

[0013] The invention discloses a nucleic acid molecule, which encodes the human-mouse chimeric monoclonal antibody of the NF186 protein.

[0014] The invention discloses a vector, which comprises a nucleotide sequence encoding the human-mouse chimeric monoclonal antibody of the NF186 protein.

[0015] The present invention discloses a host cell, characterized in that the host cell comprises the above-mentioned vector.

[0016] The present invention also discloses the use of the human-mouse chimeric monoclonal antibody, vector or host cell of the NF186 protein in preparing a reagent / kit for detecting the NF186 protein.

[0017] The present invention also discloses the use of the human-mouse chimeric monoclonal antibody, vector or host cell of the NF186 protein in preparing a chromatography column for separating and purifying the NF186 protein.

[0018] The present invention also discloses the use of the human-mouse chimeric monoclonal antibody, vector or host cell of the NF186 protein in the preparation of reagents / kits for human chronic inflammatory demyelinating polyradiculoneuropathy and / or prognosis judgment.

[0019] The present invention also discloses a method for detecting NF186 protein or NF186 polypeptide, wherein the sample to be tested is contacted with the human-mouse chimeric monoclonal antibody to detect the NF186 protein or NF186 polypeptide in the sample.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discloses a human-mouse chimeric monoclonal antibody of NF186 protein. The mouse monoclonal antibody sequence of NF186 protein is first obtained by molecular biological means, and then the human-mouse chimeric monoclonal antibody of NF186 protein is prepared by modification. The human-mouse chimeric antibody can be expressed and purified on a large scale by recombinant protein expression and purification technology, and a large amount of antibodies can be obtained at one time at a low cost. The intra-batch difference and inter-batch difference can be controlled and kept consistent. The obtained human-mouse chimeric monoclonal antibody can recognize NF186 protein, and its Fc region can also be recognized by anti-human IgG secondary antibody, and can replace the positive serum of NF186 protein as a quality control product / reference product / calibrator in a diagnostic kit to identify the situation of NF186 protein on a cell plate overexpressing NF186 protein / on a carrier coated with NF186 protein in the kit. Therefore, the present invention solves the technical problems that the current mouse / rabbit mono / polyclonal antibodies have batch differences, cannot form low-cost large-scale industrial production, and their Fc regions cannot react with anti-human IgG antibodies.

[0022] In addition, the antibody disclosed in the present invention can also be used as a ligand in an affinity layer system to prepare a chromatography column to achieve separation and purification of NF186 protein, etc. At the same time, since the signal morphology generated by the combination of the human-mouse chimeric antibody of NF186 protein and the cell slide overexpressing NF186 is similar to the signal morphology generated by positive blood, a large number of fluorescent images can be obtained through multiple experiments to enrich the image library of NF186 antibody positive, provide a large number of different types of signals for the development of the automatic processing system of immunofluorescence images, and improve the accuracy of automatic interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is the result of the determination of the binding titer of serum antibodies of three mice to NF186 protein;

[0024] Figure 2 This is the result of ELISA determination of the binding titer of hybridoma 16F12 supernatant and NF186 protein;

[0025] Figure 3 Immunofluorescence detection of the binding between the hybridoma 16F12 supernatant and the NF186 protein; A is a slide of cells overexpressing an empty vector; B is a slide of cells overexpressing NF186;

[0026] Figure 4 This is a diagram showing the purification results of the human-mouse chimeric antibody 16F12;

[0027] Figure 5 To verify the specificity of the human-mouse chimeric antibody 16F12 by CBA method;

[0028] Figure 6The specificity of the human-mouse chimeric antibody 16F12 was verified by WB; A is the result of the commercial antibody; the exposure time is 500ms; B is the result of the human-mouse chimeric antibody 16F12 of the present invention of the present application; the exposure time is 50s;

[0029] Figure 7 The figure is a comparison of the signal morphology between the human-mouse chimeric antibody 16F12 and the NF186 protein positive serum; wherein A is the result of incubating the human-mouse chimeric antibody 16F12, A-1, A-2 and A-3 are the pictures of signal amplification; B is the result of incubating the positive serum 1; C is the result of incubating the positive serum 2; and D is the result of incubating the positive serum 3. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0033] The term "antibody" includes 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 antigen, such as Fab fragments or fragments produced from a Fab expression library.

