An antibody for detecting neurofilament light chain protein and use thereof

Abstract

The application discloses an antibody for detecting neurofilament light chain protein and application thereof, and particularly relates to an antibody for detecting neurofilament light chain protein and a kit. The antibody and the kit can accurately detect the presence of neurofilament light chain protein.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and more specifically, to an antibody for detecting neurofilament light chain protein and its application. Background Technology

[0002] Neurofilaments are important structural frameworks in neurons, composed of four distinct subunits: neuroflament triplet proteins, α-internexin, or Type III peripherin. The neuroflament triplet proteins are composed of neuroflament light (NfL), neurofilament middle (NfM), and neurofilament heavy (NfH).

[0003] Neuronal proteins are highly expressed in neurons. Axonal injury and neurodegeneration lead to the release of neuronal proteins through the axonal membrane, leaking into the extracellular fluid and then into the cerebrospinal fluid, eventually seeping into the bloodstream. Neurofilament protein (NfL) is the smallest molecular weight and most abundant neurofilament protein, responsible for maintaining the function of the nervous system, and can be detected simultaneously in cerebrospinal fluid and blood. Under normal physiological conditions, axons release a small amount of NfL, but the release amount increases significantly under pathological conditions. Several studies have indicated that NfL can serve as a diagnostic, prognostic, and monitoring biomarker for brain injury and various neurological diseases, such as Alzheimer's disease, Parkinson's disease / syndrome, multiple sclerosis, and amyotrophic lateral sclerosis (ALS), making it one of the most promising biomarkers for neurodegenerative diseases. Furthermore, in cognitively normal individuals, serum NfL levels increase significantly with age, becoming more pronounced after age 65.

[0004] The concentration of non-fL (NfL) in cerebrospinal fluid is relatively high, generally less than 1800 pg / ml, but the concentration in blood is very low, generally less than 10 pg / ml. Currently, most reagent methods used for NfL detection are performed on research-only platforms, such as enzyme-linked immunosorbent assay (ELISA) and single-molecule assay (SIMOA). The gold standard NfL kit for research use (UmanDiagnostics) is based on the ELISA platform and cannot achieve large-scale batch detection. Currently, there is a lack of high-performance antibodies and paired antibodies on the market, which limits the accuracy and reliability of NfL detection. There is a strong demand in this field for antibodies and paired antibodies with strong binding and high sensitivity. Summary of the Invention

[0005] This application provides an antibody that provides an important source of raw materials for the detection of neurofilament light chain proteins, and has good detection performance, high sensitivity, strong specificity, and low cross-linking rate.

[0006] To achieve the above objective, according to a first aspect of the present invention, an antibody for detecting neurofilament light chain proteins is provided, the antibody comprising three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in any of SEQ ID NO:17 and 37, and three light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in any of SEQ ID NO:19 and 39.

[0007] To achieve the above objective, according to a second aspect of the present invention, an antibody for detecting neurofilament light chain proteins is provided, wherein the complementarity-determining region of the antibody includes any one of (a') to (b'):

[0008] (a') The amino acid sequences are as shown in SEQ ID NO:1 (SSYRWN) for HCDR1 and SEQ ID NO:2 respectively.

[0009] HCDR2 as shown in (YIQSTGTTNYNPSLKR), HCDR3 as shown in SEQ ID NO:3 (SAGTAPIWFTH), and LCDR1 as shown in SEQ ID NO:4 (KASQNVGSNVD), LCDR2 as shown in SEQ ID NO:5 (KASNRYT), and LCDR3 as shown in SEQ ID NO:6 (LQSNSYPPT); and

[0010] (b') The amino acid sequences are as shown in SEQ ID NO:21(NYWMN) for HCDR1 and SEQ ID NO:22, respectively.

[0011] HCDR2 as shown in (EIRLKSNNYATHYAESVKG), HCDR3 as shown in SEQ ID NO:23 (LDFDY), and LCDR1 as shown in SEQ ID NO:24 (QASQGTYINLK), LCDR2 as shown in SEQ ID NO:25 (GATNLED), and LCDR3 as shown in SEQ ID NO:26 (LQHSFLPYT).

