Human-mouse chimeric monoclonal antibody of NF155 protein, product of human-mouse chimeric monoclonal antibody, method for detecting NF155 protein and application of human-mouse chimeric monoclonal antibody

Abstract

The invention discloses a human-mouse chimeric monoclonal antibody of an NF155 protein, a product of the human-mouse chimeric monoclonal antibody, a method for detecting the NF155 protein and application of the human-mouse chimeric monoclonal antibody, and belongs to the technical field of monoclonal antibodies. The human-mouse chimeric monoclonal antibody of the NF155 protein disclosed by the invention is an NF155 antibody with strong specificity and high sensitivity. On one hand, the antibody disclosed by the invention can replace a human serum antibody to be used as a quality control product / reference product / calibration product in a diagnostic kit; on the other hand, the antibody disclosed by the invention can be used for visualizing and positioning the NF155 protein; in addition, the antibody disclosed by the invention can also be used as a ligand in an affinity layer system to prepare a chromatographic column, so that separation and purification of the NF155 protein are realized, and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and in particular relates to a human-mouse chimeric monoclonal antibody of NF155 protein and a product thereof, and a method for detecting NF155 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 the nodes of Ranvier (also known as the nodes of Ranvier or nerve knots, which are special structures on nerve fibers). Currently, five different types of NF polypeptides have been found, including NF140, NF155, NF166, NF180 and NF186. NF155 is mainly present in glial cells, and its expression begins with the formation of myelin sheaths and continues to be highly expressed during myelination. As oligodendrocytes gradually mature, NF155 migrates from the cell body to the end of the cell process, and is finally distributed in the lipid rafts in the lateral area of ​​the myelin sheath in the mature central nervous system. NF155 plays an important role in the early formation of the axon initial segment. In addition, NF155 also plays an important role in the formation of myelin structure and the repair process after injury. It ensures the stability of myelin structure by interacting with the caspr / contactin complex expressed on the axon membrane. Mathey et al. also found that NF155's repair function after myelin injury can be used as a new target for the treatment of autoantibody-mediated axonal injury (MATHEY EK, DERFUSS T, STORCH MK, et al. Neurofascin as a novel target for autoantibody-mediated axonal injury [J]. Journal of Experimental Medicine, 2007, 204 (10): 2363-2372).

[0003] In 2012, NG et al. detected anti-NF155 antibodies mainly of IgG4 subtype in patients with chronic inflammatory demyelinatily polyradiculo-neuropathy (CIDP) (Ng JKM, Malotka J, Kawakami N, et al. Neurofascin as a target for autoantibodies in peripheral neuropathies [J]. Neurology, 2012, 79 (23): 2241-2248.). At present, the diagnosis of NF155 antibody-related CIDP in China depends on anti-NF155 antibody detection.

[0004] The detection of autoantibodies has important clinical value for the diagnosis of autoimmune diseases, the judgment of disease progression, disease treatment and prognosis. In the detection system of autoantibodies, reference materials are an important part of the quality control of the detection system. Usually, the positive serum of patients can be used as a positive reference. However, due to the small number of positive sera for autoimmune diseases, high acquisition costs, and difficulty in determination and traceability, it is necessary to provide a positive reference that can replace the positive serum of patients. Chimeric antibodies or humanized antibodies can meet such needs. At present, there are some chimeric antibodies or humanized antibodies used as quality control products / reference materials / calibrators for in vitro diagnostic reagents in the world, such as the Toxoplasma chimeric antibodies prepared by Abbott Diagnostic Laboratories.

[0005] However, the commercialized NF155 antibodies currently available are rabbit / mouse monoclonal / polyclonal antibodies. Such antibodies: (1) inevitably have differences between different batches, and cannot guarantee the same sensitivity between each batch; (2) the antibody sequence is not public, and low-cost, large-scale industrial mass production cannot be achieved; (3) the Fc region is a mouse / rabbit sequence, which cannot react with anti-human IgG antibodies. Summary of the invention

[0006] 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 NF155 protein and its product and a method and application for detecting NF155 protein, so as to solve 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. At the same time, a new human-mouse chimeric monoclonal antibody of NF155 protein can be provided for identifying the expression and localization of NF155 protein and purifying NF155 protein.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a human-mouse chimeric monoclonal antibody of NF155 protein, comprising a mouse light chain variable region VL, a mouse heavy chain variable region VH, a human antibody light chain constant region and a human antibody heavy chain constant region, wherein:

[0009] The variable region VL of the murine light chain includes three light chain complementary determining regions, and the amino acid sequences of the three light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively;

[0010] The variable region VH of the murine heavy chain includes three heavy chain complementary determining regions, and the amino acid sequences of the three heavy chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively;

[0011] The amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID NO.13;

[0012] The amino acid sequence of the human antibody heavy chain constant region is as shown in SEQ ID NO.14.