[0034] The term "monoclonal antibody (MAb)" refers to an antibody population of only one species consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity determining region (CDR) of the monoclonal antibody is the same in all molecules of the antibody population and can bind to the antigen binding site of a specific epitope of the antigen.

[0035] Among them, the term "chimeric antibody" means an antibody in which the sequences of the H chain variable region and the L chain variable region (VH and VL) are derived from non-human animal species and the constant region sequences (CH and CL) are derived from humans. The sequence of the variable region is preferably derived from, for example, an animal species that allows easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0036] Among them, the term "humanized monoclonal antibody" refers to replacing the CDR region of a human antibody with the CDR region from a non-human animal species antibody, also known as a CDR transplanted antibody. The CDR sequence of a non-human animal species antibody is preferably derived from, for example, an animal species that allows easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0037] Among them, the term "antigen binding site" refers to the part of the antibody molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable region (V) of the heavy chain (H) and the light chain (L). Three highly branched extensions called "hypervariable regions" in the V region of the heavy chain and the light chain are inserted between multiple conserved lateral extensions called "framework regions" or "FRs". In the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs", and the three "CDRs" are usually called CDR1, CDR2 and CDR3 and exist from the N-terminus of the light chain / heavy chain. The light chain and heavy chain CDR regions are often defined by the Kabat, IMGT, Abm, Chothia or Martin (Extended Chothia) numbering system. The Kabat definition scheme is based on sequence variability; the Chothia definition scheme is based on the position of the structural loop region; and the Contact definition scheme is based on existing antibody complex crystal structure data. Using Kabat to define the CDR region of the antibody variable region, the CDR region of the light chain variable region VL is defined by the residues at the amino acid sequence numbering 44-54 (CDR1), 70-76 (CDR2), and 109-117 (CDR3) shown in SEQ ID NO: 1, and the CDR region of the heavy chain variable region VH is defined by the residues at the amino acid sequence numbering 50-54 (CDR1), 69-85 (CDR2), and 118-127 (CDR3) shown in SEQ ID NO: 6.

[0038] Wherein, the term "polypeptide" refers to a protein, a protein fragment, and a fragment or analog of a polypeptide sequence. Protein fragments and analogs are considered to be polypeptides. Further, according to the present invention, examples of polypeptides include a light chain immunoglobulin molecule represented by SEQ ID NO: 1 and a heavy chain immunoglobulin molecule represented by SEQ ID NO: 6 and CDRs represented by SEQ ID NO: 3, 4, 5, 8, 9 and 10, antibody molecules formed by a combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule, such as a kappa light chain immunoglobulin molecule (or vice versa), and fragments and analogs thereof.

[0039] Among them, NF186 (Neuro-oncological ventral antigen 1, NF186) protein: NF186 protein is the main neuropilin subtype expressed in mature neurons. The NF186 protein mentioned in the present invention refers to human NF186 protein, or refers to a polypeptide, fragment or analog that retains the binding ability of NF186 protein.

[0040] Wherein, the term "amino acid" refers to the 20 conventional amino acids and their abbreviations used in the present invention as conventionally used.

[0041] The monoclonal antibody of the present invention also includes antibodies having "equivalent binding properties" to the antibody, and the antibodies having "equivalent binding properties" can bind to the NF186 protein or polypeptide.

[0042] Specifically, changes in the amino acid sequence of antibodies with "equivalent binding properties" are considered to be covered in the present invention, provided that the changes in the amino acid sequence of antibodies with "equivalent binding properties" maintain at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99%. Including certain percentages in between, such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity.

[0043] Wherein, the term "gene" or "DNA" or "nucleic acid molecule": includes not only double-stranded DNA, but also the corresponding single-stranded DNA, the sense strand and the antisense strand constituting the double-stranded DNA. It is not particularly limited by its length. Unless otherwise specified, the gene (DNA) in this specification includes double-stranded DNA (including human genomic DNA), single-stranded DNA (positive strand) (including cDNA), single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof. According to the present invention, examples of nucleic acid molecules include the nucleotide sequence encoding the light chain variable region of the above-mentioned NF186 protein human-mouse chimeric monoclonal antibody (SEQ ID NO.2); the nucleotide sequence encoding the heavy chain variable region of the above-mentioned NF186 protein human-mouse chimeric monoclonal antibody (SEQ ID NO.7); the nucleotide sequence encoding the light chain constant region of human Ig Kappa (SEQ ID NO.11), specifically AGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG, and its corresponding amino acid sequence is SEQ ID NO.13, specifically: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC*; nucleotide sequence encoding the constant region of the heavy chain of human IgG1 (SEQ ID NO.12),Specifically, GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA,The corresponding amino acid sequence is SEQ IDNO.14, specifically: ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*. The nucleic acid molecules of the invention can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example by combining chemical synthesis and recombinant methods.