[0012] To achieve the above objectives, according to a third aspect of the present invention, an antibody for detecting neurofilament light chain proteins is provided, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in either SEQ ID NO: 17 or 37, and the amino acid sequence of the light chain variable region is shown in either SEQ ID NO: 19 or 39.

[0013] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody for detecting neurofilament light chain proteins is provided, the antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as either SEQ ID NO: SEQ ID NO: 18 or 38, and the amino acid sequence of the light chain is shown as either SEQ ID NO: 20 or 40.

[0014] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody pair for detecting neurofilament light chain proteins is provided, the antibody pair comprising a coating antibody and / or a labeled antibody, wherein the coating antibody and the labeled antibody are respectively selected from the aforementioned antibodies.

[0015] To achieve the above objectives, according to a sixth aspect of the present invention, a detection reagent or kit for detecting neurofilament light chain proteins is provided, wherein the reagent or kit comprises the aforementioned antibody or antibody pair.

[0016] To achieve the above objectives, according to a seventh aspect of the present invention, a method for detecting neurofilament light chain proteins is provided, comprising:

[0017] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody, antibody pair, or reagent or kit is brought into contact with the sample to be tested to form an immune complex; and

[0018] b) Detect the presence of the immune complex, the presence of which indicates the presence of neurofilament light chain protein in the test sample.

[0019] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody, antibody pair, or reagent or kit in the preparation of products for detecting neurofilament light chain proteins is provided. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1Correlation comparison chart between the NfL kit of this invention and commercial NfL ELISA kits. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0024] In this invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance containing an antibody CDR that lacks some amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest antibody recognition unit. The antigen-binding fragments of the aforementioned antibodies can bind to the same antigen as the parent antibody.

[0025] Antigen-binding fragments of antibodies typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments. Antigen-binding fragments can also be obtained using recombinant genetic techniques known to those skilled in the art or synthesized using, for example, automated peptide synthesizers, such as those sold by Applied BioSystems.

[0026] In some embodiments, the present invention provides an antibody pair comprising at least two antibodies that bind to neurofilament light chain proteins.

[0027] In some embodiments, the first antibody comprises at least one antibody that binds to neurofilament light chain protein, and the second antibody comprises at least one antibody that binds to neurofilament light chain protein.

[0028] In some implementations, the first antibody has only one antibody, and the second antibody has only one antibody.

[0029] In this invention, the terms "comprising" and "including" are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0030] In this invention, the term "optionally" generally means that the event or condition described below may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0031] In a first aspect, embodiments of the present invention provide an antibody for detecting neurofilament light chain proteins. The antibody comprises three heavy chain complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, in the heavy chain variable region as shown in any of SEQ ID NO:17 or 37, and three light chain complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3, in the light chain variable region as shown in any of SEQ ID NO:19 or 39.

[0032] In an optional implementation, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0033] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0034] In this invention, the heavy chain complementarity-determining region (CDR) is denoted by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is denoted by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2, and LCDR3. In this invention, the term "antibody pair" refers to the use of multiple antibodies paired together, which can be two, three, four, or more antibodies, but at least two antibodies.

[0035] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, with slight variations in labeling across different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0036] Table 1: CDR Definition 1

[0037] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0038] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0039] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0040] 3If neither H35A nor H35B exists, then HCDR1 ends at bit 35; if only H35A exists, then HCDR1 ends at bit 35A; if both H35A and H35B exist, then HCDR1 ends at bit 35B.

[0041] 4 If neither H35A nor H35B exists, then HCDR1 ends at bit 32; if only H35A exists, then HCDR1 ends at bit 33; if both H35A and H35B exist, then HCDR1 ends at bit 34.

[0042] 5 If neither H35A nor H35B exists, then HCDR1 ends at bit 33; if only H35A exists, then HCDR1 ends at bit 34; if both H35A and H35B exist, then HCDR1 ends at bit 35.

