Neutralizing antibody against vitamin-d-binding protein, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of antibodies. Provided are a neutralizing antibody against a vitamin-D-binding protein, and a preparation method therefor and the use thereof. The provided neutralizing antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region, and amino acid sequences of complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 thereof are respectively as set forth in positions 25-32, 50-56 and 93-102 of SEQ ID NO: 3; the light chain comprises a light chain variable region, and amino acid sequences of complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 thereof are respectively as set forth in positions 27-34, 52-54 and 91-101 of SEQ ID NO: 5. The provided neutralizing antibody can effectively neutralize the vitamin-D-binding protein in cells, and can be used for treating nerve damage caused by the vitamin-D-binding protein.

Description

Neutralizing antibody of vitamin D binding protein and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to a neutralizing antibody of vitamin D binding protein and a preparation method and application thereof. BACKGROUND

[0002] Vitamin D binding protein (VDBP) is a multifunctional protein that plays an important role in maintaining the total level of vitamin D and regulating the level of free vitamin D as the main transporter of vitamin D. Related studies have shown that VDBP can bind fatty acids and actin monomers to prevent their aggregation in the circulatory system and play a harmful role. At the same time, based on the immune regulation function of VDBP independent of vitamin D transport, further studies have found that VDBP is related to the occurrence and development of inflammation and tumors.

[0003] A neutralizing antibody is an antibody that can neutralize the activity of a virus, toxin or other pathogen, and is usually used for the prevention and treatment of infectious diseases. In related technologies, the preparation of neutralizing antibodies mainly includes methods such as immunizing animals, cell culture and protein expression. In the laboratory, the neutralizing antibody can be induced by injecting the target antigen into animals such as mice, rabbits, etc. to induce the production of neutralizing antibodies, and then collecting the antibodies through serum or cell culture fluid. In addition, cell culture technology can also be used to introduce genes encoding specific antibodies into mammalian cells or eukaryotic expression systems to obtain neutralizing antibodies through expression and purification. However, although there are many methods for preparing neutralizing antigens, there is still no report on neutralizing antibodies against vitamin D binding protein.

[0004] Therefore, the present application aims to provide a neutralizing antibody of vitamin D binding protein, which can effectively neutralize the expression amount of VDBP in cells and help to improve the protective effect on neurons.

[0005] SUMMARY

[0006] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application provides an antibody or antibody binding fragment of vitamin D binding protein, which has excellent affinity for vitamin D binding protein, can effectively neutralize the expression amount of VDBP in cells, and can improve the protective effect on neurons.

[0007] The present application also provides a biological material related to the above-mentioned antibody or antibody binding fragment thereof.

[0008] The present application also provides a conjugate.

[0009] The present application also provides a product.

[0010] The application further provides application of the antibody or antibody binding fragment thereof and the biological material in preparation of a reagent for detecting vitamin D binding protein or a medicine for treating neuronal injury.

[0011] The application further provides a preparation method of the antibody or antibody binding fragment thereof.

[0012] In a first aspect, the application provides an antibody or antibody binding fragment thereof against vitamin D binding protein, which comprises a heavy chain and a light chain.

[0013] The heavy chain comprises a heavy chain variable region, and the heavy chain variable region comprises a complementarity determining region CDR-H1, CDR-H2 and CDR-H3.

[0014] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO. 3, 25-32.

[0015] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO. 3, 50-56.

[0016] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO. 3, 93-102.

[0017] The light chain comprises a light chain variable region, and the light chain variable region comprises a complementarity determining region CDR-L1, CDR-L2 and CDR-L3.

[0018] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO. 5, 27-34.

[0019] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO. 5, 52-54.

[0020] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO. 5, 91-101.

[0021] The antibody or antibody binding fragment thereof against vitamin D binding protein according to the embodiments of the application has at least the following beneficial effects:

[0022] The application first screens and prepares the antibody or antibody binding fragment thereof against vitamin D binding protein, which can effectively neutralize the expression amount of VDBP in cells and can be used for treating neuronal injury caused by VDBP.

[0023] In some embodiments of the application, the amino acid sequence of the heavy chain variable region comprises any one of A1) to A3):

[0024] A1) the amino acid sequence shown in SEQ ID NO. 3.

[0025] A2) an amino acid sequence that is identical to the sequence set forth in SEQ ID NO. 3 except for one or several substitutions and / or deletions and / or additions of amino acids and that has the same function as the sequence set forth in SEQ ID NO. 3;

[0026] A3) an amino acid sequence that has 80%, 85% or 90% or more homology to SEQ ID NO. 3 and that has the same function as the sequence set forth in SEQ ID NO. 3;

[0027] the amino acid sequence of the light chain variable region comprises any one of B1) to B3):

[0028] B1) the amino acid sequence set forth in SEQ ID NO. 5;

[0029] B2) an amino acid sequence that is identical to the sequence set forth in SEQ ID NO. 5 except for one or several substitutions and / or deletions and / or additions of amino acids and that has the same function as the sequence set forth in SEQ ID NO. 5;

[0030] B3) an amino acid sequence that has 80%, 85% or 90% or more homology to SEQ ID NO. 5 and that has the same function as the sequence set forth in SEQ ID NO. 5.

[0031] Herein, the term "homology" refers to the similarity between amino acid sequences or nucleotide sequences, which can be evaluated by computer software. It is understood that an amino acid sequence that has 80% or more homology to SEQ ID NO. 3 and that has the same function as the sequence set forth in SEQ ID NO. 3 refers to an amino acid sequence that is identical to the neutralizing antibody heavy chain variable region in SEQ ID NO. 3 or that is obtained by adjusting the linker amino acids.

[0032] Herein, the 80% or more homology can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0033] In some embodiments of the present application, the antibody or antigen binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody.

[0034] In some embodiments of the present application, the heavy chain further comprises a heavy chain constant region; and / or

[0035] the light chain further comprises a light chain constant region.

