Human-mouse chimeric monoclonal antibody of nf186 protein, products thereof and method for detecting nf186 protein

Abstract

The application discloses a human-mouse chimeric monoclonal antibody of NF186 protein, a product thereof and a method for detecting the NF186 protein, and belongs to the technical field of monoclonal antibodies. The application first obtains a mouse monoclonal antibody sequence of the NF186 protein through a molecular biology method, then the mouse monoclonal antibody sequence is reformed 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 amount of the antibody can be obtained at one time at a low cost, and the batch difference and the batch-to-batch difference can be controlled and kept consistent. The application can effectively solve the technical problems that the current mouse / rabbit single / multiclonal antibodies have batch-to-batch differences, cannot form low-cost large-scale industrial production, and the Fc region cannot react with an anti-human IgG antibody.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monoclonal antibodies, and particularly relates to a human-mouse chimeric monoclonal antibody of NF186 protein, a product thereof and a method for detecting the NF186 protein. BACKGROUND

[0002] Neurofascin (NF) is a cell surface protein involved in the formation of nerve bundles discovered in vivo in chickens by Ranthjen et al. in 1987, which belongs to a cell adhesion molecule and plays a crucial role in the formation and maintenance of Ranvier nodes. Currently, there are five different NF polypeptide types reported, i.e. NF140, NF155, NF166, NF180 and NF186. Studies have shown that on the axons of mature neurons, NF186 maintains the stability of the neuron structure through interaction 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 axon-Schwann cell microvilli, that is, the main role of NF186 is to form a node complex as a molecular barrier to limit the diffusion and migration of proteins and molecules in the paranode to the node, thereby promoting the composition of the myelinated axon domain and the saltatory conduction of signals along the axon. Therefore, insufficient expression of NF186 will directly affect the insulation of the myelin paranode, and in multiple sclerosis patients, the distribution of ion channels in the corresponding axolemma of each myelin region is disordered, the Na + channel structure in the node is destroyed and cannot be aggregated, and the Kv1.2 channel originally located in the paranode crosses the paranode to the node, resulting in failure of rapid nerve conduction of myelinated fibers.

[0003] Currently, neurofascin-specific antibodies have been found in some autoimmune demyelinating diseases in humans, such as specific autoantibodies of NF155 and NF186 found in the blood or cerebrospinal fluid of multiple sclerosis patients and patients with acute and chronic inflammatory demyelinating neuropathy. The Chinese invention patent with publication number CN109734791A "Human NF186 antigen, human NF186 antibody detection kit and preparation method and application thereof" discloses the preparation of a kit for detecting NF186 autoantibodies. The "2022 version of Chinese expert consensus on diagnosis and treatment of chronic inflammatory demyelinating polyradiculoneuropathy" points out that different items can be selected according to the clinical characteristics of patients to distinguish chronic inflammatory demyelinating polyradiculoneuropathy from other diseases, and these items include detection of NF186 protein antibodies (Chinese Medical Association Neurology Branch, et al. 2022 version of Chinese expert consensus on diagnosis and treatment of chronic inflammatory demyelinating polyradiculoneuropathy [J]. Chinese Journal of Neurology, 2023, 56(2): 125-132.).

[0004] In vitro diagnostic reagents, reference products are an important part of the quality control of detection systems, and enterprises use internal reference products for product research and development and production stage quality control. Common internal reference products include antibodies and antigens. The raw material sources of antibody internal reference products mainly have two ways: positive serum and humanized monoclonal antibody. Because the cost of obtaining positive serum is high, it is difficult to value and trace. Therefore, it is necessary to provide a positive control sample or a positive reference product that can replace patient positive serum, and a chimeric antibody or a humanized antibody can meet such a demand. However, the existing commercial NF186 antibody is a rabbit / mouse monoclonal antibody, and because the mouse / rabbit polyclonal antibody is directly purified from the serum of the immunized mouse / rabbit, there are certain differences between the immunized mice / rabbits at different times, and it is difficult to ensure that the antibody types of different batches are completely consistent. Moreover, the packaging specification of the commercial mouse / rabbit monoclonal antibody is small, and the price is expensive, and the long-term purchase cost is too high. Specifically, such antibodies have 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 disclosed, and it is impossible to form a low-cost, large-scale industrialized batch production; (3) The Fc region is a mouse / rabbit sequence, and cannot react with an antibody against human IgG. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a human-mouse chimeric monoclonal antibody of NF186 protein and a product thereof and a method for detecting NF186 protein, to solve the technical problems that the existing commercial antibody has batch differences, cannot form a low-cost large-scale industrialized production, and the Fc region cannot react with an antibody against human IgG, and at the same time, a new NF186 monoclonal antibody is provided for identifying the expression and positioning of the NF186 antigen.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

