Neural fiber light chain targeting antibody as well as preparation method and application thereof

Abstract

The invention discloses an antibody targeting a nerve cellosilk light chain as well as a preparation method and application of the antibody. According to the present invention, with the HEAD structure, the prokaryotic recombinant nerve fiber filament light chain antigen has strong immunogenicity, more dominant epitopes are exposed, and the monoclonal antibody with high affinity is prepared through immunization; besides, the HEAD structural domain is fused with the specific label on the carrier, so that the stability of the antigen is greatly improved, the problem of polysinolysis caused by the property of the antigen is solved, and the success rate of antibody preparation is further improved. The antibody or the antigen binding fragment thereof can be used for early diagnosis and prognosis of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and immunology, and in particular to an antibody targeting neurofilament light chain and a preparation method and application thereof. Background Art

[0002] Neurofilament Light Chain (NFL) is a member of the neurofilament protein family and is a structural component of intermediate filament proteins. It is mainly found in the axons of neurons. The neurofilament protein family includes heavy chains, medium chains, and light chains, and NFL is the smallest subunit. Neurofilaments play a key role in maintaining neuronal morphology, structural stability, and axon diameter, thereby affecting the conduction speed of nerve signals.

[0003] NFL has been widely studied clinically as a biomarker for neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), etc. Increased NFL levels in blood or cerebrospinal fluid often indicate neuronal damage. Preparation of NFL antigens by genetic recombination and preparation of monoclonal antibodies using this as an immunogen can provide core biological raw materials for detecting NFL levels in blood or cerebrospinal fluid.

[0004] Under normal physiological conditions, NFL forms neurofilaments by co-polymerizing with NF-M and NF-H to maintain the morphology and function of neurons. Its metabolic dynamics are regulated by multiple kinases (such as PKA and PKC), which affect the assembly and disassembly of NFL. After nerve injury, the transport of NFs is abnormal, activating calcium-activated proteases (such as calpain) to lead to hydrolysis. The E3 ligase TRIM2 can mediate the ubiquitination of NFL, with NFM as its target. E3 ligases can target NFs for degradation, after which NFL is released into the blood and cerebrospinal fluid in the form of complete NFL protein and fragments produced by enzymatic hydrolysis.

[0005] Therefore, there is still a need for an antibody or an antigen-binding fragment thereof targeting NFL to develop products for detecting neurodegenerative diseases.

[0006] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0007] In order to solve at least some of the technical problems in the prior art, the present invention provides an antibody targeting neurofilament light chain and a preparation method and application thereof. Specifically, the present invention includes the following contents.

[0008] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can target the light chain of neurofilaments, and the antibody or the antigen-binding fragment thereof comprises the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or the light chain CDR1-3 shown in SEQ ID NO.4-6.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0010] In certain embodiments, according to the antibody or antigen-binding fragment thereof of the present invention, the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment comprises a Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc fragment.

[0011] The second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof described in the present invention.

[0012] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0013] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule or vector molecule of the present invention.

[0014] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which is prepared by artificial synthesis or genetic engineering.

[0015] In a sixth aspect, the present invention provides a product for detecting neurofilament light chain, which comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0016] In certain embodiments, the detection product according to the present invention comprises a kit, a test paper or a protein chip.

[0017] The seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the present invention in the preparation of a neurofilament light chain detection product.

[0018] The present invention utilizes the HEAD structure to make the prokaryotic recombinant neurofilament light chain antigen more immunogenic and more exposed to the dominant epitope, and obtains a monoclonal antibody with higher affinity through immunization preparation. In addition, the present invention greatly improves the stability of the antigen by fusing the HEAD domain to a specific tag on the carrier, solves the problem of aggregation and degradation caused by the nature of the antigen itself, and further improves the success rate of antibody preparation. The antibody or antigen-binding fragment thereof of the present invention can be used for the early diagnosis and prognosis of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the recombinant expression vector used for the preparation of the antibody targeting NFL.

[0020] Figure 2 The purity evaluation results of the purified NFL antigen are shown.

[0021] Figure 3 The results of ELISA activity verification of the monoclonal antibody of the present invention are shown.

[0022] Figure 4 The results of using the antibody of the present invention to detect serum samples of healthy people and Parkinson's patients are shown. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] Antibodies or antigen-binding fragments thereof In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can bind to the light chain of neurofilaments with high affinity and targeting. Without being bound by any theory, the antibody or the antigen-binding fragment thereof of the present invention comprises the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or the light chain CDR1-3 shown in SEQ ID NO.4-6.