[0044] The invention still further relates to the variant of above-mentioned nucleic acid molecules, and nucleic acid molecule variant can be naturally occurring variant, such as naturally occurring allele variant, or it can be the variant that non-natural exists.By mutagenesis technique, comprise those mutagenesis techniques that are applied to nucleic acid molecules, cell or organism, these non-natural existence variants of nucleic acid molecules can be prepared.

[0045] The vector of the present invention includes the above-mentioned nucleic acid molecules. Many suitable vectors are known to those skilled in the art of molecular biology, and the selection depends on the desired function. The present invention is not particularly limited to the vector, and can be a vector that can replicate and / or express polynucleotides in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacterial cells (such as E. coli). Non-limiting examples of vectors include 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, pX T1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPO RT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K and pPIC3.5K.

[0046] The host cell described in the present invention includes the above-mentioned nucleic acid molecule or vector.

[0047] In an embodiment of the present invention, the host cells used to introduce the vector include prokaryotic cells and eukaryotic cells, including but not limited to bacterial cells, such as Escherichia coli; yeast cells; fungal cells such as Pichia pastoris, Saccharomyces cerevisiae; insect cells such as Sf9 cells, Sf21 cells, Tn-368 cells, High Five cells; animal cells, such as CHO cells, COS, NSO, 293T, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, PERC.6 (human retinal cells); plant cells. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.

[0048] The present invention provides a method for detecting NF186 protein or polypeptide, which comprises contacting a sample with the human-mouse chimeric monoclonal antibody, thereby detecting the NF186 protein or polypeptide in the sample.

[0049] In the present invention, the method for detecting or measuring the amount of a protein or polypeptide can be any known immunological detection technique, including immunoagglutination assay, immunoprecipitation assay, enzyme-linked immunosorbent assay, fluorescent immunoassay, immunoelectron microscopy, immunoblotting assay, and immunomagnetic bead assay. Immunological detection techniques are methods for detecting or measuring the amount of an antibody or antigen using labeled antigens or antibodies. Biological samples that can be tested by the above-mentioned immunoassays or assays include, but are not limited to, plasma, whole blood, dried whole blood, serum, tissues, cells, or extracts of tissues and cells.

[0050] In an embodiment of the present invention, enzyme immunoassay includes but is not limited to indirect immunofluorescence, direct ELISA, dot blot (linear blot). A method is listed, which includes adsorbing human NF186 polypeptide (antigen) onto a solid support NC membrane, blocking the solid phase with a blocking protein (e.g., skim milk powder, bovine serum albumin, etc.), contacting and incubating the NF186 protein human-mouse chimeric monoclonal antibody with the solid phase, removing unreacted antibodies, and adding a labeled second antibody that specifically reacts with the NF186 protein human-mouse chimeric monoclonal antibody to the solid phase to determine the amount of the marker on the solid phase.

[0051] The product of the present invention includes the above-mentioned monoclonal antibody, nucleic acid molecule, vector or host cell.

[0052] As an optional embodiment, the product is a kit; the kit includes the antibody prepared by the present invention. As another optional embodiment, the kit of the present invention includes a diagnostic composition, the diagnostic composition includes at least one detectable labeling substance, as the labeling substance, a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, etc. can be used. The enzyme can be β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.; the fluorescent substance can be fluorescamine, fluorescein isothiocyanate, etc.; the luminescent substance can be luminol, luminol derivatives, luciferin, lucigenin, etc.

[0053] As an optional embodiment, the kit includes a protein immunoassay kit; the protein immunoassay kit includes the above-mentioned monoclonal antibody or a fragment thereof.