[0036] In some embodiments of the present application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 8 from 133 to 455; and the light chain constant region is shown in SEQ ID NO. 9 from 133 to 236.

[0037] In some embodiments of the present application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 8 from 133 to 455; and the light chain constant region is shown in SEQ ID NO. 9 from 133 to 236.

[0038] In some embodiments of the present application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 8 from 133 to 455; and the light chain constant region is shown in SEQ ID NO. 9 from 133 to 236.

[0039] C1) a nucleic acid molecule encoding the antibody or antibody binding fragment of any one of the first aspect;

[0040] C2) an expression cassette comprising the nucleic acid molecule of C1);

[0041] C3) a recombinant vector comprising the nucleic acid molecule of C1) or the expression cassette of C2);

[0042] C4) a recombinant microorganism comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3);

[0043] C5) a recombinant cell comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3), which does not comprise reproductive material.

[0044] The skilled in the art can mutate the nucleotide sequence by known methods (such as directed evolution and point mutation), and those artificially modified nucleotide sequences having certain identity with the antibody or antigen binding fragment thereof of the present application are derived from and equivalent to the nucleotide sequence of the present application, as long as they encode the antibody or antigen binding fragment thereof or variants and have the same function as the antibody or antigen binding fragment thereof.

[0045] In some embodiments of the present application, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region;

[0046] In some embodiments of the present application, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region;

[0047] In some embodiments of the present application, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain.

[0048] In some embodiments of the present application, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain.

[0049] In some embodiments of the present application, the expression cassette refers to DNA capable of expressing the antibody or antigen-binding fragment thereof in a host cell, which can include not only a promoter capable of initiating transcription of the DNA molecule encoding the antibody or antigen-binding fragment thereof, but also a terminator capable of terminating transcription of the DNA molecule encoding the antibody or antigen-binding fragment thereof. Further, the expression cassette can further comprise a replication initiation site, a transcription initiation sequence, an enhancer sequence, a selection element or a reporter gene.

[0050] In some embodiments of the present application, the recombinant vector can be a plasmid, a bacteriophage, a lentivirus, an adenovirus, an adeno-associated virus. The recombinant vector is used to achieve replication, integration, amplification and / or expression of the exogenous target gene in the recipient cell; it can be a cloning vector or an expression vector. For example, the recombinant vector in C3) can be a recombinant plasmid obtained by inserting the nucleic acid molecule into a CMV vector.

[0051] In some embodiments of the present application, the recombinant microorganism can be a bacterium (such as E. coli or B. subtilis, etc.) or a fungus (such as yeast or Aspergillus, etc.). For example, the recombinant microorganism in C4) can be E. coli DN5a.

[0052] In some embodiments of the present application, the recombinant cell can be an insect cell (such as S2 fruit fly cell or Sf9 cell, etc.), an animal cell (such as B lymphocyte, 293T cell, 293F cell, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell or HEK293 cell, etc.) or a plant cell (such as Arabidopsis thaliana or tobacco, etc.).

[0053] In some embodiments of the present application, the recombinant cell can be a hybridoma cell. The hybridoma cell is obtained by fusing a spleen cell with a SP2 / 0 cell.

[0054] In a third aspect of the present application, a conjugate is provided, comprising the antibody or antibody-binding fragment thereof of any one of the first aspect;

[0055] and a conjugating moiety, the conjugating moiety comprising at least one of a detectable label, a drug, a toxin, a radionuclide, an enzyme.

[0056] In a fourth aspect of the present application, a product comprising at least one of D1) to D3) is provided.

[0057] D1) the antibody or antibody binding fragment thereof according to any one of the first aspect;

[0058] D2) the biomaterial according to any one of the second aspect;

[0059] D3) the conjugate according to the third aspect.

[0060] The product is selected from at least one of a drug, a detection plate, a chip, a test paper, and a kit.

[0061] In a fifth aspect of the present application, any one of E1) to E3) is used in the preparation of a reagent for detecting vitamin D binding protein or a drug for treating neuronal injury:

[0062] E1) the antibody or antibody binding fragment thereof according to any one of the first aspect;

[0063] E2) the biomaterial according to any one of the second aspect;

[0064] E3) the conjugate according to the third aspect.

[0065] In some embodiments of the present application, the drug further comprises a pharmaceutically acceptable excipient. The dosage used is non-toxic to the recipient.

[0066] In some embodiments of the present application, the excipient comprises at least one of a solvent, a dispersant, a diluent, a filler, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifying agent, an excipient, a stabilizer, a buffer, an isotonic agent, a flavoring agent, a carrier. The excipient is used to facilitate the absorption of the antibody or antigen binding fragment thereof to exert a preventive and / or therapeutic effect.

[0067] In a sixth aspect of the present application, a method for preparing an antibody or antibody binding fragment thereof is provided, comprising: obtaining from the recombinant microorganism or the recombinant cell in the biomaterial according to the second aspect by culturing.

[0068] In some embodiments of the present application, the antibody or antibody binding fragment thereof of the present application can be obtained by the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), or also by recombinant DNA technology (see, for example, Journal of virological methods, 2009, 158(12):171 179).

[0069] Other features and advantages of the present application will be set forth in the following description. BRIEF DESCRIPTION OF DRAWINGS

[0070] The application will be further described below in connection with the accompanying drawings and examples, in which:

[0071] Figure 1 is a SDS-PAGE result chart of each sample in the process of purifying the protein of the present application;

[0072] Figure 2 is a SDS-PAGE result chart of the protein of the present application after transfection of 80ml XtenCHO;

[0073] Figure 3 is a SDS-PAGE result chart of the protein of the present application after transfection of 330ml XtenCHO;

[0074] Figure 4 is a SDS-PAGE result chart of the protein of the present application after transfection of 480ml XtenCHO;

[0075] Figure 5 is a SDS-PAGE result chart of the 6A2 antibody of the present application;

[0076] Figure 6 is a Western Blot analysis result of different cell processing groups of the present application;

[0077] Figure 7 is the result of the influence of the VDBP neutralizing antibody 6A2 of the present application on the complexity of neuron dendrites, in which A is the result of immunofluorescence staining, and B is the result of statistical analysis. DETAILED DESCRIPTION

[0078] The concept and technical effects of the present application will be described below in connection with the examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0079] In the description of the present application, the reference terms "preferably", "more preferably" and the like mean the embodiments of the present application which can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments can also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present application.