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

[0008] The variable region of the light chain comprises three light chain complementarity determining regions, and the amino acid sequences of the three light chain complementarity 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 comprises three heavy chain complementarity determining regions, and the amino acid sequences of the three heavy chain complementarity 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 of the NF186 protein is shown as SEQ ID NO. 2, and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody of the NF186 protein is shown as SEQ ID NO. 7.

[0012] Preferably, the human-mouse chimeric monoclonal antibody comprises a light chain of the complementary determining region of the amino acid sequence having at least 80% identity with the sequence of the variable region of the light chain, and comprises a heavy chain of the complementary determining region of the amino acid sequence having at least 80% identity with the sequence of the variable region of the heavy chain; and the complementary domain of the light chain and the complementary domain of the heavy chain maintain the NF186 binding ability.

[0013] The present application discloses a nucleic acid molecule encoding the human-mouse chimeric monoclonal antibody of the NF186 protein.

[0014] The present application discloses a vector comprising the nucleotide sequence encoding the human-mouse chimeric monoclonal antibody of the NF186 protein.

[0015] The present application discloses a host cell, characterized in that the host cell comprises the vector described above.

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

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

[0018] The present application also discloses the application of the human-mouse chimeric monoclonal antibody, the vector or the host cell of the NF186 protein described above in the preparation of a reagent / reagent kit for human chronic inflammatory demyelinating polyradiculoneuropathy and / or prognosis.

[0019] The present application also discloses a method for detecting the NF186 protein or the NF186 polypeptide, which comprises contacting a sample to be detected with the human-mouse chimeric monoclonal antibody described above, and detecting the NF186 protein or the NF186 polypeptide in the sample.

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

[0021] The application discloses a human-mouse chimeric monoclonal antibody of NF186 protein, and first obtains a mouse monoclonal antibody sequence of the NF186 protein through a molecular biology method, and then the human-mouse chimeric monoclonal antibody of the NF186 protein is prepared by modification, 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 amount of antibody can be obtained at one time at a low cost, the batch difference and the batch-to-batch difference can be controlled and kept consistent, the obtained human-mouse chimeric monoclonal antibody can recognize the NF186 protein, the Fc region thereof can also be recognized by an anti-human IgG secondary antibody, and the human-mouse chimeric monoclonal antibody can replace the positive serum of the NF186 protein and be used as a quality control product / control product / calibration product in a diagnostic kit to identify the NF186 protein on a cell slide overexpressing the NF186 protein / a carrier coated with the NF186 protein. Therefore, the application solves the technical problems that the current mouse / rabbit single / multiclonal antibody has batch-to-batch difference, cannot form large-scale industrialized production at a low cost, and the Fc region thereof cannot react with an anti-human IgG antibody.

[0022] In addition, the antibody disclosed by the application can also be used as a ligand in an affinity layer system to prepare a chromatography column, so that the NF186 protein can be separated and purified. At the same time, since the human-mouse chimeric antibody of the NF186 protein has a signal form similar to that of the positive blood when combined with the cell slide overexpressing the NF186 protein, a large amount of fluorescent pictures can be obtained through multiple experiments, the picture library of the NF186 antibody positive is enriched, a large number of different signal styles are provided for the development of an immunofluorescence image automatic processing system, and the accuracy of automatic interpretation is improved. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 3 is a result graph of determination of the binding titer of the serum antibody of three mice and the NF186 protein;

[0024] Figure 2 Figure 5 is a result graph of determination of the binding titer of the supernatant of the hybridoma 16F12 and the NF186 protein by ELISA;

[0025] Figure 3 Figure 7 is a result graph of immunofluorescence detection of the binding of the supernatant of the hybridoma 16F12 and the NF186 protein; wherein, A is a cell slide overexpressing an empty carrier; and B is a cell slide overexpressing the NF186 protein;