[0054] The present invention provides the use of the above-mentioned monoclonal antibody, nucleic acid molecule, vector, host cell or product in detecting NF186 protein or polypeptide product.

[0055] Example 1 Preparation of mouse monoclonal antibodies

[0056] 1. Preparation of NF186 recombinant protein

[0057] The human NF186 gene sequence was obtained from the GenBank sequence database, and the NCBI sequence number was NM_001005388.3. The gene sequence was synthesized by Jinweizhi Company into the pET30a vector, and the insertion sites were EcoR I and Xba I to obtain the NF186 recombinant vector. The constructed and correctly sequenced recombinant vector plasmid was transferred into Escherichia coli Arctic Express (Agilent) competent cells; a single clone was picked and inoculated into LB medium, and the culture was shaken at 37°C until the OD 600 value of 0.5 to 1, induced by adding IPTG at a final concentration of 0.5 mM, and expressed at 16°C overnight; the bacteria were collected, ultrasonically disrupted, and the supernatant was collected after centrifugation. The NF186 protein expressed by pET30a was purified using Ni-NTA (QIAGEN), and the purified protein was recorded as NF186 recombinant protein.

[0058] 2. Mouse Antigen Immunization

[0059] The NF186 recombinant protein prepared above was used to immunize three female Balb / c mice aged 6-8 weeks. The immunization required three times, and the amount of antigen used to immunize the mice was 40 μg / mouse. The first immunization was to mix 40 μg of antigen with equal volumes of Freund's adjuvant, fully grind it into a water-in-oil emulsion, and inject 100 μL (40 μg antigen) of the mixed antigen intraperitoneally. The second immunization was two weeks after the initial immunization, 40 μg of antigen was mixed with incomplete Freund's adjuvant 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally. The third immunization was performed two weeks after the second immunization, and the dose, method, and route of the injected antigen were the same as the second immunization. After an interval of 2 weeks, the antigen was injected intraperitoneally to strengthen the immunization, and the tail blood was taken from the mouse 3 days later to detect its titer using ELISA.

[0060] The specific detection process is as follows: use carbonate buffer with a pH value of 9.6 to coat the purified NF186 recombinant protein at 4°C overnight, 100 ng / well; wash with PBST three times the next day, 3 min each time, and pat dry; block the wells with 2% BSA and incubate at 37°C for 1 h; wash with PBST three times, 3 min each time, and pat dry; dilute the sera of three mice with PBS in multiple ratios, at 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, and 1:109350, add 100 μL to each well, incubate at 37°C for 1 h, wash with PBST three times, 3 min each time, and pat dry; dilute goat anti-mouse IgG-HRP 1:5000 (Cat. No. 115-035-003, Jackson) and incubate at 37°C for 30 min. Wash with PBST three times, pat dry, develop with TMB for 10 min, terminate with 2M H2SO4, and measure absorbance at 450 nm. Figure 1As shown, based on the test results and the comprehensive titer of serum antibodies, mouse No. 3 was selected for subsequent experiments.

[0061] 3. Cell fusion and hybridoma culture

[0062] Immunization was enhanced by intraperitoneal injection of antigens. The animals were killed 3 days later and spleen cells were obtained for cell fusion. A 37°C water bath was prepared in a clean bench. 5-6×10 7 Mouse myeloma SP2 / 0 cells and spleen cells in logarithmic growth phase and in good growth condition were added to a 50mL centrifuge tube at a ratio of 1:10 and mixed. Centrifuge at 500g for 10min, aspirate the supernatant, flick the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly drip 1mL of 45% PEG1450 solution preheated to 37℃ within 90s, and shake the centrifuge tube gently continuously. The centrifuge tube was in a 37℃ water bath during the whole process. Then gradually add DMEM culture medium to the cell mixture, drip 1mL dropwise in the first minute, add 2mL in the second minute, add 3mL in the third minute, add 4mL in the fourth minute, add 5mL in the fifth minute, and shake while adding in a 37℃ water bath. Then incubate at 37℃ for 15min, centrifuge at 500g for 5min, and remove the supernatant. Add 5 mL of DMEM medium containing HAT (thermo, catalog number: 21060017), gently suspend the precipitated cells, and finally add DMED medium containing HAT to about 100 mL. Dispense into a 96-well cell culture plate with macrophages, 100 μL / well, and then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0063] 4. Screening of positive hybridomas