[0080] In the description of the present application, the reference term "and / or" includes all and any combination of one or more related listed items.

[0081] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application.

[0082] When a numerical range is disclosed herein, the range is to be construed as continuous along the entire range, and inclusive of the minimum and maximum values of the range, as well as each integer within the range. Further, where a range is provided, the range is intended to include any and all sub-ranges of the range. In other words, unless it is specified otherwise, all ranges disclosed herein are to be understood to encompass any and all sub-ranges of the original range.

[0083] Unless otherwise indicated, conventional methods or those modifications known to those of ordinary skill in the art were used. Unless otherwise indicated, all reagents or instruments were commercially available and were used according to the manufacturer's instructions.

[0084] I. Acquisition of target antigen

[0085] 1. Construction of pATX2-VDBP recombinant vector

[0086] The gene sequence of vitamin D binding protein was obtained from the National Center for Biotechnology Information (NCBI) database, and the gene was optimized according to the codon bias of Escherichia coli. The optimized VDBP nucleotide sequence information is as follows:

[0087] SEQ ID NO. 1, wherein the first to 15th positions are a connection sequence, the 16th to 1445th positions are a VDBP gene coding sequence, and the 1446th to 1481st positions are a connection sequence containing a His tag sequence.

[0088] The above-mentioned optimized VDBP gene sequence was recombined into the pATX2 vector through the EcoR I and Not I enzyme cutting sites of the pATX2 vector to obtain the recombinant plasmid pATX2-VDBP. The obtained recombinant plasmid pATX2-VDBP was sequenced and verified, and the result was consistent with the expectation.

[0089] 2. Small sample purification

[0090] (1) Pre-purification analysis

[0091] The above-mentioned optimized VDBP gene sequence was recombined into the pATX2 vector through the EcoR I and Not I enzyme cutting sites of the pATX2 vector to obtain the recombinant plasmid pATX2-VDBP. The obtained recombinant plasmid pATX2-VDBP was sequenced and verified, and the result was consistent with the expectation. The transfection kit was used to transfect 50 μg of the recombinant plasmid pATX2-VDBP constructed above into 80 mL (1 x 10 6 cells / mL) XtenCHO cells in FreeStyle TM CHO expression medium. On the 6th day after transfection, 1.0 mL of the sample was taken and purified using the following steps:

[0092] S1, the sample was placed in a high-speed refrigerated centrifuge (model: Neofuge 15R) and centrifuged at 10000g. The culture supernatant and bacterial precipitate were separated to obtain the culture supernatant sample (labeled as Medium) and the bacterial precipitate.

[0093] S2, proteinase inhibitors were added to the bacterial precipitate, which was resuspended and mixed uniformly, placed on crushed ice, and subjected to ultrasonic lysis using an ultrasonic crusher (specific parameters: 250W, ultrasonic lysis for 4s, interval of 7s, 99 cycles), centrifuged at 13000g and 4°C for 5min to separate the supernatant and the precipitate. The precipitate was resuspended with an equal volume of PBS to obtain the supernatant sample after bacterial disruption (labeled as NPE) and the precipitate sample after bacterial disruption (labeled as DPE).

[0094] S3, 10 μL of 6x Protein Loading Buffer (provided by Beijing Zhenben Technology Co., Ltd.) was added to the culture supernatant sample, the supernatant sample after bacterial disruption, and the precipitate sample after bacterial disruption, respectively, and vortexed to mix uniformly, followed by instantaneous centrifugation at 100°C for 10min in a metal bath. The samples after the metal bath were subjected to SDS-PAGE electrophoresis and Coomassie blue staining.

[0095] The SDS-PAGE detection results are shown in Figure 1, which shows that the theoretical molecular weight of the target antigen is 54.77kDa (including His tag), which is mainly expressed in the culture supernatant. The target antigen is obtained by purifying the culture supernatant.

[0096] (2) Small sample purification process

[0097] Based on the above transfection system, the culture was continued to the 9th day, and then the culture supernatant was collected and purified using Ni-NTA affinity chromatography based on the His tag, and the specific steps are as follows:

[0098] The collected culture supernatant was loaded onto a chromatography column (Ni-NTA resin) equilibrated with equilibration buffer (PBS, pH 7.5, 30 mM Imidazole). After loading, the column was washed with washing buffer, followed by elution with a 0%-100% gradient. The eluted fractions were analyzed by SDS-PAGE protein electrophoresis. The eluent was collected based on the results and dialyzed back into equilibration buffer. The dialysate was then collected for SDS-PAGE electrophoresis. The equilibration buffer contained PBS (pH 7.5) and 30 mM Imidazole; the washing buffer contained PBS (pH 7.5) and 50 mM Imidazole; and the eluent contained PBS (pH 7.5) and 300 mM Imidazole.

[0099] The SDS-PAGE electrophoresis results are shown in Figure 2, indicating that the band positions are consistent with expectations. Further quantitative analysis showed that approximately 1.04 mg of the target antigen could be obtained after transfecting 80 mL of XtenCHO cells with the recombinant plasmid pATX2-VDBP and culturing for 9 days. The purified target antigen was stored in PBS buffer at pH 7.5 for later use.