[0043] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0044] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0045] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0046] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0047] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0048] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0049] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0050] Secondly, embodiments of the present invention provide an antibody for detecting neurofilament light chain proteins, wherein the complementarity-determining region of the aforementioned antibody includes any one of (a') to (b'):

[0051] (a') The amino acid sequences are as shown in SEQ ID NO:1 (SSYRWN) for HCDR1 and SEQ ID NO:2 respectively.

[0052] HCDR2 as shown in (YIQSTGTTNYNPSLKR), HCDR3 as shown in SEQ ID NO:3 (SAGTAPIWFTH), and LCDR1 as shown in SEQ ID NO:4 (KASQNVGSNVD), LCDR2 as shown in SEQ ID NO:5 (KASNRYT), and LCDR3 as shown in SEQ ID NO:6 (LQSNSYPPT); and

[0053] (b') The amino acid sequences are as follows: HCDR1 (SEQ ID NO:21(NYWMN), HCDR2 (SEQ ID NO:22(EIRLKSNNYATHYAESVKG), HCDR3 (SEQ ID NO:23(LDFDY)), and LCDR1 (SEQ ID NO:24(QASQGTYINLK), LCDR2 (SEQ ID NO:25(GATNLED), LCDR3 (SEQ ID NO:26(LQHSFLPYT)).

[0054] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0055] The antibody of the present invention also includes a frame region. In the present invention, the "frame region" or "FR" region includes a heavy chain frame region and a light chain frame region, which refers to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR. The heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3 and LFR4 frame regions.

[0056] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In optional embodiments, the first antibody, the second antibody, or the antibody further comprises the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0058] In an optional embodiment, the framework region of the first antibody is selected from any one of (a) to (b):

[0059] (a) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and

[0060] (b) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:27 to SEQ ID NO:30, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:31 to SEQ ID NO:34; or the amino acid sequences having at least 80% identity with the sequences of the respective frame regions.

[0061] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0062] In other embodiments, the amino acid sequences of the first antibody, second antibody, or each frame region of the antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions described above.

[0063] Thirdly, embodiments of the present invention provide an antibody for detecting neurofilament light chain proteins. The aforementioned antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in either SEQ ID NO:17 or 37, and the amino acid sequence of the light chain variable region is shown in either SEQ ID NO:19 or 39.

[0064] In an optional embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of (A) to (B):

[0065] (A) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:19; and

[0066] (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:39.

[0067] In an optional implementation, the antibody described in the first, second, and third aspects above further includes a constant region.

[0068] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0069] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0070] In optional embodiments, the heavy chain constant region of the antibody is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and / or; the light chain constant region is selected from the κ-type or λ-type light chain constant region; the λ-type light chain constant region can be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0071] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0072] In an optional embodiment, the constant region of the antibody is selected from any group of (C) to (D):

[0073] (C) The heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:15; and the light chain constant region with an amino acid sequence as shown in SEQ ID NO:16; or an amino acid sequence having at least 80% identity with each of the constant regions; and

[0074] (D) The heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:35; and the light chain constant region with an amino acid sequence as shown in SEQ ID NO:36; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0075] It should be noted that, in some embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region.

[0076] In this paper, the division of the variable region and constant region sequences is based on the IMGT partitioning method, see Lefranc, M.-P. and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGHC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGLC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.orgCreated: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0077] Fourthly, embodiments of the present invention provide an antibody for detecting neurofilament light chain proteins, the antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as either SEQ ID NO: SEQ ID NO: 18 or 38, and the amino acid sequence of the light chain is shown as either SEQ ID NO: 20 or 40.

[0078] In an optional embodiment, the heavy chain and light chain of the antibody are selected from any combination of the following:

[0079] combination Heavy chain Light chain 1 SEQ ID NO:18 SEQ ID NO:38 2 SEQ ID NO:20 SEQ ID NO:40 .

[0080] Fifthly, embodiments of the present invention provide an antibody pair for detecting neurofilament light chain proteins, wherein the antibody pair includes a coating antibody and / or a labeled antibody, and the coating antibody and the labeled antibody are respectively selected from the antibodies described in the first aspect, the second aspect, the third aspect, and the fourth aspect.

