An antibody targeting neurofilament light chain, its preparation method and application

Abstract

The present invention discloses an antibody targeting neurofilament light chain, its preparation method and application. The present invention utilizes the HEAD structure to make the prokaryotic recombinant neurofilament light chain antigen have stronger immunogenicity and more exposed dominant epitopes, and monoclonal antibodies with higher affinity are obtained through immunization. In addition, by fusing a specific tag on the HEAD domain of the vector, the stability of the antigen is greatly improved, solving the problem of precipitation and degradation caused by the nature of the antigen itself, and further improving the success rate of antibody preparation. The antibody or its antigen-binding fragment of the present invention can be used for the early diagnosis and prognosis of neurodegenerative diseases.

Description

Technical Field

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

[0002] Neurofilament Light Chain (NFL) is a member of the neurofilament protein family and belongs to the intermediate filament protein structural component, mainly present in the axons of neurons. The neurofilament protein family includes heavy chain, medium chain, and light chain, and NFL is the smallest subunit. Neurofilaments play a key role in maintaining the morphology, structural stability, and axon diameter of neurons, 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. An increase in the level of NFL in blood or cerebrospinal fluid often indicates neuronal damage. Preparing NFL antigen by genetic recombination and using it as an immunogen to prepare monoclonal antibodies can provide core biological raw materials for detecting the level of NFL in blood or cerebrospinal fluid.

[0004] Under normal physiological conditions, NFL forms neurofilaments by polymerizing with NF-M and NF-H together to maintain the morphology and function of neurons. Its metabolic dynamics are regulated by various kinases (such as PKA and PKC), affecting the assembly and disassembly of NFL. After nerve injury, the transport of NFs is abnormal, activating calcium-activated proteases (such as calpain) to cause hydrolysis. The E3 ligase TRIM2 can mediate the ubiquitination of NFL, with NFM as its target. The E3 ligase can target NFs for degradation. After that, NFL will be released into the blood and cerebrospinal fluid in two forms: intact NFL protein and fragments generated by enzymatic hydrolysis.

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

[0006] The information in the background art is only for explaining the general background of the present invention and should not be regarded as admitting or implying in any form that this information constitutes the prior art known to those of ordinary skill in the art. Summary of the Invention

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

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

[0009] In certain embodiments, for the antibody or the antigen-binding fragment thereof according to the present invention, wherein the antibody or the antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0010] (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;

[0011] (II) An amino acid sequence having at least 90% homology with the amino acid sequence shown in (I) and having the same function;

[0012] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0013] In certain embodiments, for the antibody or the antigen-binding fragment thereof according to the present invention, wherein the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes a Fab, Fab’, F(ab)2, F(ab’)2, scFv or scFv Fc fragment.

[0014] In a second aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or the antigen-binding fragment thereof according to the present invention.

[0015] In a third aspect of the present invention, there is provided a vector molecule comprising the nucleic acid molecule according to the present invention.

[0016] In a fourth aspect of the present invention, there is provided a host cell comprising the nucleic acid molecule or the vector molecule according to the present invention.

[0017] In a fifth aspect of the present invention, there is provided a method for preparing the antibody or the antigen-binding fragment thereof according to the present invention, which is prepared by artificial synthesis or genetic engineering methods.

[0018] In a sixth aspect of the present invention, there is provided a detection product for neurofilament light chain, which comprises the antibody or the antigen-binding fragment thereof according to the present invention.

[0019] In certain embodiments, for the detection product according to the present invention, wherein the detection product includes a kit, a test strip or a protein chip.

[0020] In the seventh aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment of the present invention in the preparation of a product for detecting neurofilament light chain.

[0021] The present invention utilizes the HEAD structure to make the prokaryotic recombinant neurofilament light chain antigen have stronger immunogenicity and more exposed dominant epitopes, and obtains monoclonal antibodies with higher affinity through immunization. In addition, by fusing a specific tag on the HEAD domain of the fusion vector, the present invention greatly improves the stability of the antigen, solves the problem of precipitation and degradation caused by the nature of the antigen itself, and further improves the success rate of antibody preparation. The antibody or its antigen-binding fragment of the present invention can be used for the early diagnosis and prognosis of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows the recombinant expression vector used in the preparation of the antibody targeting NFL.

[0023] Figure 2 Shows the purity identification result of the purified NFL antigen.

[0024] Figure 3 Shows the ELISA activity verification result of the monoclonal antibody of the present invention.