[0100] 3. Scale-up purification

[0101] use The transfection kit was used to transfect 200 μg of the recombinant plasmid pATX2-VDBP obtained above into 330 mL (1×10⁻⁶) of plasma. 6 XtenCHO cells (cells / mL) were used in FreeStyle TM XtenCHO cells were cultured in CHO expression medium. On day 9 post-transfection, the culture supernatant was collected for purification and SDS-PAGE protein electrophoresis. The specific purification method is the same as described in the small-sample purification procedure above. The SDS-PAGE protein electrophoresis results are shown in Figure 3, indicating that the band positions were consistent with expectations. Quantitative analysis showed that approximately 1.76 mg of target antigen could be obtained after 9 days of culture using the recombinant plasmid pATX2-VDBP transfection into 330 mL XtenCHO cells. The purified target antigen was stored in PBS buffer at pH 7.5 for later use.

[0102] use The transfection kit was used to transfect 300 μg of the recombinant plasmid pATX2-VDBP obtained above into 480 mL (1×10⁻⁶) of plasma. 6 XtenCHO cells (6 groups, 80 mL each) were collected and processed in FreeStyle. TMThe CHO expression medium was cultured, and the culture supernatant was collected on the 9th day after transfection for purification and SDS-PAGE protein electrophoresis detection. The specific purification method is referred to the small sample purification process described above. The SDS-PAGE protein electrophoresis detection result is shown in FIG. 4, which shows that the band position is consistent with the expected one. Quantitative analysis shows that about 2.70 mg of target antigen (VDBP) can be obtained after 9 days of culture of 330 mL Xten CHO cells transfected with the recombinant plasmid pATX2-VDBP. The target antigen obtained by the above purification is stored in PBS buffer with pH 7.5 for standby.

[0103] II. Antigen immunization and serum titer determination

[0104] (1) Immunization of New Zealand white rabbits with VDBP

[0105] Three 4-5 month old female New Zealand white rabbits were randomly divided into two groups. One group was a control group, including one New Zealand white rabbit (without immunization treatment); one group was an immunization group, including two New Zealand white rabbits.

[0106] The Pierce BCA Protein Assay Kit (product number: 23227, Thermo) was used to determine the concentration of VDBP, and then the VDBP was diluted to the target concentration with PBS, and mixed with Freund's adjuvant at a volume ratio of 1:1. After sufficient emulsification, the New Zealand white rabbits were immunized, and the specific immunization scheme is shown in Table 1. TM BCA Protein Assay Kits (product number: 23227, Thermo) was used to determine the concentration of VDBP, and then the VDBP was diluted to the target concentration with PBS, and mixed with Freund's adjuvant at a volume ratio of 1:1. After sufficient emulsification, the New Zealand white rabbits were immunized, and the specific immunization scheme is shown in Table 1.

[0107] Table 1: Immunization scheme

[0108] According to the above immunization scheme, the emulsified reagent was injected into the New Zealand white rabbits intradermally (2 points) and subcutaneously (3 points) on the abdomen, with 200 μL injected at each point. The New Zealand white rabbit serum was collected after the 3rd and 4th immunization, respectively.

[0109] (2) ELISA detection of serum titer

[0110] The collected New Zealand white rabbit serum was detected by ELISA method, and the specific steps were as follows:

[0111] S1, coating: dilute VDBP to a concentration of 5 μg / mL with PBS, add 100 μL / well to the enzyme-labeled plate, and incubate at 37°C for 2 h;

[0112] S2, blocking: shake off the coating liquid in the enzyme-labeled plate, add 300 μL PBS (containing 3% BSA) per well, cover the cover, and incubate in a 37°C incubator for 1 hour (or overnight at 4°C);

[0113] S3, washing: take out the coated and sealed enzyme-labeled plate, pour the blocking solution into the sink, add 300 μL PBST to each well, wash 3 times;

[0114] S4, primary antibody incubation: dilute the serum 2000, 4000, 8000, 16000, 32000, 64000, 128000 times with the blocking solution, add 100 μL / well to the enzyme-labeled plate, incubate at 37°C for 1 h, then wash with PBST 3 times (300 μL per well) and dry on a water-absorbing towel;

[0115] S5, secondary antibody incubation: dilute the enzyme-labeled antibody Anti-Rabbit IgG (H+L) (min X Hu Sr Prot) (brand Antibody System, product number PTB96431) with the blocking solution at 1:10000, add 100 μL / well to the enzyme-labeled plate, incubate at 37°C for 1 h, then remove the secondary antibody and wash with PBST 3 times (300 μL per well), and dry on a water-absorbing towel;

[0116] S6, TMB color development: pour the prepared TMB color development solution into a clean sample addition tank covered with a PE glove, use a gun to add 100 μL per well to the enzyme-labeled plate in turn, cover the lid and react at 37°C for 15 min in the dark, and observe the color development;

[0117] S7, termination: open the enzyme-labeled instrument, preheat for 1 minute, take out the enzyme-labeled plate from the incubator, and add 2M HCl to each well of the enzyme-labeled plate in turn at a volume of 50 μL per well;

[0118] S8, reading: open the Thermo enzyme-labeled instrument software, select 450 nm wavelength, wipe the bottom of the enzyme-labeled plate with a water-absorbing towel, and then put it into the enzyme-labeled instrument slot, click start to read the OD value. The blank control is the blocking solution without serum.

[0119] The maximum dilution of rabbit serum titer is (immune rabbit serum OD value-blank control OD value) / (control group rabbit serum OD value-blank control OD value)≥2.1. The test results are shown in Table 2.

[0120] Table 2: Serum titer after the third and fourth immunization

[0121] The test results show that as the number of immunizations increases, the titer of rabbit serum also increases, and the highest titer after the third immunization can reach 1:128000.