[0081] In an optional administration method, the above-mentioned labeled antibody is labeled with a marker and / or a binding partner.

[0082] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0083] In optional embodiments, the markers are selected from, but not limited to, metal ions, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, electron-dense markers, adamantane, and nanoparticle markers.

[0084] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0085] In optional embodiments, the fluorescent dyes are selected from, but not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0086] In optional embodiments, the enzymes are selected from, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0087] In optional embodiments, the radioactive isotopes mentioned above are selected from, but not limited to, those selected from, and others. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0088] In optional embodiments, the chemiluminescent reagent is selected from, but not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0089] In an optional embodiment, the marker is acridine ester.

[0090] In optional embodiments, the above-mentioned nanoparticle markers are selected from, but not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0091] In optional embodiments, the colloid is selected from, but not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0092] In an optional embodiment, the colloidal metal is selected from, but not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0093] In an optional implementation, the labeled antibody is labeled by binding the coupler to the labeler.

[0094] It should be noted that when an antibody is labeled with a marker to become a labeled antibody, it does not affect the characteristics of the antibody's immune response, nor does it change the activity of the marker itself.

[0095] In an optional embodiment, the coated antibody is linked to a solid phase and / or a binding partner.

[0096] In an optional embodiment, the solid phase is selected from microspheres, latex particles, microfluidic chips, magnetic beads, microporous plates, or nitrocellulose membranes.

[0097] In an optional embodiment, the solid phase is a magnetic bead.

[0098] In an optional embodiment, the binding coupler includes biotin / avidin or biotin derivative / avidin derivative protein.

[0099] In this invention, the term "binding pair" refers to a pair of molecules capable of binding together through non-covalent interactions. This binding is typically specific and can be temporary or form a more stable complex.

[0100] In this invention, the binding of the aforementioned binding couple to the marker or solid phase can take various forms, such as physical adsorption, electrostatic adsorption, or covalent bonding. Such binding / combining methods are known to those skilled in the art.

[0101] In a sixth aspect, embodiments of the present invention provide a reagent or kit for detecting neurofilament light chain proteins, wherein the aforementioned reagent or kit includes the antibody or antibody pair described above.

[0102] It should be noted that the meaning of "reagent" in this application can be considered equivalent to the meaning of "kit".

[0103] In some embodiments, the kit described above is used to detect neurofilament light chain proteins from subject samples, and the kit includes the antibody pairs or antibodies described above.

[0104] The antibody pairs or antibodies in some specific embodiments or examples of this invention can bind to neurofilament light chain proteins. Therefore, reagents or kits containing said antibody pairs or antibodies can effectively perform qualitative or quantitative detection of neurofilament light chain proteins. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of neurofilament light chain proteins and their antibodies, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies of this invention have improved neurofilament light chain protein binding activity, affinity, stability, or specificity; therefore, reagents or kits containing said antibodies have improved detection sensitivity, specificity, and / or reduced false negatives.

[0105] The kit of the present invention includes a neurofilament light chain protein detection reagent card (test strip).

[0106] In an alternative implementation, immunofluorescence technology can be used to label antibodies onto detectable markers such as fluorescent microspheres, and the neurofilament light chain protein in a subject, such as a patient, can be detected using the double antibody sandwich method.

[0107] In an optional embodiment, the present invention can utilize antibodies that bind to different amino acid fragments to recognize multiple sites on an antigen, thereby reducing the risk of missed detection and improving the detection rate.

[0108] In optional embodiments, the present invention provides a method for detecting neurofilament light chain proteins, a detection kit, and a preparation method. The kit of the present invention has improved sensitivity and specificity. In some embodiments, the antibody of the present invention can be a monoclonal antibody or a polyclonal antibody.

[0109] In optional embodiments, the antibody pairs or antibodies of the present invention can be prepared using methods known in the art.

[0110] In an optional embodiment, the antibody pair or antibody of the present invention can be prepared by immunizing animals with an antigen comprising the amino acid fragments described herein. To increase immunogenicity, a carrier protein (including but not limited to BSA, ovalbumin, KLH, etc.) can be conjugated to an immunoreactive substance (e.g., epitope peptide). The carrier protein may include a protein or polypeptide that can act as an immunogenic carrier. These types of polypeptides include albumins, serum proteins, globulins, lens proteins, lipoproteins, and / or fragments thereof.