[0026] Figure 4 Figure 9 is a result graph of purification of the human-mouse chimeric antibody 16F12;

[0027] Figure 5 Figure 11 is a result graph of verification of the specificity of the human-mouse chimeric antibody 16F12 by CBA;

[0028] Figure 6To verify the specificity of the human-mouse chimeric antibody 16F12 by WB; Wherein, A is the result of commercialized antibody; exposure time 500 ms; B is the result of the human-mouse chimeric antibody 16F12 of the present application, exposure time 50 s;

[0029] Figure 7 Comparison of the signal morphology of 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 pictures of signal amplification; B is the result of incubating positive serum 1; C is the result of incubating positive serum 2; D is the result of incubating positive serum 3. DETAILED DESCRIPTION

[0030] In order to make the technical personnel in the art better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

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

[0032] The present application will be further described in detail below in conjunction with the drawings:

[0033] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized monoclonal antibodies, fully humanized monoclonal antibodies, single-chain antibodies or parts thereof capable of binding to their antigens, such as Fab fragments or fragments generated from Fab expression libraries.

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

[0035] wherein the term "chimeric antibody" means an antibody wherein the sequences of the variable regions of the H and L chains (VH and VL) are derived from an antibody of a non-human animal species and wherein the constant region sequences (CH and CL) are derived from a human antibody. The sequences of the variable regions are preferably derived from an animal species which allows easy preparation of hybridomas, such as mouse, rat, rabbit, etc.

[0036] wherein the term "humanized monoclonal antibody" means an antibody wherein the CDR regions of a human antibody are replaced by CDR regions from an antibody of a non-human animal species, also known as CDR-grafted antibodies. The CDR sequences of the non-human animal species antibody are preferably derived from an animal species which allows easy preparation of hybridomas, such as mouse, rat, rabbit, etc.

[0037] wherein the term "antigen binding site" means the part of an antibody molecule that is involved in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable regions (V) of the heavy (H) and light (L) chains. Within the V regions of the heavy and light chains, three highly branched stretches, called "hypervariable regions", are inserted between several conserved side- wards stretches, called "framework regions" or "FRs". In an 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 with respect 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 and light chain are called "complementarity determining regions" or "CDRs". The three "CDRs" are usually referred to as CDR1, CDR2 and CDR3 and are present from the N-terminus of the light / heavy chain. The light and heavy chain CDR regions are often defined by the Kabat, IMGT, Abm, Chothia or Martin (extended Chothia) numbering systems. The Kabat definition scheme is based on sequence variability; the Chothia definition scheme is based on the location of structural loop regions; the Contact definition scheme is based on available antibody complex crystal structure data. Using the Kabat definition of CDR regions of an antibody variable region, the CDR regions of the light chain variable region VL are defined by residues at amino acid sequence numbers 44-54 (CDR1), 70-76 (CDR2), 109-117 (CDR3) as shown in SEQ ID NO: 1, and the CDR regions of the heavy chain variable region VH are defined by residues at amino acid sequence numbers 50-54 (CDR1), 69-85 (CDR2), 118-127 (CDR3) as shown in SEQ ID NO: 6.

[0038] wherein the term "polypeptide" refers to proteins, protein fragments and fragments or analogs of polypeptide sequences. Protein fragments and analogs are considered to be polypeptide species. Further, according to the present application, examples of polypeptides include the light chain immunoglobulin molecule denoted SEQ ID NO: 1 and the heavy chain immunoglobulin molecule denoted SEQ ID NO: 6 as well as the CDRs denoted SEQ ID NO: 3, 4, 5, 8, 9 and 10, by antibody molecules formed by the combination of a heavy chain immunoglobulin molecule with a light chain immunoglobulin molecule, e.g. a kappa light chain immunoglobulin molecule, and vice versa, as well as fragments and analogs thereof.

[0039] wherein the NF186 (Neuro-oncological ventral antigen 1, NF186) protein is the major neurofilament protein subtype expressed in mature neurons, and the NF186 protein according to the present application refers to the human NF186 protein, or to a polypeptide, fragment or analog that maintains the NF186 protein binding ability.

[0040] wherein the term "amino acid" refers to the 20 conventional amino acids and their abbreviations as used according to the present application.