[0122] Three, monoclonal cell screening

[0123] 1, single B lymphocyte sorting

[0124] The New Zealand white rabbits with the highest titer after four immunizations were injected intraperitoneally with 500 μL of PBS containing 100 μg of VDBP three days in advance for booster immunization. The spleen tissue was aseptically isolated and peripheral blood was taken, and PBMC cells were isolated from the two materials by lymphocyte separation medium. Then about 960 single B lymphocyte clones (10*96-well plate, antigen-enriched) were isolated and cultured by B lymphocyte staining and flow sorting. After 7 days of culture, the supernatant was detected for the titer of each monoclonal cell strain by the method for detecting the titer of rabbit serum by ELISA as described above, and positive monoclonal cells with relatively high titer were screened.

[0125] 2. Monoclonal cell culture

[0126] The positive monoclonal cells with high titer as described above were blown down, washed once with complete culture medium, resuspended, and the cell density was adjusted to 10 4 The cells were diluted 1000-fold by limited dilution method, and added to a 96-well plate at 100 μL / well for monoclonalization. After 7-10 days of culture, whether there were monoclonal cell strains was observed under a microscope, and preserved for later use.

[0127] Four, amplification and sequencing analysis of the variable region of the positive monoclonal antibody

[0128] The antibody variable region genes VH and VL fragments of the positive monoclonal cells were amplified by PCR method, and then subcloned into an expression vector containing rabbit IgG constant region for sequencing analysis. The sequence information of the VH and VL fragments of the positive monoclonal antibody was obtained by sequence alignment analysis in the IMGT database. The nucleotide sequence information of the heavy chain variable region VH is as follows:

[0129] The amino acid sequence information of the heavy chain variable region VH is as follows:

[0130] QAVEESGGRLVTPGTPLTLTCTVSGFSLSNFYMQWVRQAPGKGLEWLGVIATGGNTYYASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCVRGDPAYPYLWGQGTLVTVSS (SEQ ID NO. 3). Among them, the amino acid sequence of the heavy chain variable region CDR-H1 is GFSLSNFY (as shown in SEQ ID NO. 3, positions 25-32); the amino acid sequence of the heavy chain variable region CDR-H2 is IATGGNT (as shown in SEQ ID NO. 3, positions 50-56); and the amino acid sequence of the heavy chain variable region CDR-H3 is VRGDPAYPYL (as shown in SEQ ID NO. 3, positions 93-102).

[0131] The nucleotide sequence information of the light chain variable region VL is as follows:

[0132] The amino acid sequence information of the light chain variable region VL is as follows:

[0133] AQVLTQTPSSVSAAVGGTVTISCQASQSVRSSNNLAWFQQKPGQPPKLLIYWASKLVSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGTYYNSGWSNTFGGGTEVVVK (SEQ ID NO. 5). Among them, the amino acid sequence of the light chain variable region CDR-L1 is QSVRSSNN (as shown in SEQ ID NO. 5 27-34); the amino acid sequence of the light chain variable region CDR-L2 is WAS (as shown in SEQ ID NO. 5 52-54); and the amino acid sequence of the light chain variable region CDR-L3 is QGTYYNSGWSN (as shown in SEQ ID NO. 5 91-101).

[0134] Five, expression, purification and sequencing analysis of neutralizing antibodies

[0135] 1. Antibody expression

[0136] Based on the VH and VL fragment information obtained above, a recombinant vector containing the heavy chain and light chain sequences was constructed, and then XtenCHO cells were co-transfected, wherein the heavy chain encoding nucleotide sequence information is as follows:

[0137] Among them, the 1-57 of SEQ ID NO. 6 is the coding nucleotide sequence of the heavy chain signal peptide, the 58-396 is the coding nucleotide sequence of the heavy chain variable region, and the 397-1368 is the coding nucleotide sequence of the heavy chain constant region.

[0138] The light chain encoding nucleotide sequence information is as follows:

[0139] Among them, the 1-60 of SEQ ID NO. 7 is the coding nucleotide sequence of the light chain signal peptide, the 61-396 is the coding nucleotide sequence of the light chain variable region, and the 397-711 is the coding nucleotide sequence of the light chain constant region.

[0140] After transfecting XtenCHO cells, recombinant cells were obtained, and after culture, the culture supernatant and bacterial body precipitate were separated and prepared for use.

[0141] 2. Antibody purification and sequencing analysis

[0142] (1) Purification sample pretreatment: collect the above-mentioned cell culture supernatant, further clarify by high-speed centrifugation at 8500 rpm for 10 min, then filter and clarify the high-speed centrifuged culture medium again by using a pretreated hollow fiber filter device, and finally concentrate the filtered culture medium by using a pretreated tangential flow concentration device for standby, during which 80 μl of sample (IN) is taken for SDS-PAGE detection;

[0143] (2) Column passing: add purification filler (rProtein A filler, purchased from GE healthcare, item number 17547402) to the gravity column, drop the 20% ethanol storage solution, add 10 times the column volume of ultrapure water to wash away the ethanol, and finally add 10 times the column volume of PBS pH 7.5 to balance the gravity column; block the lower end of the gravity column with a plug, and suspend the filler uniformly with the sample to be purified, mix them, put them into the binding shaker, and place the binding shaker in the 4°C refrigerator, the binding time should not be less than 60 min; take the combined sample from the binding shaker, stand it in the 4°C refrigerator for 5-10 min, then use a pipette to add the sample and filler mixture to the empty column, and collect the flow-through sample (FT), take 40 μl of sample (FT) for SDS-PAGE detection;

[0144] (3) Elution: use PBS pH 7.5 to wash the column, elute the non-specifically bound host proteins, collect the sample (W1), and take 40 μl of sample (W1) for SDS-PAGE detection; then wash each column volume with one tube of citric acid, add the amount of neutralizing liquid tested before, and mix gently, a total of 11 tubes, take 40 μl of sample from each tube and mark as E1-E11 for SDS-PAGE detection;

[0145] (4) Protein quantification of the purified antibody (labeled as 6A2 antibody) by BCA method, and electrophoretic detection of the purified antibody by SDS-PAGE.