[0064] ELISA was used to select positive clones. The growth of hybridoma cells was observed. After seven days, when the cell culture supernatant turned yellow, an appropriate amount of cell supernatant was taken for ELISA antibody detection. According to the ELISA results, clones with high OD values ​​were selected, which were more than twice that of the negative control, and 96-well plates were spread for the first subclone screening. After seven to ten days, ELISA was performed again to detect antibodies, clones with high OD values ​​were selected, 96-well plates were spread, and the second subclone screening was performed, so that there was about 1 cell per well. After seven to ten days of culture, ELISA was performed again to detect antibodies, clones with high OD values ​​were selected, 96-well plates were spread, and the third subclone screening was performed, so that there was about 1 cell per well. After three screenings, a clone with good signal was initially screened out and named hybridoma 16F12. The supernatant of cultured hybridoma 16F12 was diluted to 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, and the titer was detected by ELISA. At the same time, the supernatant of cells transfected with empty pCDNA3.1 was used as a control. The results are shown in Figure 2 As shown by Figure 2It can be seen that NF186 protein can bind to the antibody in the supernatant of hybridoma 16F12, but not react with the supernatant of cells transfected with empty pCDNA3.1. Therefore, positive hybridomas that can secrete NF186 protein antibodies were screened by ELISA method.

[0065] The positive hybridomas screened by ELISA were verified by indirect immunofluorescence. Preparation of cell slides overexpressing NF186 protein: The NF186 gene was linked to pCDNA3.1 by molecular biology methods to obtain the recombinant plasmid pCDNA3.1-NF186. The recombinant plasmid was transfected into 293T cells with a cell density of 30% to 40% using PEI transfection reagent (transfection reagent purchased from Thermo Company, refer to the instructions for transfection) (a 6cm*6cm cell slide has been placed in advance in a 10cm cell culture dish). After 48 hours of transfection, acetone was added to fix at 4°C for 5 minutes, washed twice with PBS, and cut the 6cm*6cm cell slide into a size of 0.25cm*0.25cm after drying for later use. The cell slide transfected with empty pCDNA3.1 was used as the control cell slide, and its preparation method was the same as that of the cell slide overexpressing NF186 protein. The supernatant of hybridoma 16F12 was diluted 100 times as the primary antibody and incubated on the cell slides overexpressing NF186 protein and control cell slides, incubated at room temperature for 1 hour, washed 3 times with PBST, each time for 5 minutes; incubated with Alexa 594-labeled goat anti-mouse secondary antibody (Jackson) was used for 40 min, and the cells were washed 3 times with PBST, each time for 5 min. The fluorescence microscope was used to take pictures. The results are shown in the figure. Figure 3 As shown in the figure, compared with the control cell slide, the cell slide overexpressing NF186 protein binds to the antibody in the supernatant of hybridoma 16F12 to produce a significant signal. Therefore, the antibody secreted by hybridoma 16F12 can not only recognize the prokaryotic NF186 protein coated in the ELISA plate, but also recognize the NF186 protein expressed in eukaryotic cells.

[0066] 5. Identification of antibody typing

[0067] The identification of antibody typing was performed by indirect ELISA. The specific process was as follows: overnight coated purified NF186 protein (100 ng / well), blocked with 2% BSA, incubated with the hybridoma 16F12 supernatant obtained in Example 1 at 37°C for 1 hour, washed 3 times with PBST, and then incubated with different types of commercial 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, washed 3 times with PBST, and then developed with TMB for 5 minutes. The indirect ELISA results showed that the heavy chain subtype of the 16F12 antibody was IgG1, and the light chain subtype was kappa.

[0068] Example 2 Identification of the sequence of mouse monoclonal antibody against NF186 protein

[0069] The positive hybridoma 16F12 used for antibody production was grown 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 SMARTer RACE 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 then sequenced by a sequencing company. The Kabat method was used to mark the CDR region of the antibody amino acid sequence.