[0025] Figure 4 Shows the results of detecting serum samples of healthy people and Parkinson's patients using the antibody of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Antibody or its antigen-binding fragment

[0030] In one aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof that can bind to neurofilament light chain with high affinity and targeting. Without being bound by any theory, the antibody or the antigen-binding fragment thereof of the present invention comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0031] In a specific embodiment, the antibody or the antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0032] (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;

[0033] (II) An amino acid sequence having at least 90% homology with the amino acid sequence shown in (I) and having the same function;

[0034] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0035] As used herein, "neurofilament light chain", "neurofilament protein L", "neurofilament light polypeptide", "neurofilament light chain", "neurofilament light chain protein" and "NFL" are used interchangeably and all refer to the constituent subunit of the neuronal neurofilament protein (Neurofilaments, NF).

[0036] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. An antibody typically contains variable and constant regions in each heavy and light chain. The variable regions of the antibody heavy and light chains contain the 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. Thus, 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.

[0037] The "variable light chain region (VL)" or "variable heavy chain region (VH)" of the present invention consists of "framework" regions that intersperse three "complementary determining regions (CDRs)". The framework regions are used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are mainly 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.

[0038] 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. To determine sequence identity, sequence alignment can be performed, which can be carried out in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0039] As used herein, antibody sequences obtained by modification also fall within the scope of protection 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 with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence that results in an increase in one or more amino acids compared to the naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in regions other than the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional characteristics of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding characteristics.

[0040] In the present invention, the antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies, or murine antibodies.

[0041] As used herein, the term "monoclonal antibody", sometimes also referred to as "mAb" or Ab, refers to an immunoglobulin obtained from a pure line of cells, having the same structure and chemical properties, and being specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically are a mixture of different antibodies directed 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 by culturing hybridomas or recombinant engineered cells and are not contaminated with other immunoglobulins. This property is in contrast to polyclonal antibody preparations, which generally include antibodies directed against different antigenic determinants. The modifier "monoclonal" indicates the nature of the antibody, which is obtained from a homogeneous population of antibodies, but this should not be construed as requiring any particular method for producing the antibody.

[0042] As used herein, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence in an antibody from a particular species or belongs to a particular class, while the remaining segments of that chain are homologous to the corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of an antibody from one animal species (such as a mouse, rat, etc.), while the constant portions are homologous to the antibody sequences from another species (such as a human). For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells can be used to generate the variable regions, and the constant regions combined therewith are from a human. The variable regions have the advantage of being easily prepared, and their specificity is not affected by the origin of the constant regions combined therewith. At the same time, since the constant regions of chimeric antibodies can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than that of an antibody with a non-human-derived constant region.

[0043] As used herein, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from non-human immunoglobulins, thereby reducing the immunogenicity when the xenogeneic antibody is introduced into humans, while maintaining the full antigen-binding affinity and specificity of the antibody.

[0044] As used herein, the term "murine antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from murine germline immunoglobulin sequences. In addition, if the antibody contains constant regions, they are also derived from murine germline immunoglobulin sequences. In the present invention, a murine antibody may contain amino acid residues not encoded by murine germline immunoglobulin sequences, for example, it may include mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or by in vivo somatic mutagenesis.

[0045] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that perform the function of specifically binding an antigen. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by: the heavy chain of a fragment including Fv, scFv, dsFv, Fab, Fab' or F(ab')2, or, the light chain of a fragment including Fv, scFv, dsFv, Fab, Fab' or F(ab')2.

[0046] In the present invention, the terms "against", "bind", "immunobind", "specifically bind" and "target" are used interchangeably and generally refer to non-covalent interactions that occur between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed by the dissociation constant (Kd), where a smaller Kd represents a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, when used herein, "binding affinity" refers to the intrinsic binding affinity reflecting the 1:1 interaction between the members of a binding pair (such as an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the prior art and described herein.

[0047] Unless otherwise specified, the antibodies or their antigen-binding fragments described herein are isolated antibodies or their antigen-binding fragments. The term "isolated" as used herein refers to an antibody or its antigen-binding fragment that has been removed from its natural environment. An antibody or its fragment that has been "isolated" thus includes an antibody or its fragment purified by standard purification methods. The term also includes antibodies or their fragments prepared by recombinant expression in host cells and chemically synthesized antibodies or their fragments.

[0048] Nucleic acid molecule

[0049] In one aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment described in the present invention.