[0146] The SDS-PAGE detection result is shown in Figure 5, which is consistent with the expectation, indicating that the 6A2 antibody is successfully purified.

[0147] Further, the 6A2 antibody purified above is subjected to sequencing analysis, and the result shows that the heavy chain amino acid sequence information of the 6A2 antibody is as follows:

[0148] Among them, the sequence shown in SEQ ID NO. 8 from 1st to 19th is the heavy chain signal peptide sequence, the sequence shown from 20th to 132nd is the heavy chain variable region sequence, and the sequence shown from 133rd to 455th is the heavy chain constant region sequence.

[0149] The light chain amino acid sequence information of the 6A2 antibody is as follows:

[0150] wherein the sequence shown in positions 1-20 in SEQ ID NO. 9 is a light chain signal peptide sequence, the sequence shown in positions 21-132 is a light chain variable region sequence, and the sequence shown in positions 133-236 is a light chain constant region sequence.

[0151] Six, detection of the titer of neutralizing antibodies

[0152] The binding ability of the supernatant of cells containing the 6A2 antibody to the antigen protein was detected by ELISA, and the specific detection method was as follows:

[0153] S1, coating: dilute VDBP to a concentration of 5 μg / mL with PBS to obtain a coating solution, add 100 μL / well to the enzyme-labeled plate, and incubate at 37°C for 2 h;

[0154] S2, blocking: shake off the coating solution in the enzyme-labeled plate, add 300 μL of PBS (containing 3% BSA) per well, cover the lid, and incubate in a 37°C incubator for 1 h (or overnight at 4°C);

[0155] S3, washing: remove the coated and blocked enzyme-labeled plate, shake the blocking solution into the sink, add 300 μL of PBST per well, and wash 3 times;

[0156] S4, primary antibody incubation: dilute the supernatant of cells containing the 6A2 antibody obtained above with the blocking solution to 7 gradients (6A2 antibody concentrations are 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 μg / mL), and the negative control group does not contain 6A2 antibody (0 μg / mL), add 100 μL / well to the enzyme-labeled plate, incubate at 37°C for 1 h, shake dry with PBST for 3 times (300 μL per well), and then pat dry on a water-absorbing paper towel;

[0157] S5, secondary antibody incubation: dilute the enzyme-labeled antibody Anti-Rabbit IgG (H+L) (min X Hu Sr Prot) with the blocking solution at 1:10000, add 100 μL / well to the enzyme-labeled plate, incubate at 37°C for 30 min, shake off the secondary antibody, wash with PBST for 3 times (300 μL per well), and then pat dry on a water-absorbing paper towel;

[0158] S6, TMB color development: pour the prepared TMB color development solution into a clean sample addition tank covered with a PE glove, add 100 μL per well to the enzyme-labeled plate in turn with a gun, cover the lid, and react at 37°C in the dark for 10-15 min to observe the color development;

[0159] S7, termination: open the enzyme label instrument, preheat for 1 minute, take the enzyme label plate from the incubator, add 2M HC1 to the enzyme label plate hole in the amount of 50 μL per hole in turn;

[0160] S8, reading: open the Thermo enzyme label instrument software, select 450 nm wavelength, wipe the bottom of the enzyme label plate with a water-absorbing towel and put it into the enzyme label instrument slot, click start to read to measure the OD value. The cell supernatant without VDBP neutralizing antibody is used as negative control. The sample OD450 value greater than 2.1 times of the negative control OD450 value is used as positive determination.

[0161] The detection results are shown in Table 3:

[0162] Table 3: Antibody titer detection results

[0163] The results show that as the concentration of 6A2 antibody increases, the value of OD450 also increases, proving that the titer is higher.

[0164] Seven, Western Blot analysis of neutralizing antibodies

[0165] The VDBP neutralizing antibody (6A2 antibody) obtained above is used to treat cells and perform Western Blot analysis, wherein the cell treatment groups are as follows:

[0166] Control group: primary neuron cells are cultured in normal culture medium without treatment.

[0167] VDBP treatment group: primary neuron cells are cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) is added.

[0168] VDBP+6A2 antibody treatment group: primary neuron cells are cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) and 6A2 neutralizing antibody are added.

[0169] The protein extraction and Western Blot detection steps are as follows:

[0170] (1) Total protein extraction and quantification:

[0171] ① Sample preparation and protein extraction: collect the cell samples of each group, aspirate the culture medium and rinse twice with PBS, add RIPA lysis buffer with protease inhibitor, place the culture bottle / dish on ice, use a cell scraper to scrape the cells, and aspirate the mixture into a centrifuge tube.

[0172] ② Protein quantification: Refer to the BCA protein quantification kit instructions for the corresponding operation to determine the protein concentration. Specifically, dilute the protein standard with a concentration of 2 pg / pL with ddH20 to 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 pg / pL, respectively, and use 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0 pg / pL as the detection points of the protein standard curve. Dilute the protein sample to be tested 10 times with ddH20. Mix the A and B liquids in the kit at a ratio of 50:1, and add 200 pL to each well of a 96-well plate. Add the diluted protein standard and the sample to be tested to the AB mixture in the 96-well plate, and incubate at 37°C for 30 min. Use a microplate reader to read the absorbance at 562 nm. According to the concentration of the protein standard and the corresponding absorbance, a standard curve is drawn, and the concentration of each sample to be tested is calculated using the curve.

[0173] ③ Protein denaturation: According to the measured concentration of each sample to be tested, adjust the concentration of each sample to the same with RIPA lysis buffer, add 1 / 4 volume of 5x SDS loading buffer, shake well, and heat in a metal bath at 100°C for 5 min to denature the protein. After denaturation, the sample can be used for subsequent experiments or stored at -20°C.