[0027] In a specific embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0028] In this article, "neurofilament light chain", "neurofilament protein L", "neurofilament protein light chain", "neurofilament light chain", "neurofilament light chain protein" and NFL are used interchangeably and all refer to the constituent subunits of neuronal neurofilaments (NF).

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0030] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region interspersed with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0031] As used herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required for achieving optimal alignment in the full-length sequence being compared.

[0032] Herein, the antibody sequences obtained by modification also belong to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0033] In the present invention, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a mouse antibody.

[0034] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoantibody" or Ab, refers to an immunoglobulin obtained from a pure cell line, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. This characteristic is in contrast to polyclonal antibody preparations, which generally include antibodies against different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0035] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light chain and the heavy chain are all from the variable regions of antibodies of one animal species (such as mice, rats, etc.), while the constant portion is homologous to the antibody sequence from another species (such as humans). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, while the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response during injection is lower than that of using antibodies with non-human constant regions.

[0036] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.

[0037] Herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, the murine antibody may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo.

[0038] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2.

[0039] In the present invention, the terms "directed against", "binding", "immunobinding", "specific binding" and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific for the immunoglobulin. The strength or affinity of the immunological binding interaction can be expressed in terms of a dissociation constant (Kd), with a smaller Kd representing a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be described in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the art and described herein.

[0040] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. An antibody or fragment thereof that has been "isolated" thus includes an antibody or fragment thereof that has been purified by standard purification methods. The term also includes antibodies or fragments thereof prepared by recombinant expression in a host cell and chemically synthesized antibodies or fragments thereof.

[0041] Nucleic acid molecules In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0042] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0043] Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities by recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then separate it from the propagated host cells by conventional methods.

[0044] Carrier molecules One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0045] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0046] Host cells In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0047] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. A host cell includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0048] Preparation method In one aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0049] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention or antigen-binding fragments thereof are obtained by direct amino acid synthesis.

[0050] In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems for genetic engineering expression include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0051] In a preferred embodiment, the antibody of the present invention can be prepared by the following steps: (1) constructing a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector expressing the antibody light chain of the amino acid sequence shown in SEQ ID NO.8; (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression; (3) Collect and purify antibodies.

[0052] In another preferred embodiment, the antibody of the present invention is prepared by immunization with a recombinant antigen, wherein the recombinant antigen has the amino acid sequence shown in SEQ ID No. 9. In a specific embodiment, the method for preparing the antibody of the present invention comprises: (1') constructing a recombinant vector expressing the antigen sequence shown in SEQ ID No.9, and obtaining a recombinant antigen after expression; (2') inoculating the antigen into mice to prepare monoclonal antibodies; (3') Using the antigen to screen positive hybridoma cells.

[0053] In the step (1') of the present invention, the full-length NFL gene is inserted into vectors such as pET28a, pET32a, pET22b, pET30a, etc., preferably pET32a. The nucleotide sequence of the constructed NFL prokaryotic expression vector is shown in SEQ ID NO.10.

[0054] In the step (1') of the present invention, the host bacteria used to express the recombinant antigen include BL21 (DE3), BL21Star, Rosetta, etc., preferably Rosetta.

[0055] Preferably, the step (1') of the present invention further comprises the step of purifying the recombinant antigen using a buffer comprising 10% glycerol, Tris, NaCl and DTT.

[0056] In the step (2') of the present invention, the amount of antigen inoculated into the experimental mice is 250 μg, and the immune adjuvant used for the antigen is Freund's complete adjuvant.

[0057] In the steps (1') and (3') of the present invention, the protein sequence of the full-length antigen used is shown in SEQ ID NO.9.

[0058] Testing products The present invention further provides a detection product for detecting (or quantifying) neurofilament light chains, which comprises the antibody or antigen-binding fragment thereof described in the present invention, and instructions on how to implement the detection method of the present invention on neurofilament light chains.

[0059] In a preferred embodiment, the detection product includes a kit, a test paper or a protein chip.

[0060] Herein, the term "kit" refers to a combination of reagents and other materials. The kit is expected to include reagents, such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or their antigen binding fragments, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials, for example, the kit may also include instructions for using the reagents. The kit may be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for performing detection instructions. The kit may be prepared by a variety of methods known in the art.

[0061] In certain embodiments, the kit may further include at least one of a cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. The substrate solution may use a known substrate, and examples thereof include, but are not limited to, a chromogenic substrate, a fluorescent substrate, a luminescent substrate, and the like. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, a BSA solution, a trehalose solution, an animal serum, and the like.