[0013] Furthermore, the amino acid sequence of the light chain variable region VL of the human-mouse chimeric monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody is shown in SEQ ID NO.6.

[0014] Furthermore, the nucleotide sequence encoding the light chain variable region VL of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.2; the nucleotide sequence encoding the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.7.

[0015] Furthermore, the amino acid sequence of the light chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown as SEQ ID NO.15; the amino acid sequence of the heavy chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown as SEQ ID NO.16.

[0016] Furthermore, 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 VL 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 VH of the variable region of the heavy chain; and the complementary domains of the light chain and the heavy chain maintain the ability to bind to the NF155 protein.

[0017] Furthermore, the above antibody has three light chains comprising complementarity determining regions with amino acid sequences having at least 90% sequence identity with sequences including QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4) and LQFYEFPFT (SEQ ID NO.5), and three heavy chains comprising complementarity determining regions with amino acid sequences having at least 90% sequence identity with sequences including SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9) and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domain and the heavy chain complementary domain maintain NF155 binding ability.

[0018] Furthermore, the above antibody has three light chains comprising complementarity determining regions with amino acid sequences having at least 95% sequence identity with sequences including QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4) and LQFYEFPFT (SEQ ID NO.5), and three heavy chains comprising complementarity determining regions with amino acid sequences having at least 95% sequence identity with sequences including SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9) and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domain and the heavy chain complementary domain maintain the ability to bind to the NF155 protein.

[0019] Furthermore, the above antibody has three light chains comprising complementarity determining regions with amino acid sequences having at least 99% sequence identity with sequences including QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4) and LQFYEFPFT (SEQ ID NO.5), and three heavy chains comprising complementarity determining regions with amino acid sequences having at least 99% sequence identity with sequences including SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9) and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domain and the heavy chain complementary domain maintain the ability to bind to the NF155 protein.

[0020] The invention also discloses a nucleic acid molecule, which encodes the human-mouse chimeric monoclonal antibody of the NF155 protein.

[0021] The present invention also discloses a vector, which comprises a nucleotide sequence encoding the human-mouse chimeric monoclonal antibody of the NF155 protein.

[0022] The present invention also discloses a host cell, which comprises the above-mentioned vector.

[0023] The present invention discloses the use of the human-mouse chimeric monoclonal antibody, vector or host cell of the NF155 protein in preparing a reagent / kit for detecting the NF155 protein.

[0024] The present invention discloses the use of the human-mouse chimeric monoclonal antibody, vector or host cell of the NF155 protein in preparing reagents / kits for human chronic inflammatory demyelinating polyradiculoneuropathy and / or prognosis judgment.

[0025] The present invention also discloses a method for detecting NF155 protein or NF155 polypeptide, wherein the sample to be tested is contacted with the human-mouse chimeric monoclonal antibody of the NF155 protein to detect the NF155 protein or NF155 polypeptide in the sample.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses a human-mouse chimeric monoclonal antibody of NF155 protein, which is a NF155 antibody with strong specificity and high sensitivity. In the prior art, since mouse-derived / rabbit-derived polyclonal antibodies are directly purified from the serum of mice / rabbits after immunization, there are certain differences between 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-derived / rabbit-derived monoclonal / polyclonal antibodies are small, expensive, and the long-term purchase cost is too high. Therefore, the human-mouse chimeric monoclonal antibody of NF155 protein prepared by the present invention by molecular biological means can be expressed and purified on a large scale by genetic engineering technology, and a large amount of antibodies can be obtained at one time to solve the technical problems that the current mouse-derived / rabbit-derived monoclonal / polyclonal antibodies have batch differences, cannot form low-cost large-scale industrial production, and their Fc regions cannot react with anti-human IgG antibodies. From the application point of view, on the one hand, the antibody disclosed in the present invention can replace human serum antibody as a quality control / reference substance / calibrator in the diagnostic kit; on the other hand, the antibody disclosed in the present invention can visualize and locate the NF155 protein; in addition, the antibody disclosed in the present invention can also be used as a ligand in the affinity layer system to prepare a chromatography column to achieve the separation and purification of NF155 protein. At the same time, since the signal morphology generated by the human-mouse chimeric antibody 4c7 of NF155 protein combined with the NF155 overexpressing cell slide is similar to the signal morphology generated by positive blood, a large number of fluorescent images can be obtained through multiple experiments, enriching the anti-NF155 antibody positive image library, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the NF155 recombinant protein purified by prokaryotic expression;