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

[0042] In particular, variations in the amino acid sequence of the antibodies having "equivalent binding properties" are considered to be encompassed by the present application, provided that the variations in the amino acid sequence of the antibodies having "equivalent binding properties" maintain at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99%. Including certain percentages therebetween, 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] The term "gene" or "DNA" or "nucleic acid molecule" includes not only double-stranded DNA but also 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 the present 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. Examples of the nucleic acid molecule according to the present application include a nucleotide sequence (SEQ ID NO. 2) encoding the above-mentioned light chain variable region of the human mouse chimeric monoclonal antibody against the NF186 protein; a nucleotide sequence (SEQ ID NO. 7) encoding the above-mentioned heavy chain variable region of the human mouse chimeric monoclonal antibody against the NF186 protein; a nucleotide sequence (SEQ ID NO. 11) encoding the human Ig Kappa light chain constant region, specifically AGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG, the corresponding amino acid sequence of which is SEQ ID NO. 13, specifically: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC*; a nucleotide sequence (SEQ ID NO. 12) encoding the human IgG1 heavy chain constant region,Specifically, GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA,The corresponding amino acid sequence is SEQ ID NO. 14, in particular: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*. The nucleic acid molecules of the application can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example, by combinatorial chemical synthesis and recombinant methods.

[0044] The present application also relates to variants of the above-mentioned nucleic acid molecules, which can be naturally occurring variants, such as naturally occurring allelic variants, or which can be non-naturally occurring variants. These non-naturally occurring variants of the nucleic acid molecules can be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms.

[0045] The vectors described herein include the nucleic acid molecules described above. Many suitable vectors are known to those skilled in the art of molecular biology, the selection of which depends on the desired function. The present application is not particularly limited to the vectors, which can be a vector capable of replicating and / or expressing a polynucleotide 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 (e.g., 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, 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.

[0046] The host cells described herein include the nucleic acid molecules or vectors described above.

[0047] In embodiments of the present application, the host cells for introducing the vectors include prokaryotic cells and eukaryotic cells, including but not limited to bacterial cells such as E. 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 application provides a method for detecting an NF186 protein or polypeptide, the method comprising contacting a sample with the human-mouse chimeric monoclonal antibody described above, thereby detecting the NF186 protein or polypeptide in the sample.

[0049] In the present application, the method for detecting or determining the amount of protein or polypeptide can be any known immunological detection technique, including immunological agglutination experiment, immunological precipitation experiment, enzyme-linked immunoassay, fluorescent immunoassay, immunoelectron microscopy, immunoblotting experiment, immunomagnetic bead experiment. The immunological detection technique is a method for detecting or determining the amount of antibody or antigen using a labeled antigen or antibody. The biological sample that can be tested by the above immunological detection or determination includes, but is not limited to, plasma, whole blood, dried whole blood, serum, tissue, cells or extracts of tissues and cells.

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

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

[0052] As an alternative embodiment, the product is a kit; the kit includes the antibody prepared in the present application. As another alternative embodiment, the kit of the present application includes a diagnostic composition including at least one detectable labeling substance, as the labeling substance, a radioisotope, 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 derivative, luciferin, lucigenin, etc.

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

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

[0055] Example 1 Preparation of murine monoclonal antibody

[0056] 1. Preparation of NF186 recombinant protein

[0057] The human NF186 gene sequence was searched from the GenBank sequence database, NCBI sequence number NM_001005388.3, and the gene sequence was synthesized into a pET30a vector by Jinweizhi Company, and the insertion site was EcoR I and Xba I, to obtain a NF186 recombinant vector. The constructed and sequenced recombinant vector plasmid was transformed into an E. coli Arctic Express (Agilent) competent cell; a single colony was picked and inoculated in LB medium, and the bacteria were shaken at 37°C to an OD 600 value of 0.5-1, and the final concentration of IPTG was 0.5 mM for induction, and the expression was performed at 16°C overnight; the bacterial cells were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The NF186 protein expressed by the pET30a was purified by Ni-NTA (QIAGEN), and the purified protein was recorded as NF186 recombinant protein.