[0062] use The present invention further provides the use of a detection reagent in the preparation of a neurofilament light chain detection product, wherein the detection product includes a kit, a test paper or a protein chip. The detection product is used for the prediction, diagnosis and / or prognosis of NFL-related diseases, and "NFL-related diseases" refer to abnormal expression of NFL in the subject, or an increase in the amount of NFL released in the sample to be tested (including but not limited to blood, cerebrospinal fluid), or an increase in NFL activity. For example, in some cases, compared with the normal expression of NFL in healthy people, the expression of NFL in the sample to be tested of the subject has a significantly upregulated expression.

[0063] In the present invention, NFL-related diseases include neurodegenerative diseases, examples of which include but are not limited to Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, Lewy body dementia, Huntington's disease, cerebral ischemia, epilepsy, frontotemporal dementia, Creutzfeldt-Jakob disease, multiple sclerosis, etc.

[0064] Example 1 This example demonstrates the synthesis of a monoclonal antibody.

[0065] 1. Obtaining the Immunogen The antigen expression vector NFL-HEAD-pET32a was transferred into the Rosetta (DE3) strain, and the correctly sequenced strain was inoculated into 10 ml of LB medium (Amp antibiotics) at a ratio of 1:100, and cultured overnight at 37°C and 220 rpm. The bacterial solution was then inoculated into 1 L of LB medium (Amp antibiotics) at a ratio of 1:100, and cultured at 37°C and 220 rpm until OD600≈0.6, then 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and cultured overnight at 20°C and 220 rpm. The next day, the cells were collected by centrifugation, and the collected cells were resuspended in 250 ml of lysis buffer (50 mM Tris-HCl pH8.0, 500 mM NaCl), precooled on ice for 10 min, and then ultrasonically lysed, with an ultrasonic power of 300 W, an ultrasonic time of 1 s, an ultrasonic interval of 1 s, and a total ultrasonic time of 30 min. It was identified that NFL was expressed in a soluble form in prokaryotic cells.

[0066] The inclusion body precipitate after bacterial cell disruption was resuspended with 25 mM Tris-HCl pH8.0, 125 mM NaCl, 0.5% Tween-20, 1 mM EDTA, and 1 M UREA, vertically mixed for 30 min, centrifuged at 12000 rpm for 10 min, discarded the supernatant, and repeated washing twice. The precipitate was washed with 25 mM Tris-HCl pH8.0 and 125 mM NaCl, and after sufficient resuspension, centrifuged at 12000 rpm for 10 min to fully remove the residual UREA.

[0067] The cell supernatant after bacterial cell disruption was purified by affinity chromatography using a Ni-NTA affinity chromatography column. The purification buffer was 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 10% glycerol + 1 mM DTT. The final optimal washing concentration was 40 mM imidazole, and the optimal elution concentration was 250 mM imidazole. The target protein eluate was finally dialyzed into 50 mM Tris-HCl pH8.0 + 500 mM NaCl + 10% glycerol + 1 mM DTT. The purity of the purified NFL protein was 90% ( Figure 2 ).

[0068] 2. Mouse Immunization Purebred BALB / C mice were selected for the first immunization with 50 μg of NFL antigen, which was emulsified by adding Freund's complete adjuvant and injecting subcutaneously at multiple points. Three weeks later, the second immunization dose was the same as above, and Freund's incomplete adjuvant was added for subcutaneous injection. Three weeks later, the third immunization dose was the same as above, without adjuvant, and injected intraperitoneally. Blood was collected 5-7 days later to measure its titer. After another 2-3 weeks, booster immunization was performed with a dose of 100 μg of NFL antigen, injected intraperitoneally. Three days later, spleen cells were taken for hybridoma cell screening and antibody library construction.

[0069] This example is a titer test for NFL experimental mice, and the experimental mouse serum is tested, wherein the coating conditions are: cold pack and heat seal, NFL-Ag, coating concentration: 1 μg / ml, 10 μg / plate, coating solution: 0.05 M CB, 100 μl / well, blocking solution: CEA blocking solution 130 μl / well, reactant concentration: as shown in Table 1, enzyme concentration: 1:1000, reaction mode: sample addition for 30 min, secondary antibody for 30 min, color development for 10 min, stop, and read.

[0070] Table 1 Results of the third immune titer determination The experimental results showed that the detection titer of 3# mice was around 100,000, the detection titer of 5# mice was around 200,000, and the detection titer of 1#, 2# and 4# mice was around 400,000 on average. 2# was selected for subsequent experiments.