[0174] (2) Western blotting experiment:

[0175] S1, Gel preparation: According to the molecular weight of the detected protein, prepare the corresponding concentration separation gel according to the above ratio, and add an appropriate amount of isopropanol to press the gel. After the lower gel solidifies, prepare the upper concentrated gel and insert the comb. Place the prepared gel in the electrophoresis tank, add the electrophoresis liquid, and remove the comb.

[0176] S2, SDS-PAGE electrophoresis: Take the sample out at -20°C, shake well after warming. According to the experimental purpose, add protein marker indicator and protein sample in turn. Electrophorese at 80V constant voltage, and after the bromophenol blue indicator enters the separation gel, adjust to 120V constant voltage, continue to electrophorese until the bromophenol blue reaches the lower edge of the separation gel, and the protein marker indicator is completely separated, and the electrophoresis is completed.

[0177] S3, Membrane transfer: Pre-cool the membrane transfer solution, and place the sponge and filter paper in it for standby. Activate the PVDF membrane with methanol for 30s. Take out the gel and cut off the concentrated gel. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the membrane transfer clamp in order, ensuring that there are no air bubbles between the layers. After clamping and fixing, place it in the membrane transfer tank. According to the size of the molecular weight of the protein to be tested, adjust the current intensity and membrane transfer time.

[0178] S4, Blocking: Place the PVDF membrane after membrane transfer into 5% skimmed milk, and slowly shake on a shaker for 1-2 hours to completely remove the influence of non-specific antigens.

[0179] S5, primary antibody incubation: wash the membrane with TBST three times, and prepare the primary antibody with 5% BSA-TBST, put the PVDF membrane into the primary antibody, incubate at 4°C overnight. The antibody dilution ratio is: VDBP (1:1000), β-Actin (1:1000).

[0180] S6, secondary antibody incubation: take the PVDF membrane out of the primary antibody, wash the membrane with TBST on the shaker for 3 times, 10 min each time. Put the PVDF membrane into the corresponding secondary antibody prepared with 5% skimmed milk-TBST, incubate at room temperature for 1-1.5 hours. Wash the membrane with TBST on the shaker again for 3 times, 10 min each time.

[0181] S7, exposure: refer to the ECL reagent instruction, mix A and B evenly in a ratio of 1:1 to prepare the developing solution. Drop the developing solution evenly on the PVDF membrane, and expose using the chemiluminescence imaging system.

[0182] The detection results are shown in Figure 6, where A is the WB band of each group, and B is the statistical results of each group, showing that compared with the control group, the expression of synaptic proteins PSD95 and Synapsin I in the VDBP treatment group decreased significantly, and compared with the VDBP treatment group, the synaptic proteins PSD95 and Synapsin I in the neutralizing antibody 6A2 treatment group recovered, indicating that VDBP can damage the synaptic plasticity of neurons, and the neutralizing antibody 6A2 can block VDBP to avoid damage to the synaptic plasticity of neurons.

[0183] Eight, evaluation of neutralizing activity of neutralizing antibody

[0184] The neutralizing effect of the VDBP neutralizing antibody (6A2 antibody) obtained above was evaluated by MAP2 immunofluorescence staining method, wherein a control group, a VDBP treatment group and a VDBP+6A2 antibody treatment group were set up, wherein:

[0185] Control group: the primary neuron cells were cultured in normal culture medium without treatment.

[0186] VDBP treatment group: the primary neuron cells were cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) was added.

[0187] VDBP+6A2 antibody treatment group: the primary neuron cells were cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) and 6A2 neutralizing antibody were added.

[0188] The MAP2 immunofluorescence staining method is as follows:

[0189] (1) Reagent consumables preparation

[0190] Rabbit anti-MAP2 antibody (primary antibody), CoraLite 488 labeled goat anti-rabbit IgG (secondary antibody, purchased from Proteintech), 4% paraformaldehyde, triton (membrane breaking agent), BSA or serum of the animal from which the secondary antibody is derived, usually goat serum (for blocking), PBS, DAPI and anti-fluorescence quenching mounting medium (optional with DAPI), cover glass or special cell slide, glass slide, etc.

[0191] (2) Cell slide preparation

[0192] Pre-treatment of the slide before use: cut the slide or purchase the finished product, soak it in concentrated sulfuric acid overnight, rinse it with tap water the next day and place it in a sterilized lunch box. Put gauze on the bottom of the lunch box and place the slide at an angle against the side wall of the lunch box, making sure that the front of the slide does not touch the gauze. Then sterilize it with high-pressure steam and dry it in an oven.

[0193] For primary cells or cells that do not adhere well, treat the slide with polylysine or laminin or collagen solution before the cell slide to increase cell adhesion, so as not to wash the cells off when washing the slide with PBS later. After treating the slide with the above cell adhesion-promoting solution, rinse it with culture medium 1-3 times before placing it in the culture medium.

[0194] (3) Cell slide

[0195] Count the primary neuron cells after trypsin digestion and resuspend them in complete culture medium. Before adding cells, according to the size of the slide, first prepare the position where the slide will be placed in each well by dropping 100 μL of culture medium, so that the slide and the culture dish are adhered together by the tension of the culture medium, then place the slide to prevent the slide from floating up when adding the cell suspension, causing double-layer cell patches (the whole process should be sterile). After the cells adhere, divide them into the control group, VDBP treatment group and VDBP + 6A2 antibody treatment group according to the above method. The VDBP in the VDBP treatment group and the VDBP + 6A2 antibody treatment group is added to the culture medium at a volume of 20 μL (concentration of 50 ng / mL); the 6A2 neutralizing antibody in the VDBP + 6A2 antibody treatment group is added to the culture medium at a volume of 10 μL (concentration of 0.56 mg / mL), and the slide is taken after 24 hours of treatment.