[0029] Figure 2 The figure is the result of the determination of the binding titer of serum antibodies of three mice to NF155 protein;

[0030] Figure 3 ELISA was used to determine the binding titer of hybridoma 4c7 supernatant to NF155 protein;

[0031] Figure 4 Immunofluorescence detection of the binding between hybridoma 4c7 supernatant and NF155 protein; the primary antibody incubated in A is the serum of rat No. 3 after immunization with NF155 protein; the primary antibody incubated in B is the commercial antibody of NF155 protein; the primary antibody incubated in C is the supernatant of hybridoma 4c7;

[0032] Figure 5 Purification of human-mouse chimeric antibody 4c7;

[0033] Figure 6It is the typing and identification of human-mouse chimeric antibody 4c7;

[0034] Figure 7 Verification of the specificity of the human-mouse chimeric antibody 4c7;

[0035] Figure 8 Comparison of signal morphology between human-mouse chimeric antibody 4c7 and serum. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0039] 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.

[0040] The term "primary antibody" refers to an antibody that can specifically bind to a specific antigen (non-antibody antigen). It is usually produced by B cells under antigen stimulation, has a high degree of specificity, and can recognize and bind to a specific antigen epitope on the target antigen. The primary antibody can be a monoclonal antibody (mAb) or a polyclonal antibody (pAb). Primary antibodies are widely used in immunoassays, disease diagnosis, treatment, and scientific research. For example, in an ELISA experiment, the primary antibody can be used as a probe to specifically bind to the target protein, thereby achieving the detection of the target protein.

[0041] The term "secondary antibody" refers to an antibody that can bind to a primary antibody, that is, an antibody to an antibody. It is usually produced by the immune system of a xenogeneic animal and targets the immunoglobulin of the primary antibody. The main function of the secondary antibody is to detect the presence of the primary antibody and amplify the signal of the primary antibody. In practical applications, a secondary antibody that specifically matches the species of the target primary antibody is usually selected. Secondary antibodies are widely used in immunoassay techniques such as immunohistochemistry, flow cytometry, and Western blot. For example, in immunohistochemistry experiments, the secondary antibody can bind to the primary antibody and emit a signal through its coupled labeling substance (such as a fluorescent dye), thereby achieving the localization and detection of the target antigen.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Among them, NF155 (Neuro-oncological ventral antigen 1, NF155) protein is a protein present in glial cells and mainly expressed in the central nervous system. The NF155 protein described in the present invention refers to a polypeptide, fragment or analog that retains the binding ability of NF155 protein.

[0047] Wherein, the term "amino acid" refers to the 20 conventional amino acids and their abbreviations used in the present invention as conventionally used.

[0048] 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 NF155 protein or polypeptide.

[0049] 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.

[0050] Among them, the term "gene" or "DNA" or "nucleic acid molecule": includes not only double-stranded DNA, but also corresponding single-stranded DNA, sense strand and antisense strand constituting 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 VL of the human-mouse chimeric monoclonal antibody of the above-mentioned NF155 protein (SEQ ID NO.2); the nucleotide sequence encoding the heavy chain variable region VH of the above-mentioned NF155 monoclonal antibody (SEQ ID NO.7); the nucleotide sequence encoding the human Ig Kappa light chain constant region is shown in SEQ ID NO.11, and its corresponding amino acid sequence is shown in SEQ ID NO.13; the nucleotide sequence encoding the human IgG4 heavy chain constant region is shown in SEQ ID NO.12, and its corresponding amino acid sequence is shown in SEQ ID NO.14. 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.

[0051] 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.

[0052] The vectors of the present invention include the above-mentioned nucleic acid molecules. Many suitable vectors are known to those skilled in the art of molecular biology, and their 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 Escherichia coli (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.

[0053] The host cell described in the present invention includes the above-mentioned nucleic acid molecule or vector.

[0054] 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.

[0055] The present invention provides a method for detecting NF155 protein or polypeptide, which comprises contacting a sample with the human-mouse chimeric monoclonal antibody, thereby detecting the NF155 protein or polypeptide in the sample.