[0111] In an optional implementation, an immunoreactive substance (such as, but not limited to, neurofilament light chain protein amino acid fragments) can be used to generate antibodies with affinity for neurofilament light chain protein.

[0112] In alternative embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc., can be used to detect the effects of the antibody pair or antibody, such as binding activity and / or cross-reactivity.

[0113] In optional embodiments, the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, etc.

[0114] In an optional embodiment, the kit of the present invention includes reagents suitable for performing immunoassays.

[0115] In optional embodiments, the kit of the present invention can be used for immunoassays, such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods.

[0116] In a seventh aspect, embodiments of the present invention provide a method for detecting neurofilament light chain proteins, comprising:

[0117] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0118] b) Detect the presence of the immune complex, the presence of which indicates the presence of neurofilament light chain protein in the test sample.

[0119] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody or reagent in detecting neurofilament light chain protein or in preparing products for detecting neurofilament light chain protein is provided.

[0120] Unless otherwise defined, 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 disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0121] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0122] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0123] Based on long-term, innovative research on neurofilament light chain proteins, the inventors discovered an antibody that meets the detection requirements for neurofilament light chain proteins. The preparation, activity identification, and performance testing of the antibody are illustrated in the examples.

[0124] Example 1: Antibody Discovery of Monoclonal Antibodies

[0125] 1. Animal immunization

[0126] Recombinant neurofilament light chain protein (from Phypon Biotech) was mixed with an equal volume of Freund's complete adjuvant to obtain an oily emulsion. This emulsion was injected subcutaneously at multiple sites into BALB / c mice at a dose of 0.2 ml per mouse. Fourteen days later, mice were immunized intraperitoneally with the same antigen and adjuvant. Immunization continued until the fourth injection, and tail blood was collected for titer testing. When the titer met the fusion requirements, a booster immunization was administered intraperitoneally three days before fusion, using the same dose of antigen mixed with an equal volume of 0.9% sodium chloride injection.

[0127] 2. Preparation of hybridoma cell lines

[0128] On the third day after booster immunization of mice, spleens were removed under sterile conditions. Mouse tumor cells and immune spleen cells were mixed at a ratio of 1:5, fused, and cultured. The culture medium was changed twice on the sixth day. On the seventh day after fusion, the cell supernatant was collected for antibody detection to screen for hybridoma cell lines secreting specific antibodies. Two lines were identified and named 4G9 and 6D3.

[0129] Example 2 Antibody Preparation

[0130] 1. Antibody gene preparation

[0131] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen.

[0132] (1) Antibody gene preparation

[0133] mRNA was extracted from two hybridoma cell lines, 4G9 and 6D3, which secrete monoclonal antibodies against neurofilament light chain proteins. DNA products were obtained by RT-PCR. The products were then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of heavy chain and light chain genes were collected and sent to a gene sequencing company for sequencing.

[0134] (2) Sequence analysis of the variable region gene of neurofilament light chain protein antibody

[0135] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct.

[0136] (3) Construction of recombinant antibody expression plasmid

[0137] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The heavy chain gene fragment and the light chain gene fragment were amplified by PCR.

[0138] The heavy chain and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the heavy chain gene and light chain gene were ligated into the 3.4A expression vector, respectively, to obtain recombinant expression plasmid DNA of the heavy chain and light chain.

[0139] 2. Recombinant antibody production

[0140] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 3.2 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Two plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed, and the cells were incubated in a 35°C incubator at 120 rpm and 8% CO2 concentration.

[0141] 3. Indirect ELISA method for detecting expression supernatant of recombinant cell lines

[0142] 1) Coating: NFL antigen (from Feipeng Biotechnology) was coated at a concentration of 0.25ug / ml and incubated overnight at 4℃.

[0143] 2) Wash twice with PBST, blot dry, block with 120 μL / well of 20% bovine serum, incubate at 37°C for 1 h, and blot dry.