[0058] 2. Mouse antigen immunization

[0059] Three 6-8 week old female Balb / c mice were immunized with the NF186 recombinant protein prepared above, and the immunization was required for three times. The antigen amount used for the immunized mice was 40 μg per mouse. The first immunization was performed by mixing 40 μg of the antigen with Freund's adjuvant in the same volume, grinding to an oil-in-water emulsion, and intraperitoneally injecting 100 μL (40 μg of the antigen) of the mixed antigen. The second immunization was performed two weeks after the first immunization, and 40 μg of the antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1, and 100 μL (40 μg of the antigen) of the mixed antigen was intraperitoneally injected. The third immunization was performed two weeks after the second immunization, and the antigen injection dose, method, and route were the same as those of the second immunization. Two weeks later, the antigen was intraperitoneally injected for booster immunization, and the mouse tail blood was collected three days later for ELISA detection of the titer.

[0060] The specific detection process was as follows: the purified NF186 recombinant protein was coated with a carbonate buffer at pH 9.6 at 4°C overnight, 100 ng / well; the next day, PBST was washed for 3 times, 3 min each time, and dried; 2% BSA was fully blocked, 37°C incubation for 1 h; PBST was washed for 3 times, 3 min each time, and dried; the serum of the three mice was diluted with PBS, and the dilution was 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, and 1:109350, 100 μL was added to each well, 37°C incubation for 1 h, and PBST was washed for 3 times, 3 min each time, and dried; goat anti-mouse IgG-HRP was diluted at 1:5000 (product number 115-035-003, Jackson), 37°C incubation for 30 min. PBST was washed for 3 times, dried, TMB was colored for 10 min, 2M H2SO4 was used for termination, and the absorbance value was measured at 450 nm. The results are shown in Figure 1The results showed that according to the detection results, the titer of serum antibodies was integrated, and No. 3 mouse was selected for subsequent experiments.

[0061] 3. Culture of cell fusion and hybridoma

[0062] The animals were killed three days after intraperitoneal injection of antigen for booster immunization, and the spleen cells were taken for cell fusion. A 37°C water bath was prepared in a clean bench, and 5-6x10 7 The mouse myeloma cells SP2 / 0 cells and spleen cells in the logarithmic growth phase and in good growth condition were added to a 50 mL centrifuge tube at a ratio of 1:10, and mixed well. Centrifugation was performed at 500g for 10 min, and the supernatant was aspirated. The bottom of the centrifuge tube was tapped to slightly loosen the cell sediment. 1 mL of 45% PEG1450 solution pre-warmed to 37°C was slowly dropped in 90 s, and the centrifuge tube was constantly shaken. The whole process was in a 37°C water bath. Then DMEM medium was gradually added to the cell mixture, 1 mL was added dropwise in the first minute, 2 mL was added in the second minute, 3 mL was added in the third minute, 4 mL was added in the fourth minute, 5 mL was added in the fifth minute, and the shaking was performed in the 37°C water bath. Then 37°C incubation was performed for 15 min, centrifugation was performed at 500g for 5 min, and the supernatant was removed. 5 mL of DMEM medium containing HAT (thermo, item number: 21060017) was added, the sedimented cells were suspended, and finally DMED medium containing HAT was added to about 100 mL. The cells were dispensed into 96-well cell culture plates coated with macrophages, 100 μL / well, and then the culture plates were placed in a 37°C, 5% CO2 incubator for culture.

[0063] 4. Screening of positive hybridoma

[0064] ELISA detection was performed to select positive clones. The growth of hybridoma cells was observed, and after seven days when the cell culture supernatant turned yellow, an appropriate amount of cell supernatant was aspirated for ELISA detection of antibodies. According to the ELISA results, clones with high OD values, more than twice that of the negative control, were selected, plated in 96-well plates, and subjected to the first subcloning screening. After seven to ten days, ELISA detection of antibodies was performed again, and clones with high OD values were selected, plated in 96-well plates, and subjected to the second subcloning screening, so that there was about 1 cell per well. After seven to ten days of culture, ELISA detection of antibodies was performed again, and clones with high OD values were selected, plated in 96-well plates, and subjected to the third subcloning screening, so that there was about 1 cell per well. After three screenings, a clone with a good signal was preliminarily screened, and was named hybridoma 16F12. The supernatant of the cultured hybridoma 16F12 was diluted at 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, and then used for ELISA detection of titer. 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 Figure 2 ​It can be seen that the NF186 protein can be combined with the antibody in the supernatant of hybridoma 16F12, and does not react with the supernatant of cells transfected with empty pCDNA3.1. Therefore, the positive hybridoma secreting NF186 protein antibody is screened by ELISA method.