[0056] 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.

[0057] 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 NF155 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 anti-human NF155 human-mouse chimeric monoclonal antibody (test antibody) with the solid phase, removing unreacted antibodies, and adding a labeled second antibody that specifically reacts with the test antibody to the solid phase to determine the amount of the label on the solid phase.

[0058] The product of the present invention includes the above-mentioned human-mouse chimeric monoclonal antibody, nucleic acid molecule, vector or host cell.

[0059] 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.

[0060] 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.

[0061] The present invention provides the use of the human-mouse chimeric monoclonal antibody, nucleic acid molecule, vector, host cell or product in detecting NF155 protein or polypeptide products.

[0062] The term "recombinant plasmid extraction" refers to the method of inserting the target gene into the multiple cloning site of the plasmid, constructing a new recombinant plasmid, and extracting these plasmids in large quantities through a series of steps.

[0063] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0064] Example 1 Preparation of mouse monoclonal antibodies

[0065] 1. Preparation of NF155 recombinant protein

[0066] The human NF155 gene sequence was obtained from the GenBank sequence database, with the NCBI sequence number NM_001160331.2. The gene sequence was synthesized by Jinweizhi Company into the pET30a vector, with the insertion sites of EcoR I and Xba I to obtain the NF155 recombinant vector. The constructed and correctly sequenced recombinant plasmid was transferred into the expression competent cells of Escherichia coli Arctic Express (Agilent); a single clone was picked and inoculated into LB medium, and the bacteria were shaken at 37°C to an OD value of 0.5-1, and induced with a final concentration of 0.5mM IPTG, and expressed at 16°C overnight; the bacteria were collected, ultrasonically disrupted, and the supernatant was collected after centrifugation. The NF155 protein expressed by pET-30a was purified using Ni-NTA (QIAGEN), and the purified recombinant protein was recorded as NF155. The purification results are shown below. Figure 1 shown.

[0067] 2. Mouse Antigen Immunization

[0068] Three Balb / c mice were immunized with the NF155 recombinant protein prepared as above as antigen. Three immunizations were required, and the amount of antigen used for immunizing mice was 40 μg / mouse. For the first immunization, the antigen was mixed with equal volumes of Freund's adjuvant, fully ground into a water-in-oil emulsion, and 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally; for the second immunization, two weeks after the first immunization, 40 μg antigen was mixed with incomplete Freund's adjuvant in a 1:1 ratio, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle. The third immunization was performed two weeks after the second immunization, and the dose, method, and route of antigen injection were the same as those of 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.

[0069] The specific detection process is as follows: use carbonate buffer with a pH value of 9.6 to coat the purified NF155 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 2 As shown, based on the test results and the comprehensive titer and specificity data of serum antibodies, mouse No. 3 was selected for subsequent experiments.

[0070] 3. Cell fusion and hybridoma culture

[0071] 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 is 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.

[0072] 4. Screening of positive hybridomas

[0073] 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 a good signal was initially screened out and named hybridoma 4c7. The supernatant and stock solution of the cultured hybridoma 4c7 were diluted at 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, and the titer was detected by ELISA. The results showed that the supernatant of the screened hybridoma 4c7 could still be detected after being diluted 2187 times, and the antibody in the supernatant secreted by the hybridoma had a good binding ability with the NF155 protein ( Figure 3 ). The antibody in the supernatant secreted by hybridoma 4c7 was named as mouse monoclonal antibody 4c7.

[0074] The positive clones screened by ELISA were verified by indirect immunofluorescence. Preparation of cell slides overexpressing NF155 protein: The NF155 gene was linked to pCDNA3.1 by molecular biology methods to obtain a recombinant plasmid. The recombinant plasmid was transfected into 293T cells with a cell density of 30% to 40% using PEI transfection reagent (the transfection reagent was purchased from thermo company, refer to the instructions for transfection). A 6cm*6cm cell slide had 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 after drying, the 6cm*6cm cell slide was cut into a size of 0.25cm*0.25cm for later use. The hybridoma 4c7 supernatant, the commercial antibody against NF155 (abcam), and the immunized No. 3 mouse blood were diluted 200 times as the primary antibody and incubated on the cell slides overexpressing NF155 protein. Incubate at room temperature for 1 hour, wash 3 times with PBST, each time for 5 minutes; incubate The cells were incubated with 594-labeled goat anti-mouse secondary antibody (Jackson) for 40 min, washed 3 times with PBST, 5 min each time, and photographed under a fluorescence microscope. The results are shown in Figure 4 As shown, the hybridoma 4c7 supernatant produced a signal similar to that of the commercial antibody, further indicating that the mouse monoclonal antibody in the hybridoma 4c7 supernatant can not only recognize the prokaryotic NF155 protein coated in the ELISA well plate, but also recognize the NF155 protein expressed in eukaryotic cells.