[0144] 3) Dilute the cell supernatant with 20% bovine serum to different concentrations. Add the supernatant of 4G9 and 6D3 to an ELISA plate coated with NFL antigen and incubate at 37°C for 30 min.

[0145] 4) Wash 5 times with PBST, pat dry, add 100 μL / well of goat anti-rat IgG-HRP (1% casein diluted 20,000 times), and incubate at 37°C for 30 min.

[0146] 5) Wash 5 times with PBST, pat dry, add 50 μL each of solution A and solution B, incubate in the dark for 10 min, add 50 μL of stop solution, and read the results using a microplate reader. The results are shown in Table 2. The results show that the antibodies in the supernatant of 4G9 and 6D3 can effectively bind to NFL antigen, and the binding activity is strong.

[0147] Table 2: Cell supernatant binding activity data by indirect ELISA

[0148] Dilution factor 5 10 50 100 200 400 800 1600 3200 6400 12800 4G9 antibody supernatant 2.085 2.113 1.949 2.033 1.874 1.217 0.839 0.502 0.299 0.18 0.113 6D3 antibody supernatant 2.276 2.2 2.23 2.188 2.143 1.564 1.022 0.551 0.3 0.163 0.098

[0149] 4. Antibody supernatant purification

[0150] The supernatant of the recombinant expressed antibody was purified by affinity chromatography using a protein A column. The resulting antibodies were named 4G9Rmb and 6D3Rmb. The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in Table 3.

[0151] Table 3: Antibody Sequences

[0152] Antibody name Heavy chain Light chain 4G9Rmb SEQ ID NO:18 SEQ ID NO:20 6D3Rmb SEQ ID NO:38 SEQ ID NO:40

[0153] Example 3 Performance Testing

[0154] This embodiment exemplifies and demonstrates some antibody performance testing data.

[0155] The 6D3Rmb antibody obtained above was used as the capture antibody, and the 4G9Rmb antibody was used as the labeling antibody. The experimental procedure is as follows:

[0156] (1) Biotinylated antibody process:

[0157] Antibody 6D3Rmb and control antibody 1B11 (from EnCor Biotechnology) were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10 KMWCO). Biotin was prepared into a 4 mM solution using DMSO. The antibody and biotin solutions were mixed at a 1:10 molar ratio and reacted at 25°C for 2 hours. After desalting to remove excess reagents, glycerol was added to a final concentration of 50% and stored at -20°C for later use.

[0158] (2) Acridinium ester labeled antibody process:

[0159] Antibody 4G9Rmb and control antibody 1B11 (from EnCor Biotechnology) were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10 KMWCO). Acridinium ester was prepared into a 4 mM solution using DMSO. The antibody solution and acridinium ester solution were mixed at a 1:10 molar ratio and reacted at 25°C for 2 hours. After desalting to remove excess reagents, glycerol was added to a final concentration of 50% and stored at -20°C for later use.

[0160] (3) The process of coating magnetic beads with streptavidin:

[0161] Wash 10 mg of carboxyl magnetic beads three times with MES (100 μM MES, pH 5.5) buffer. Resuspend the magnetic beads in MES buffer. Add EDC to a final concentration of 1 mg / mL, mix thoroughly on a shaker at 25°C, and react for 30 min. Magnetic separation is performed, the supernatant is discarded, and the magnetic beads are resuspended in MES buffer. Streptavidin is added, and the mixture is mixed on a shaker at 25°C, and reacted for 120 min. Magnetic separation is performed again, the supernatant is discarded, and the mixture is resuspended in Tris buffer to a final concentration of 10 mg / mL solids. Store at 2–8°C for later use.

[0162] (4) On-machine testing process:

[0163] The streptavidin magnetic beads in step (3) were diluted to a working solution of 0.5 mg / mL using diluent (150 mM PB, 150 mM sodium chloride, 1% BSA, pH 7.2). The antibody 6D3Rmb and the control antibody 1B11 in step (1) were diluted to a working solution of 1 μg / mL biotin. The antibody 4G9Rmb and the control antibody 1B11 in step (2) were diluted to a working solution of 1 μg / mL acridinium ester.