[0071] 3. Hybridoma Antibody Preparation ① Cell fusion Myeloma cells SP2 / 0 and spleen cells were mixed at a ratio of 1:10, placed in a 50 ml centrifuge tube, washed once with serum-free incomplete culture medium, centrifuged at 1200 rpm for 8 min, discarded the supernatant, and the remaining liquid was sucked up with a pipette. The bottom of the centrifuge tube was flicked to slightly disperse the cell pellet. Within 90 s, 1 ml of 45% PEG (molecular weight 3250) solution preheated at 37°C was added, and the tube was gently shaken while adding, and placed in a 37°C water bath for 90 s.

[0072] Add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml of incomplete culture medium preheated at 37°C every 2 min to terminate the PEG reaction, and centrifuge at 800 rpm for 6 min. Discard the supernatant and resuspend in HAT selection culture medium (containing 20% ​​calf serum). Add 100 μl of resuspended cells to each well of a 96-well plate with a feeder cell layer and culture in a 37°C, 5% CO2 incubator.

[0073] ② Clone screening of hybridoma cells and acquisition of antibodies The hybridoma cells were monocloned using the limiting dilution method, and the reactivity of the cell supernatant of each clone with the NFL recombinant antigen was detected. The cells in the positive wells were transferred to a 24-well plate for expansion culture.

[0074] BALB / C mice were intraperitoneally injected with 0.5 ml of liquid paraffin oil. One to two weeks later, 1×10 6 After 7-10 days, the ascites was collected and purified to obtain antibodies.

[0075] ③ Monoclonal antibody activity verification Coating: Dilute NFL antigen to 1 μg / ml in PBS, 100 μl / well, incubate at 37℃ for 2 h.

[0076] Blocking: Use CEA blocking solution for blocking, 200 μl per well, incubate at 37℃ for 2 h.

[0077] Primary antibody: dilute the NFL monoclonal antibody from 10 μg / ml using PBS, 100 μl / well, incubate at 37°C for 1 h, and wash the plate.

[0078] Secondary antibody: HRP-labeled goat anti-mouse was diluted with CCP enzyme at a ratio of 1:1000, 100 μl / well, and incubated at 37°C for 30 min.

[0079] Color development: Mix solution A and solution B in a 1:1 ratio, 100 μl / well, and leave at room temperature for 5 min.

[0080] Stop: 50 μl / well of stop solution.

[0081] Reading: Read using an ELISA reader.

[0082] Table 2 Antibody activity verification The results showed that the EC50 value of antibody #66 was the lowest, indicating that it had the strongest ability to bind to the antigen.

[0083] Example 2 This example shows the detection of serum samples of healthy people and Parkinson's disease patients using the antibody targeting neurofilament light chain screened in Example 1 by enzyme-linked immunosorbent assay.

[0084] The prepared NFL antibody was diluted to 2 μg / ml with coating buffer, 100 μl was added to each well, and placed at 4°C overnight. 200 μl of blocking solution was added and placed at 37°C for 2 h. The NFL antigen was diluted to 1 ng / ml, three-fold gradient dilution, 7 gradients were diluted, and placed at 37°C for 1 h. The plate was washed, and 100 μl of NFL-HRP secondary antibody with a dilution ratio of 1000 times was added to each well. The plate was washed, and the color development solution was added for 5 min. The stop solution was added, and the ELISA plate was used for reading. The standard curve was drawn according to the reading. The sample detection absorbance value OD (450-630) nm was substituted into the standard equation to calculate the sample antigen concentration. The serum NFL content of healthy people and Parkinson's patients was detected, and the number of samples was 30 people each.

[0085] The sample test results are as follows Figure 4 As shown, the results indicate that the NFL antibody of the present invention can sensitively detect the level of NFL in serum, can be used for scientific research or clinical detection of NFL expression, and can further be used for early diagnosis and prognosis prediction of various neurological diseases such as Parkinson's disease and Parkinson's plus syndrome.

[0086] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that It can target the light chain of neurofilaments, and the antibody or its antigen-binding fragment comprises the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or the light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies or murine antibodies, and the antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc fragments.

4. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4 or the vector molecule according to claim 5.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: Prepared by artificial synthesis or genetic engineering.

8. A product for detecting neurofilament light chain, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. The detection product according to claim 8, characterized in that: The detection product includes a test kit, a test paper or a protein chip.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a neurofilament light chain detection product.

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