[0075] 5. Identification of the typing of mouse monoclonal antibody 4c7

[0076] The identification of mouse monoclonal antibody typing was performed by indirect ELISA. The specific process of indirect ELISA was as follows: coating with NF155 antigen (100 ng / well), blocking with 2% BSA, incubating with the hybridoma 4c7 supernatant obtained in Example 1 at 37°C for 1 hour, washing with PBST three times, and then incubating 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, washing with PBST three times, and developing with TMB for 5 minutes. The indirect ELISA results all showed that the heavy chain subtype of the mouse monoclonal antibody in the hybridoma 4c7 supernatant was IgG1, and the light chain subtype was Kappa (Table 1).

[0077] Table 1 Identification of mouse monoclonal antibody 4c7 typing

[0078]

[0079] Example 2 Identification of the sequence of anti-NF155 mouse monoclonal antibody 4c7

[0080] The hybridoma 4c7 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 mouse monoclonal antibody 4c7 amino acid sequence.

[0081] Table 2 Sequence of mouse monoclonal antibody 4c7

[0082]

[0083]

[0084]

[0085]

[0086] Example 3 Preparation and typing identification of anti-NF155 human-mouse chimeric antibody 4c7

[0087] Since the anti-NF155 antibodies present in CIDP patient samples are mainly expressed as IgG4 subtype, in order to prepare quality control substances / reference substances / calibrators in the anti-NF155 autoantibody detection kit, in this example, when constructing a recombinant vector, the heavy chain variable region VH of the mouse monoclonal antibody 4c7 was connected to the human IgG4 heavy chain constant region sequence for recombinant expression.

[0088] With reference to the sequences in the Kabat database, molecular biology methods were used to Seamless Cloning and Assembly Kit, the nucleotide sequence of the light chain variable region VL of the mouse monoclonal antibody 4c7 encoding the NF155 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-NF155-VL, the nucleotide sequence of the heavy chain variable region VH of the mouse monoclonal antibody 4c7 encoding the NF155 protein SEQ ID NO.7 and the human IgG4 heavy chain constant region sequence SEQ ID NO.12 were connected to the vector pcDNA3.1, marked as pcDNA3.1-NF155-VH, the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing, and the sequenced recombinant plasmid was collected for use. The human-mouse chimeric antibody recombinant plasmids pcDNA3.1-NF155-VL and pcDNA3.1-NF155-VH were co-transfected into Expi293F cells (thermo) using PEI according to the thermo instructions for transient expression. The supernatant was collected 3 days after transfection and purified using protein A filler. The protein concentration was 0.1 mg / mL (Solebio) measured by the BCA method. The obtained recombinant antibody was named NF155 human-mouse chimeric antibody 4c7 ( Figure 5 ). In order to further confirm that the obtained recombinant human-mouse chimeric antibody is of IgG4 type, the indirect immunofluorescence method was used to identify the typing of NF155 human-mouse chimeric antibody 4c7: the human-mouse chimeric antibody 4c7 was incubated with the cell slides overexpressing NF155, incubated at room temperature for 1 hour, and then incubated with different types of FITC-labeled rabbit anti-human secondary antibodies (total IgG, IgG1, IgG2, IgG3, IgG4) diluted 1:200 at room temperature for 30 minutes, washed 3 times with PBST and photographed under a fluorescence microscope. The results of the indirect immunofluorescence method showed that the heavy chain subtype of the recombinant human-mouse chimeric antibody 4c7 was IgG4 ( Figure 6 ).

[0089] Example 4 Application of anti-NF155 human-mouse chimeric antibody

[0090] 1. Verification of the specificity of anti-NF155 human-mouse chimeric antibody by indirect immunofluorescence assay

[0091] 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 NF155 human-mouse chimeric antibody prepared by the present invention can be used to distinguish NF155 protein from NF186 protein.