[0065] The positive hybridoma screened by ELISA is verified by indirect immunofluorescence method. Cell slide preparation overexpressing NF186 protein: by molecular biology method, NF186 gene is connected to pCDNA3.1 to obtain recombinant plasmid pCDNA3.1-NF186, and the recombinant plasmid is transfected into 293T cells (6cm*6cm cell slides have been placed in 10cm cell culture dishes in advance) with a cell density of 30%-40% using PEI transfection reagent (transfection reagent is purchased from thermo company, and transfection is carried out according to the instruction), 48h after transfection, add acetone to fix at 4℃ for 5min, wash with PBS for 2 times, dry, then cut the 6cm*6cm cell slide into 0.25cm*0.25cm size, and use it, and the cell slide transfected with empty pCDNA3.1 is used as control cell slide, and the preparation method is the same as that of the cell slide overexpressing NF186 protein. Dilute the supernatant of hybridoma 16F12 100 times as primary antibody, incubate on the cell slide overexpressing NF186 protein and the control cell slide, incubate at room temperature for 1h, wash with PBST for 3 times, 5min each time; incubate Alexa 594 labeled goat anti-mouse secondary antibody (Jackson) for 40min, wash with PBST for 3 times, 5min each time, take pictures under fluorescence microscope, and the results are shown in Figure 3 As shown in the results, compared with the control cell slide, the cell slide overexpressing NF186 protein combined with the antibody in the supernatant of hybridoma 16F12 produced obvious signal. Therefore, the antibody secreted by hybridoma 16F12 can not only recognize prokaryotic NF186 protein coated in ELISA plate, but also recognize NF186 protein expressed in eukaryotic cells.

[0066] 5. Identification of antibody typing

[0067] ​The identification of antibody typing was performed by indirect ELISA. The detailed procedure was as follows: purified NF186 protein (100 ng / well) was coated overnight, after blocking with 2% BSA, the supernatant of hybridoma 16F12 obtained in Example 1 was incubated at 37°C for 1 hour, washed with PBST for 3 times, then incubated with 1:5000 diluted different kinds of commercial 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 indirect ELISA showed that the heavy chain subtype of 16F12 antibody was IgG1, and the light chain subtype was kappa.

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

[0069] After the positive hybridoma 16F12 for producing antibodies was grown to a certain scale (cell number > 3 x 10 6 After the positive hybridoma 16F12 for producing antibodies was grown to a certain scale (cell number > 3 x 10 Max DNA Polymerase was used 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.

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

[0071]

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

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

[0074] Example 4 Application of NF186 Protein Human-mouse Chimeric Monoclonal Antibody

[0075] 1. Indirect immunofluorescence method to verify the specificity of the NF186 protein human-mouse chimeric monoclonal antibody

[0076] NF186 protein and NF155 protein are different types of nerve bundle proteins, and the amino acid sequence similarity of the two proteins is 89.72%, therefore, this embodiment investigates whether the NF186 protein human-mouse chimeric antibody prepared by the application can be used to distinguish NF155 protein and NF186 protein.

[0077] A recombinant vector pCDNA3.1-NF155 overexpressing NF155 (sequence number: NM_001160331.2) was constructed using molecular biology methods: the process of preparing overexpressing NF186 cell slides in Reference Example 1 was used to prepare overexpressing NF155 cell slides. The human-mouse chimeric antibody 16F12 purified in Example 3 was diluted to 1 mg / ml, then diluted at a ratio of 1:100 as a primary antibody to incubate on the cell slides overexpressing pCDNA3.1, overexpressing NF155, and overexpressing NF186, respectively, and 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. It was found that the human-mouse chimeric antibody 16F12 could specifically bind to the NF186 protein, but not to the NF155 protein, indicating that the human-mouse chimeric antibody 16F12 prepared by the application could be used to distinguish NF186 protein and NF155 protein. Figure 5It can be known that the human-mouse chimeric monoclonal antibody 6F12 after modification can recognize the NF186 protein, and does not react with cell climbing sheets overexpressing pCDNA3.1 and NF155, so it can be known that the human-mouse chimeric antibody 16F12 has the ability to recognize the NF186 protein and good specificity.