[0050] The term "nucleic acid" as used in the present invention is intended to include polymeric forms of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides and / or their analogs, which include DNA, RNA and DNA / RNA hybrids, which also include DNA or RNA analogs, such as those containing modified backbones (such as peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0051] Once the coding sequence of the antibody of the present invention is obtained by separation, the antibody can be obtained in large quantities by recombinant techniques. An exemplary method is to clone its coding gene into a vector, then transfer it into cells, and then isolate it from the proliferated host cells by conventional methods.

[0052] Carrier molecule

[0053] In one aspect of the present invention, there is provided a vector molecule comprising the nucleic acid molecule of the present invention.

[0054] 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 encoding the antibody of the present invention, a promoter, a terminator, or optionally further comprises a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0055] Host cell

[0056] In one aspect of the present invention, there is provided a host cell comprising the nucleic acid molecule of the present invention or the vector molecule of the present invention.

[0057] 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 the nucleic acid molecule of the present invention. The host cell includes any progeny of the parental cell that is different from the parental cell due to mutations that occur during replication.

[0058] Preparation method

[0059] In one aspect of the present invention, there is provided a method for preparing the antibody of the present invention or its antigen-binding fragment. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering methods.

[0060] In certain embodiments, the antibody of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art. For example, the antibody of the present invention or its antigen-binding fragment can be obtained by direct amino acid synthesis.

[0061] In certain embodiments, the antibody of the present invention is 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 the Escherichia coli expression system. Eukaryotic cell expression systems include yeast expression systems, insect cell expression systems, and mammalian cell expression systems.

[0062] In a preferred embodiment, the antibody of the present invention can be prepared by the following steps:

[0063] (1) Construct a recombinant vector expressing the heavy chain of the antibody with the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector expressing the light chain of the antibody with the amino acid sequence shown in SEQ ID NO.8;

[0064] (2) Transform the vector into a host cell and culture it under conditions suitable for antibody expression;

[0065] (3) Collect the antibody and purify it.

[0066] 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 includes:

[0067] (1’) Construct a recombinant vector expressing the antigen sequence shown in SEQ ID No.9, and obtain the recombinant antigen after expression;

[0068] (2’) Inoculate the antigen into mice to prepare monoclonal antibodies;

[0069] (3’) Use the antigen to screen for positive hybridoma cells.

[0070] 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 prokaryotic expression vector of NFL is shown in SEQ ID NO.10.

[0071] In the step (1’) of the present invention, the host bacteria used for expressing the recombinant antigen include BL21(DE3), BL21Star, Rosetta, etc., preferably Rosetta.

[0072] Preferably, the step (1’) of the present invention further includes a step of purifying the recombinant antigen using a buffer solution containing 10% glycerol, Tris, NaCl, and DTT.

[0073] In the step (2’) of the present invention, the amount of the antigen inoculated into the experimental mice is 250 μg, and the immunoadjuvant used for the antigen is Freund's complete adjuvant.

[0074] In the steps (1’) and (3’) of the present invention, the protein sequence of the full-length antigen is as shown in SEQ ID NO.9.

[0075] Detection product

[0076] The present invention further provides a detection product for detecting (or quantifying) neurofilament light chain, which comprises the antibody or its antigen-binding fragment described in the present invention, and instructions on how to perform the detection method of the present invention on neurofilament light chain.

[0077] In a preferred embodiment, the detection product includes a kit, a test strip or a protein chip.

[0078] As used herein, the term "kit" refers to a combination of reagents and other materials. A kit is expected to contain reagents such as buffers, protein stabilizing reagents, signal generation systems (e.g., fluorescence signal generation 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, a kit may also include instructions for using the reagents. A kit can be packaged in any suitable manner. Generally, it has components in a single container or (if necessary) in multiple containers, as well as instructions for performing the detection. Kits can be prepared by a variety of methods known in the art.

[0079] In certain embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. Among them, the composition of the washing solution is not particularly limited, and its examples include but are not limited to buffers, surfactants, and preservatives. Known substrates can be used for the substrate solution, and its examples include but are not limited to chromogenic substrates, fluorescent substrates, chemiluminescent substrates, etc. The composition of the diluent is not particularly limited, and its examples include but are not limited to buffers, surfactants, etc. The composition of the calibration solution is not particularly limited, and its examples include but are not limited to BSA solutions, trehalose solutions, animal sera, etc.

[0080] Use

[0081] The present invention further provides the use of a detection reagent in the preparation of a detection product for neurofilament light chain, wherein the detection product includes a kit, a test strip or a protein chip. The detection product is used for the prediction, diagnosis, and / or prognosis of NFL-related diseases. "NFL-related diseases" refers to the abnormal expression of NFL in a subject, or an increase in the amount of NFL released in a sample to be tested (including but not limited to blood, cerebrospinal fluid), or an increase in NFL activity. For example, in certain cases, compared with the normal expression of NFL in healthy individuals, the expression level of NFL in the sample to be tested of the subject is significantly up-regulated.