[0070] Table 1 Sequence of mouse monoclonal antibody 16F12

[0071]

[0072] Example 3 Preparation of human-mouse chimeric antibody against NF186 protein

[0073] With reference to the sequences in the Kabat database, molecular biology methods were used to -Uni Seamless Cloning and Assembly Kit, the nucleotide sequence of the light chain variable region VL of the mouse monoclonal antibody 16F12 encoding the NF186 protein (shown in SEQ ID NO.2) and the human kappa light chain constant region sequence SEQ ID NO.11 were connected to the vector pcDNA3.1, marked as pcDNA3.1-NF186-VL, the nucleotide sequence of the heavy chain variable region VH of the mouse monoclonal antibody 16F12 encoding the NF186 protein SEQ ID NO.7 and the human IgG1 heavy chain constant region sequence SEQ ID NO.12 were connected to the vector pcDNA3.1, marked as pcDNA3.1-NF186-VH, the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing, and the sequenced recombinant plasmid was collected for use. The recombinant plasmids pcDNA3.1-NF186-VL and pcDNA3.1-NF186-VH were co-transfected into Expi293F cells for transient expression using PEI according to the thermo instructions. The supernatant was collected 3 days after transfection and purified using protein A filler. The protein concentration was 2.7 mg / mL (Solybol) measured by the BCA method. The obtained recombinant antibody was named NF186 human-mouse chimeric antibody 16F12 ( Figure 4 ).

[0074] Example 4 Application of human-mouse chimeric monoclonal antibody against NF186 protein

[0075] 1. Verification of the specificity of human-mouse chimeric monoclonal antibody against NF186 protein by indirect immunofluorescence assay

[0076] NF186 protein and NF155 protein are different types of neural bundle proteins, and the amino acid sequence similarity of the two proteins is 89.72%. Therefore, this example investigates whether the NF186 protein human-mouse chimeric antibody prepared by the present invention can be used to distinguish NF155 protein from NF186 protein.

[0077] A recombinant vector overexpressing NF155 (sequence number: NM_001160331.2) was constructed using molecular biological methods: pCDNA3.1-NF155. The cell slides overexpressing NF155 were prepared by referring to the process of preparing cell slides overexpressing NF186 in Example 1. The human-mouse chimeric antibody 16F12 purified in Example 3 was diluted to 1 mg / ml, and then diluted at a ratio of 1:100 and incubated as the primary antibody on cell slides overexpressing pCDNA3.1, overexpressing NF155, and overexpressing NF186. The secondary antibody was incubated with FITC-labeled goat anti-human secondary antibody (jackson) to verify the specificity of the human-mouse chimeric antibody 16F12. Figure 5It can be seen that the modified human-mouse chimeric monoclonal antibody 6F12 can recognize NF186 protein and does not react with cell slides overexpressing pCDNA3.1 and NF155. Therefore, it can be seen that the human-mouse chimeric antibody 16F12 has the ability to recognize NF186 protein and good specificity.

[0078] 2. WB verification of the binding between NF186 protein human-mouse chimeric monoclonal antibody and NF186 protein

[0079] The recombinant vector pCDNA3.1-NF186 was transferred into 1 dish (10 cm) of 293T cells with a cell density of 30% to 40%. After 48 hours, the cells were collected, added with 200 μL PBS, and ultrasonically disrupted (the disruption conditions were: 10% power, disruption for 3 seconds, stop for 6 seconds, and ultrasonication for a total of 1 minute) as the WB loading protein; the control protein was prepared using cells transfected with empty pcDNA3.1, and the preparation method was the same as above.

[0080] The prepared NF186 protein and control protein were loaded for electrophoresis at a loading amount of 1 μg / well. After the electrophoresis was completed, wet transfer was used at 200 mA for 60 min, and 5% skim milk powder was used for blocking at room temperature for 1 h. The membranes were incubated with a 1:3000 dilution of commercial NF186 antibody (abcam, catalog number ab31457) and a 1:1000 dilution of human-mouse chimeric antibody 16F12 (final concentration of 1 μg / ml), respectively, at room temperature for 2 h, and washed 3 times with TBST, each time for 5 min; HRP-labeled goat anti-rabbit secondary antibody (Jackson) was added to the membrane incubated with commercial antibody, and HRP-labeled goat anti-human secondary antibody (Jackson) was added to the membrane incubated with human-mouse chimeric antibody 16F12, at room temperature for 1 h, and washed 3 times with TBST, each time for 5 min; chemiluminescent solution was added for color development and photography, and the results are shown as follows Figure 6 As shown. Figure 6 It can be seen that in WB, both the commercial NF186 antibody and the human-mouse chimeric antibody 16F12 can recognize the NF186 protein.