[0092] The cell slides overexpressing NF186 (sequence number: NM_001005388.3) were prepared by referring to the process of preparing the cell slides overexpressing NF155 in Example 1. The chimeric human-mouse antibody 4c7 purified in Example 3 and the No. 3 mouse serum in Example 1 were taken, and the chimeric human-mouse antibody 4c7 (incubated with FITC-labeled mouse anti-human IgG4 secondary antibody, Sigma) and the No. 3 mouse serum (incubated with Alexa Fluor) were incubated with the 4c7 antibody ... 594-labeled goat anti-mouse secondary antibody, Jackson) was diluted at a ratio of 1:200 and incubated as primary antibody on cell slides overexpressing pCDNA3.1, overexpressing NF155, and overexpressing NF186 to verify the specificity of the human-mouse chimeric antibody 4c7. Figure 7 It can be seen that although there is a signal when mouse serum No. 3 is combined with cell slides overexpressing NF186, the human-mouse chimeric antibody 4c7 does not react with cell slides overexpressing pCDNA3.1 and NF186. This shows that the human-mouse chimeric antibody 4c7 has good specificity.

[0093] 2. Comparison of the signals produced by anti-NF155 human-mouse chimeric antibody and positive serum by indirect immunofluorescence

[0094] The NF155 overexpressing cell slides prepared in Example 1 were incubated with sera from three patients with positive anti-NF155 antibodies and the anti-NF155 human-mouse chimeric antibody 4c7 obtained in Example 3 of the present application, and the secondary antibody was incubated with FITC-labeled mouse anti-human IgG4 secondary antibody from Sigma. The staining results were as follows: Figure 8 As shown. Figure 8It can be seen that the anti-NF155 human-mouse chimeric antibody 4c7 can not only recognize the overexpressed NF155 protein and bind to the anti-human IgG4 antibody, but also the signal morphology generated is similar to that of the anti-NF155 antibody positive serum, both of which are irregular flakes. Therefore, the modified human-mouse chimeric antibody can replace the human serum antibody as a quality control / reference substance / calibrator in the diagnostic kit; on the other hand, the antibody disclosed in the present invention can visualize and locate the NF155 protein; in addition, the antibody disclosed in the present invention can also be used as a ligand in the affinity layer system to prepare a chromatography column to achieve the separation and purification of NF155 protein. At the same time, given that the signal morphology generated by the anti-NF155 human-mouse chimeric antibody 4c7 combined with the overexpressed NF155 cell slide is similar to the signal morphology generated by positive blood, a large number of fluorescent images can be obtained through multiple experiments, enriching the anti-NF155 antibody positive image library, 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.

[0095] Therefore, the novel human-mouse / human-rabbit chimeric antibody of NF155 protein developed by the present invention can replace the function of serum to a certain extent, and is helpful for developing a quality-controlled neuroimmune autoantibody detection kit; at the same time, the mouse monoclonal antibody or human-mouse / human-rabbit chimeric antibody of NF155 protein can also be used to visualize and locate the NF155 protein; the mouse monoclonal antibody or human-mouse / human-rabbit chimeric antibody of NF155 protein can also be used as a ligand in an affinity layer system to prepare a chromatography column to achieve separation and purification of NF155 protein and other applications.

[0096] 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 NF155 protein, characterized in that: It includes a mouse light chain variable region VL, a mouse heavy chain variable region VH, a human antibody light chain constant region and a human antibody heavy chain constant region, wherein: The variable region VL of the murine light chain includes three light chain complementary determining regions, and the amino acid sequences of the three light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; The variable region VH of the murine heavy chain includes three heavy chain complementary determining regions, and the amino acid sequences of the three heavy chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; The amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID NO.13; The amino acid sequence of the human antibody heavy chain constant region is as shown in SEQ ID NO.

14.

2. The human-mouse chimeric monoclonal antibody of NF155 protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region VL of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region VL of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.6, and the nucleotide sequence encoding the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against the NF155 protein is shown in SEQ ID NO.

7.

3. The human-mouse chimeric monoclonal antibody of NF155 protein according to claim 1, characterized in that: The amino acid sequence of the light chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.15; the amino acid sequence of the heavy chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.

16.

4. The human-mouse chimeric monoclonal antibody of NF155 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 VL of the light chain, and the complementary domain of the light chain maintains the ability to bind to the NF155 protein; 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 VH of the heavy chain, and the complementary domain of the heavy chain maintains the ability to bind to the NF155 protein.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the human-mouse chimeric monoclonal antibody of the NF155 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 NF155 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 NF155 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 NF155 protein.

9. Use of the human-mouse chimeric monoclonal antibody against NF155 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 NF155 protein or NF155 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 NF155 protein or NF155 polypeptide in the sample.

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