[0196] (4) Fixation and permeabilization

[0197] The climbing piece is removed, washed with PBS, and then fixed. The confocal dish is directly removed from the culture medium and washed with PBS for 1-3 times. When washing, it is important to avoid violent shaking. After washing, a certain volume of 4% paraformaldehyde is added for fixation. Usually, it is fixed at room temperature (RT) for 15-20 min. After fixation, it is washed with pre-cooled PBS for three times, and then treated with 0.1% Triton X-100 (permeabilizing agent) for 10 min.

[0198] (5) Blocking

[0199] The concentration of BSA can be 1% or 5% (dissolved in PBS), and the concentration of serum can be 10%. RT blocking is performed for 30 min. If the binding force of your primary antibody is strong or the secondary antibody has non-specific binding, 0.1% Tween 20 can be added.

[0200] (6) Incubation

[0201] Primary antibody incubation: After blocking, the primary antibody is incubated after washing with PBS for 3 times. The primary antibody can be diluted with blocking solution, or PBS or antibody diluent. Incubate overnight at 4°C. The dilution ratio of the rabbit anti-MAP2 antibody (1:250) is as follows:

[0202] Secondary antibody incubation: The next day, after removing the primary antibody (which can be recovered), wash with PBS for 3 times, each time for 5-10 min, and then incubate the secondary antibody in the dark for 1 h.

[0203] (7) DAPI staining and mounting

[0204] Remove the secondary antibody, wash with PBS for 3 times, add DAPI, and incubate at room temperature for 5 min. Remove the DAPI, wash with PBS for 3 times. Add one drop of mounting medium to the slide, slowly place the cover glass on the slide, with the cell side close to the slide. Use a water-absorbing paper to remove the excess mounting medium, and then perform fluorescence microscopic observation.

[0205] The neutralization effect of the VDBP neutralizing antibody (6A2 antibody) is shown in FIG. 7, where A is the MAP2 immunofluorescence staining of the control group, the VDBP treatment group, and the VDBP+6A2 neutralizing antibody treatment group, and B is the corresponding statistical analysis. The results show that the neuron dendritic complexity of the VDBP treatment group decreases, indicating that VDBP can damage the dendritic structure of neurons, and the 6A2 neutralizing antibody of the application can offset the effect of VDBP on neuron damage, and has a protective effect on neuron damage.

[0206] The above describes the embodiments of the application in detail, but the application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antibody or antibody binding fragment thereof to vitamin D binding protein characterized in that, The antibody or antibody binding fragment thereof comprises a heavy chain and a light chain; The heavy chain comprises a heavy chain variable region comprising a complementarity determining region CDR-H1, CDR-H2 and CDR-H3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO. 3, positions 25-32; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO. 3, positions 50-56; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO. 3, positions 93-102; The light chain comprises a light chain variable region comprising a complementarity determining region CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO. 5, positions 27-34; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO. 5, positions 52-54; The amino acid sequence of the CDR-L3 is shown in SEQ ID NO. 5, positions 91-101.

2. The antibody or antibody-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region comprises any one of A1) to A3): A1) the amino acid sequence shown in SEQ ID NO. 3; A2) an amino acid sequence which is identical to the sequence shown in SEQ ID NO. 3, except for one or several substitutions and / or deletions and / or additions of amino acids, and which has the same function as the sequence shown in SEQ ID NO. 3; A3) an amino acid sequence which has 80%, 85% or 90% or more homology to SEQ ID NO. 3, and which has the same function as the sequence shown in SEQ ID NO. 3; The amino acid sequence of the light chain variable region comprises any one of B1) to B3): B1) the amino acid sequence shown in SEQ ID NO. 5; B2) an amino acid sequence which is identical to the sequence shown in SEQ ID NO. 5, except for one or several substitutions and / or deletions and / or additions of amino acids, and which has the same function as the sequence shown in SEQ ID NO. 5; B3) an amino acid sequence which has 80%, 85% or 90% or more homology to SEQ ID NO. 5, and which has the same function as the sequence shown in SEQ ID NO.

5.

3. The antibody or antibody-binding fragment thereof of claim 1 or 2, wherein, The antibody or antigen binding fragment thereof comprises at least one of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv, a bispecific antibody, a multispecific antibody.

4. The antibody or antibody-binding fragment thereof of claim 3, wherein, The heavy chain further comprises a heavy chain constant region; and / or The light chain further comprises a light chain constant region.

5. Biomaterials associated with the antibody or antibody binding fragment thereof of any one of claims 1 to 4, characterized in that, The biological material comprises any one of C1) to C5): C1) a nucleic acid molecule encoding the antibody or antibody binding fragment thereof according to any one of claims 1 to 4; C2) an expression cassette comprising the nucleic acid molecule of C1); C3) a recombinant vector comprising the nucleic acid molecule of C1) or the expression cassette of C2); C4) a recombinant microorganism comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3); C5) a recombinant cell comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3), which recombinant cell does not comprise reproductive material.

6. The biomaterial of claim 5, wherein, The nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region; The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown as SEQ ID NO. 2; and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO.

4.

7. A conjugate, characterized in that, comprising the antibody or antibody binding fragment thereof of any one of claims 1 to 4; and a conjugation moiety comprising at least one of a detectable label, a drug, a toxin, a radionuclide, an enzyme.

8. A product characterized by, The product comprises at least one of D1) to D3): D1) the antibody or antibody binding fragment thereof of any one of claims 1 to 4; D2) the biomaterial of claim 5 or 6; D3) the conjugate of claim 7; The product is selected from at least one of a drug, a detection plate, a chip, a test paper and a kit.

9. Use of any one of E1) to E3) in the preparation of a reagent for detecting vitamin D binding protein or in the preparation of a medicament for treating neuronal injury: E1) the antibody or antibody binding fragment thereof of any one of claims 1 to 4; E2) the biomaterial of claim 5 or 6; E3) the conjugate of claim 7.

10. A method of producing an antibody or antibody-binding fragment thereof according to any one of claims 1 to 4, characterized in that, obtained by culturing the recombinant microorganism or the recombinant cell as described in claim 5 or 6.

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