[0082] 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, dementia with Lewy bodies, Huntington's disease, cerebral ischemia, epilepsy, frontotemporal dementia, Creutzfeldt-Jakob disease, multiple sclerosis, etc.

[0083] Example 1

[0084] This example shows the synthesis of monoclonal antibodies.

[0085] 1. Obtain the immunogen

[0086] Transfer the antigen expression vector NFL-HEAD-pET32a into the Rosetta(DE3) strain. The strain with correct sequencing is inoculated into 10 ml of LB medium (Amp antibiotic) at a ratio of 1:100 and cultured overnight at 37°C and 220 rpm. Then, the bacterial solution is inoculated into 1 L of LB medium (Amp antibiotic) at a ratio of 1:100 and cultured at 37°C and 220 rpm until OD600≈0.6. Then, 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) is added and cultured overnight at 20°C and 220 rpm. The next day, the bacteria are collected by centrifugation. The collected bacteria are resuspended in 250 ml of lysis buffer (50 mM Tris-HCl pH8.0, 500 mM NaCl), pre-cooled on ice for 10 min, and then lysed by ultrasound. The ultrasound power is 300 W, the ultrasound time is 1 s, the ultrasound interval is 1 s, and the total ultrasound time is 30 min. After identification, NFL is soluble in prokaryotic expression.

[0087] The inclusion body precipitate after cell disruption is resuspended in 25 mM Tris-HCl pH8.0, 125 mM NaCl, 0.5% Tween-20, 1 mM EDTA, 1 M UREA, mixed vertically for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant is discarded. The washing is repeated twice. The precipitate is washed with 25 mM Tris-HCl pH8.0, 125 mM NaCl, resuspended sufficiently, and centrifuged at 12000 rpm for 10 min to completely remove the residual UREA.

[0088] The cell supernatant after 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 eluate of the target protein 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 ).

[0089] 2. Mouse immunization

[0090] Purebred BALB / C mice were selected. For the primary immunization, 50 μg of NFL antigen was used. After adding Freund's complete adjuvant and emulsifying by reciprocating injection with a syringe, it was subcutaneously injected at multiple points. Three weeks later, the second immunization dose was the same as above, and it was subcutaneously injected with Freund's incomplete adjuvant. Another three weeks later, the third immunization dose was the same as above, without adjuvant, and it was intraperitoneally injected. Five to seven days later, the blood was collected to measure its titer. Two to three weeks later, a booster immunization was given with a dose of 100 μg of NFL antigen by intraperitoneal injection. Three days later, spleen cells were taken for screening of hybridoma cells and construction of the antibody library.

[0091] This example was for the titer detection of NFL experimental mice. The serum of the experimental mice was detected. The coating conditions were: cold coating and heat sealing, NFL-Ag, coating concentration: 1 μg / ml, 10 μg / well, 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, termination, reading.

[0092] Table 1 Results of the third immunization titer determination

[0093]

[0094] The experimental results showed that the detected titer of mouse #3 was about 100,000, the detected titer of mouse #5 was about 200,000, and the average detected titer of mice #1, #2, and #4 was about 400,000. Mouse #2 was selected for subsequent experiments.

[0095] 3. Preparation of hybridoma antibodies

[0096] ① Cell fusion

[0097] Mix myeloma cells SP2 / 0 and splenocytes at a ratio of 1:10, place them in a 50 ml centrifuge tube, wash once with serum-free incomplete culture medium, centrifuge at 1200 rpm for 8 min, discard the supernatant, and aspirate the residual liquid with a pipette tip. Gently flick the bottom of the centrifuge tube to slightly disperse the cell pellet. Add 1 ml of 45% PEG (molecular weight 3250) solution preheated to 37°C within 90 s, add it while gently shaking, and place it in a 37°C water bath for 90 s.

[0098] Add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 6 ml of incomplete culture medium preheated to 37°C every 2 min to terminate the action of PEG, and centrifuge at 800 rpm for 6 min. Discard the supernatant and resuspend with HAT selection culture medium (containing 20% calf serum). In a 96-well plate with a feeder cell layer, add 100 μl of resuspended cells to each well and culture in a 37°C, 5% CO2 incubator.