[0164] The assay was performed on the Shine i2910 fully automated chemiluminescence immunoassay analyzer using a double-antibody sandwich method. Specifically, 50 μL of sample, 50 μL of magnetic microparticle working solution, 50 μL of biotin working solution, and 50 μL of acridinium ester working solution were added sequentially to the instrument. After mixing and incubation for 15 minutes, the reaction mixture was rinsed, and pre-excitation and excitation solutions were added. The relative light unit (RLU) intensity was measured, and the corresponding concentration value was calculated using a four-parameter fitting method.

[0165] Remark:

[0166] 1. Antibody pairing detection is performed in 3 groups:

[0167] Group 1: Acridinium ester working solution: antibody 4G 9RMB, Biotin working solution: antibody 6D 3RMB

[0168] Group 2: Acridinium ester working solution: antibody 4G9Rmb; Biotin working solution: control antibody 1B11

[0169] Group 3: Acridinium ester working solution: control antibody 1B11, biotin working solution: antibody 6D3Rmb

[0170] 2. The sample includes:

[0171] 1) Samples without NfL

[0172] 2) Samples containing different concentrations of NfL

[0173] 3) Glial fibrillary acidic protein (GFAP), vimentin, desmin, alpha-internexin, peripherin, neurofibrillary medium chain (NfM), and neurofibrillary heavy chain (NfH) recombinant proteins (all from Feipeng Biotechnology).

[0174] The reagent testing performance evaluation methods and results are as follows:

[0175] (1) Blank limit: The sample without NfL was tested 20 times repeatedly, and its mean (M) and standard deviation (SD) were calculated. The RLU value corresponding to M+2SD was obtained, and the corresponding concentration value was obtained by substituting it into the calibration curve. This value is the blank limit. The detection results are shown in Table 4. The results show that the blank limit of Group 1 of the present invention is 0.33 pg / mL, indicating that the detection using antibodies 4G9Rmb and 6D3Rmb has high sensitivity.

[0176] Table 4: Sensitivity Detection Results

[0177]

[0178] (2) Clinical Relevance: Our laboratory collected 45 clinical serum samples. Using the NfL commercial ELISA kit (Uman Diagnostics) as a control reagent, we compared clinical samples using groups 1, 2, and 3 of this invention with the control reagent (the detection range of the control kit is 0.5 pg / mL-40 pg / mL, and all samples were tested after 10-fold dilution). The test results are shown in Table 5, and the correlation comparison graph between groups 1, 2, and 3 of this invention and the control reagent is shown below. Figure 1 As shown in the figure. The results showed that the reagent composed of antibodies 4G9Rmb and 6D3Rmb had a linear correlation of 0.98 with the control reagent, indicating a good linear correlation.

[0179] Table 5: Clinical Test Results

[0180]

[0181]

[0182] (3) Cross-reactivity: The cross-reactivity detected using antibodies 4G9Rmb and 6D3Rmb is shown in Table 6. The results show that the detection group 1 composed of antibodies 4G9Rmb and 6D3Rmb has extremely weak binding to glial fibrillary acidic protein (GFAP), vimentin, desmin, alpha-internexin, peripherin, neurofibrillary medium chain (NfM), and neurofibrillary heavy chain (NfH), with low cross-reactivity and good detection specificity.

[0183] Table 6: Cross-reactivity results

[0184]

[0185] The partial amino acid sequences involved in this application are shown in Table 7:

[0186] Table 7: Amino Acid Sequence List

[0187]

[0188]

[0189]