[0078] 2, WB verifies the binding of the human-mouse chimeric monoclonal antibody of NF186 protein to the NF186 protein

[0079] The recombinant vector pCDNA3.1-NF186 is transferred into 1 dish (10 cm) of 293T cells with a cell density of 30%-40%, and the cells are collected after 48 hours, 200 μL of PBS is added, and ultrasonic crushing (crushing conditions: 10% power, crushing for 3s, stopping for 6s, and ultrasonic crushing for 1 min in total) is used as the protein sample for WB; the cells transfected with empty pcDNA3.1 are used to prepare a control protein, and the preparation method is the same as above.

[0080] The prepared NF186 protein and the control protein are subjected to electrophoresis, and the sample amount is 1 μg / well; after the electrophoresis is completed, wet transfer is used, the transfer conditions are 200 mA, 60 min, 5% skimmed milk powder is blocked at room temperature for 1 h, 1:3000 diluted commercial NF186 antibody (ab31457 of abcam) and 1:1000 diluted human-mouse chimeric antibody 16F12 (the final concentration is 1 μg / ml) are respectively incubated at room temperature for 2 h, and TBST is washed for 3 times, each time for 5 min; the membrane piece incubated with the commercial antibody is added with HRP-labeled goat anti-rabbit secondary antibody (Jackson), the membrane piece incubated with the human-mouse chimeric antibody 16F12 is added with HRP-labeled goat anti-human secondary antibody (Jackson), and incubation is carried out at room temperature for 1 h, TBST is washed for 3 times, each time for 5 min; chemical luminescence solution is added for color development and photography, and the results are as shown in Figure 6 It can be known that on the WB, the commercial NF186 antibody and the human-mouse chimeric antibody 16F12 can recognize the NF186 protein. Figure 6

[0081] It can be known from the results of Figure 5 and Figure 6 that the human-mouse chimeric antibody 16F12 prepared in the application can recognize both the conformational epitope NF186 protein and the linear epitope NF186 protein.

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

[0083] ​Take the over-expressed NF186 cell slides prepared in Example 1, incubate with 3 NF186 antibody positive patient sera respectively and the NF186 protein human-mouse chimeric antibody 16F12 obtained in Example 3 of the present application, and incubate the secondary antibody with FITC-labeled goat anti-human IgG secondary antibody (Jackson), and the staining results are shown in Figure 7 Figure 7 It can be seen that the NF186 protein human-mouse chimeric antibody 16F12 can not only recognize the over-expressed NF186 antigen and bind the antibody of anti-human IgG, but also produce a signal pattern similar to the NF186 antibody positive serum. Therefore, the human-mouse chimeric antibody NF186 of the present application, on the one hand, can be expressed and purified on a large scale, and a large amount of antibody can be obtained at a lower cost at one time, and the batch difference and batch difference can be controlled and kept consistent; on the other hand, it can replace the positive serum of NF186 protein as the quality control / control / calibration of the diagnostic kit, to identify the over-expressed NF186 protein cell slides / coated with NF186 protein carrier NF186 protein in the kit. At the same time, in view of the fact that the signal pattern produced by the binding of the NF186 protein human-mouse chimeric antibody 16F12 and the over-expressed NF186 cell slides is similar to the signal pattern produced by the positive serum, a large number of fluorescence pictures can be obtained through multiple experiments, enriching the picture library of NF186 protein antibody positive, providing a large number of different signal styles for the development of the immunofluorescence image automatic processing system, and improving the accuracy of automatic interpretation.

[0084] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.​

Claims

1. A human-mouse chimeric monoclonal antibody to an NF186 protein, characterized in that, comprise a light chain and a heavy chain, wherein: the variable region of the light chain comprises three light chain complementarity determining regions, the amino acid sequences of the three light chain complementarity determining regions are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of the heavy chain comprises three heavy chain complementarity determining regions, the amino acid sequences of the three heavy chain complementarity determining regions are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO.

10.

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

6.

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

7.

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

6. A vector, 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 1-4.

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 of the NF186 protein according to any one of claims 1-4, the vector according to claim 6 or the host cell according to claim 7 in the preparation of a reagent / kit for detecting the NF186 protein.

9. Use of the human-mouse chimeric monoclonal antibody of the NF186 protein according to any one of claims 1-4 in the preparation of a chromatography column for isolating and purifying the NF186 protein.

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