[0099] ② Cloning and screening of hybridoma cells and antibody acquisition

[0100] Monoclonalize hybridoma cells using the limiting dilution method, detect the reactivity of the cell supernatant of each clone with the NFL recombinant antigen, and transfer the cells in the positive wells to a 24-well plate for expansion culture.

[0101] Take BALB / C mice, intraperitoneally inject 0.5 ml of liquid paraffin oil, and 1 - 2 weeks later, intraperitoneally inject 1×10 6 positive hybridoma cells. Collect ascites 7 - 10 days later and purify to obtain the antibody.

[0102] ③ Verification of monoclonal antibody activity

[0103] Coating: Dilute NFL antigen with PBS at 1 μg / ml, 100 μl / well, and incubate at 37°C for 2 h.

[0104] Blocking: Block using CEA blocking solution, 200 μl / well, and incubate at 37°C for 2 h.

[0105] Primary antibody: Gradient dilute the NFL monoclonal antibody from 10 μg / ml with PBS, 100 μl / well, incubate at 37°C for 1 h, and wash the plate.

[0106] Secondary antibody: Dilute HRP-labeled goat anti-mouse with CCP enzyme at a ratio of 1:1000, 100 μl / well, and incubate at 37°C for 30 min.

[0107] Chromogenic reaction: Mix solution A and solution B at a ratio of 1:1, 100 μl / well, and place at room temperature for 5 min.

[0108] Termination: Add 50 μl / well of termination solution.

[0109] Reading value: Read using an enzyme-linked immunosorbent assay (ELISA) reader.

[0110] Table 2 Verification of Antibody Activity

[0111]

[0112] The results showed that the EC50 value of antibody 66# was the lowest, indicating that it had the strongest binding ability to the antigen.

[0113] Example 2

[0114] This example shows the detection of serum samples from healthy individuals and Parkinson's disease patients using the enzyme-linked immunosorbent assay (ELISA) method with the antibody targeting neurofilament light chain screened in Example 1.

[0115] Dilute the prepared NFL antibody to 2 μg / ml with coating buffer, add 100 μl of the sample to each well, incubate overnight at 4°C, add 200 μl of blocking solution and incubate at 37°C for 2 h. Dilute the NFL antigen to 1 ng / ml, perform three-fold serial dilution for 7 gradients, incubate at 37°C for 1 h, wash the plate, add 100 μl of NFL-HRP secondary antibody diluted 1000-fold in dilution ratio to each well, wash the plate, add chromogenic solution for color development for 5 min, add stop solution, read using an ELISA plate reader, draw a standard curve based on the readings, substitute the absorbance OD(450 - 630)nm of the sample detection into the standard equation, and the antigen concentration of the sample can be calculated. Detect the serum NFL content of healthy individuals and Parkinson's patients, with 30 people in each group.

[0116] The sample detection results are as Figure 4 shown. The results showed that the NFL antibody of the present invention can sensitively detect the NFL level in serum, can be used for scientific research or clinical detection of NFL expression, and further can be used for the early diagnosis and prognosis prediction of various neurological diseases such as Parkinson's disease and Parkinson's plus syndrome.

[0117] 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. Without departing from the scope or spirit of the present invention, various adjustments or changes can be made to the exemplary embodiments of the present invention specification. The scope of the claims should be interpreted 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 neurofilament light chain, and the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The amino acid sequence of the antibody or its antigen-binding fragment has at least 90% homology with the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.8 and has the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment has an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.8 and has the same function.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or its antigen-binding fragment has the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.

8.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody includes monoclonal antibody, and the antigen-binding fragment includes Fab, Fab’, F(ab’)2, scFv or scFv Fc fragment.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody includes chimeric antibody, humanized antibody or murine antibody.

7. A nucleic acid molecule, characterized in that, It comprises a nucleotide sequence encoding the antibody or its antigen-binding fragment according to any one of claims 1-6.

8. Carrier molecule, characterized in that, It comprises the nucleic acid molecule according to claim 7.

9. A host cell, characterized in that, It comprises the nucleic acid molecule according to claim 7 or the vector molecule according to claim 8.

10. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-6, characterized in that, Prepared by artificial synthesis or genetic engineering methods.

11. A detection product for neurofilament light chain, characterized in that, It comprises the antibody or its antigen-binding fragment according to any one of claims 1-6.

12. The detection product according to claim 11, wherein, The detection product includes kit, test strip or protein chip.

13. Use of the antibody or its antigen-binding fragment according to any one of claims 1-6 in the preparation of a neurofilament light chain detection product.

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