[0190] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody for detecting neurofilament light chain proteins, characterized in that, The antibody contains three heavy chain complementarity-determining regions, HCDR1, HCDR2 and HCDR3, in the heavy chain variable region as shown in any of SEQ ID NO:17 and 37, and three light chain complementarity-determining regions, LCDR1, LCDR2 and LCDR3, in the light chain variable region as shown in any of SEQ ID NO:19 and 39. Optionally, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody for detecting neurofilament light chain proteins, characterized in that, The complementarity-determining regions of the antibody include any one of (a') to (b'): (a') The amino acid sequences are as follows: HCDR1 (SEQ ID NO:1 (SSYRWN)), HCDR2 (SEQ ID NO:2 (YIQSTGTTNYNPSLKR)), and HCDR3 (SEQ ID NO:3 (SAGTAPIWFTH)); and the amino acid sequences are as follows: LCDR1 (SEQ ID NO:4 (KASQNVGSNVD)), LCDR2 (SEQ ID NO:5 (KASNRYT)), and LCDR3 (SEQ ID NO:6 (LQSNSYPPT)); and (b') The amino acid sequences are as shown in SEQ ID NO:21(NYWMN) for HCDR1 and SEQ ID NO:22, respectively. HCDR2 as shown in (EIRLKSNNYATHYAESVKG), HCDR3 as shown in SEQ ID NO:23 (LDFDY), and LCDR1 as shown in SEQ ID NO:24 (QASQGTYINLK), LCDR2 as shown in SEQ ID NO:25 (GATNLED), and LCDR3 as shown in SEQ ID NO:26 (LQHSFLPYT).

3. The antibody according to claim 1 or 2, characterized in that, The antibody also includes the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from any group of (a) to (b): (a) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and (b) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:27 to SEQ ID NO:30, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:31 to SEQ ID NO:34; or the amino acid sequences having at least 80% identity with the sequences of the respective frame regions.

4. An antibody for detecting neurofilament light chain proteins, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in either SEQ ID NO:17 or 37, and the amino acid sequence of the light chain variable region is shown in either SEQ ID NO:19 or 39. Optionally, the amino acid sequences of the heavy chain variable region and the light chain variable region shown are selected from any one of (A) to (B): (A) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:17, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:19; (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:

39.

5. The antibody according to any one of claims 1 to 4, characterized in that, The antibody also includes a constant region; Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, or a combination of multiple constant regions; and / or; the light chain constant region is selected from κ-type or λ-type light chain constant regions; Optionally, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the constant region of the antibody is selected from any group of (C) to (D): (C) The heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:15; and the light chain constant region with an amino acid sequence as shown in SEQ ID NO:16; or an amino acid sequence having at least 80% identity with each of the constant regions; (D) The heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:35; and the light chain constant region with an amino acid sequence as shown in SEQ ID NO:36; or an amino acid sequence having at least 80% identity with each of the constant regions.

6. An antibody for detecting neurofilament light chain proteins, characterized in that, The antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in either SEQ ID NO:18 or 38, and the amino acid sequence of the light chain is shown in either SEQ ID NO:20 or 40.

7. An antibody pair for detecting neurofilament light chain proteins, characterized in that, The antibody pair comprises a coating antibody and / or a labeled antibody; the coating antibody and the labeled antibody are respectively selected from the antibodies of any one of claims 1 to 6; Optionally, the labeled antibody is labeled with a marker and / or a binding partner. Optionally, the marker is selected from metal ions, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent markers, electron-dense markers, adamantane, and nanoparticle markers; Optionally, the marker is an acridine ester; Optionally, the coated antibody is linked to a solid phase and / or a binding partner; Optionally, the solid phase is selected from microspheres, magnetic beads, latex particles, microfluidic chips, plates, or membranes; Optionally, the solid phase is selected from magnetic beads; Optionally, the binding coupler includes biotin / avidin or biotin derivative / avidin derivative protein.

8. A detection reagent or kit for detecting neurofilament light chain proteins, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 6 or the antibody pair as described in claim 7.

9. A method for detecting neurofilament light chain proteins, characterized in that, include: a) Under conditions sufficient to allow an antibody / antigen binding reaction to occur, the antibody of any one of claims 1 to 6, the antibody pair of claim 7, or the reagent or kit of claim 8 is brought into contact with the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of neurofilament light chain protein in the test sample.

10. Use of the antibody of any one of claims 1 to 6, the antibody pair of claim 7, or the reagent or kit of claim 8 in the preparation of a product for detecting neurofilament light chain protein.

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