[0081] comprehensive Figure 5 and Figure 6 The results show that the human-mouse chimeric antibody 16F12 prepared by the present invention can recognize both the conformational epitope and the linear epitope of the NF186 protein.

[0082] 3. Comparison of the signals produced by NF186 protein human-mouse chimeric antibody and positive serum by indirect immunofluorescence method

[0083] The NF186 overexpressing cell slides prepared in Example 1 were incubated with sera from three patients with positive NF186 antibodies and the NF186 protein human-mouse chimeric antibody 16F12 obtained in Example 3 of the present invention, and the secondary antibody was incubated with FITC-labeled goat anti-human IgG secondary antibody (Jackson). The staining results were as follows: Figure 7 As shown. Figure 7 It can be seen that the NF186 protein human-mouse chimeric antibody 16F12 can not only recognize the overexpressed NF186 antigen and bind to the anti-human IgG antibody, but also the signal morphology generated is similar to that of the NF186 antibody positive serum. Therefore, the modified human-mouse chimeric antibody NF186 of the present invention can be expressed and purified on a large scale, and a large amount of antibodies can be obtained at a low cost at one time, and the intra-batch difference and inter-batch difference can be controlled and kept consistent; on the other hand, it can replace the positive serum of NF186 protein as a quality control product / reference product / calibrator in the diagnostic kit to identify the NF186 protein on the cell slides overexpressing NF186 protein / the carrier coated with NF186 protein in the kit. At the same time, since the signal morphology generated by the combination of the NF186 protein human-mouse chimeric antibody 16F12 and the overexpressed NF186 cell slides is similar to the signal morphology generated by positive blood, a large number of fluorescent images can be obtained through multiple experiments, enriching the image library of NF186 protein antibody positive, providing a large number of different styles of signals for the development of the automatic processing system of immunofluorescence images, and improving the accuracy of automatic interpretation.

[0084] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A human-mouse chimeric monoclonal antibody against NF186 protein, characterized in that: It consists of light and heavy chains, of which: The variable region of the light chain includes three light chain complementary determining regions, and the amino acid sequences of the three light chain complementary determining regions are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively; The variable region of the heavy chain includes three heavy chain complementary determining regions, and the amino acid sequences of the three heavy chain complementary determining regions are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The human-mouse chimeric monoclonal antibody of NF186 protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.

6.

3. The human-mouse chimeric monoclonal antibody of NF186 protein according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody against the NF186 protein is shown in SEQ ID NO.2; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody against the NF186 protein is shown in SEQ ID NO.

7.

4. The human-mouse chimeric monoclonal antibody of NF186 protein according to claim 1, characterized in that: The human-mouse chimeric monoclonal antibody comprises a light chain having a complementarity determining region of an amino acid sequence that is at least 80% identical to the sequence of the variable region of the light chain, and the human-mouse chimeric monoclonal antibody comprises a heavy chain having a complementarity determining region of an amino acid sequence that is at least 80% identical to the sequence of the variable region of the heavy chain; and the complementary domains of the light chain and the heavy chain maintain NF186 binding ability.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the human-mouse chimeric monoclonal antibody of the NF186 protein according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises a nucleotide sequence encoding the human-mouse chimeric monoclonal antibody of the NF186 protein according to any one of claims 1 to 4.

7. A host cell, characterized in that The host cell comprises the vector according to claim 6.

8. Use of the human-mouse chimeric monoclonal antibody against NF186 protein according to any one of claims 1 to 4, the vector according to claim 6, or the host cell according to claim 7 in the preparation of a reagent / kit for detecting NF186 protein, or in the preparation of a chromatography column for separating and purifying NF186 protein.

9. Use of the human-mouse chimeric monoclonal antibody against NF186 protein according to any one of claims 1 to 4, the vector according to claim 6, or the host cell according to claim 7 in the preparation of reagents / kits for human chronic inflammatory demyelinating polyradiculoneuropathy and / or prognosis judgment.

10. A method for detecting NF186 protein or NF186 polypeptide, characterized in that: The sample to be tested is contacted with the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 4 to detect the NF186 protein or NF186 polypeptide in the sample.

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