Recombinant antibody targeting neurofilament light chain and kit for detecting neurofilament light chain

Abstract

The present application discloses a recombinant antibody targeting neurofilament light chain, a nucleic acid molecule encoding the recombinant antibody, a kit for detecting neurofilament light chain, and the use of the recombinant antibody or the nucleic acid molecule in preparing a kit for diagnosing nervous system diseases. The recombinant antibody of the present application has high affinity, high sensitivity, and significantly shortened production cycle, and is more suitable as a core raw material for application in the field of in vitro diagnostic reagents.

Description

Technical Field

[0001] The present application belongs to the technical field of immunoassay, and relates to a recombinant antibody targeting neurofilament light chain, a nucleic acid molecule encoding the recombinant antibody, a kit for detecting neurofilament light chain, and uses thereof. Background Art

[0002] Neurofilaments (NFs) are cylindrical proteins located exclusively in the cytoplasm of neurons. They exist in the form of 10nm filaments in dendrites, neuronal cell bodies and axons, and give neurons structural stability. NFs are mainly composed of three polypeptides, which are 200kDa, 150kDa and 68kDa in size, namely neurofilament heavy chain (NF-H), neurofilament medium chain (NF-M) and neurofilament light chain (NF-L).

[0003] NF-L is the skeleton of NFs. It is the most abundant subunit and the most soluble subunit. Under normal conditions, NF-L is continuously released from axons in an age-dependent manner, and higher levels of NF-L are produced in the elderly. When the body is in response to central nervous system (CNS) axonal damage caused by inflammation, neurodegenerative diseases, trauma or vascular injury, the release of NF-L increases sharply. The concentration of NF-L in CSF of patients with neurological diseases is higher than that of the control group, and existing results have confirmed that serum NF-L levels are closely related to NF-L levels in CSF. The potential diagnostic value of NF-L lies in the degree of damage to central nervous system diseases, disease progression, and the distinction between neurodegenerative diseases and non-neurodegenerative lesions. Compared with imaging methods such as PET-CT and MRI, immunological monitoring of NF-L helps doctors to strengthen the dynamic tracking of CNS damage, improve the diagnosis and treatment effect, optimize prognosis management, and bring significant social and economic benefits.

[0004] However, the existing NF-L antibody preparation technology has the following two main shortcomings:

[0005] (1) Traditional monoclonal antibody preparation technology is inherently deficient

[0006] Traditional monoclonal antibodies are antibodies produced by a single B cell clone and include specific antigenic epitopes. In the actual production process, the antibody titer is easily affected by the individual differences of animals. At the same time, the volume of ascites of a single mouse is generally less than 5mL. Therefore, it is difficult for traditional monoclonal antibodies to ensure the batch-to-batch difference of antibody titers. In addition, it takes about 10-11 days from hybridoma cell recovery to obtaining mouse ascites and purifying the target monoclonal antibody. Therefore, the use of traditional monoclonal antibody preparation has the disadvantages of relatively large batch differences and long production cycles.

[0007] (2) Existing antibodies have insufficient affinity

[0008] Existing published or authorized patent data show that the sensitivity of NF-L antibodies is 1-1000nM. For example, the affinity of NEFL mouse monoclonal antibody by Tang Yude et al. is 1.4nM (ELISA assay). Insufficient antibody sensitivity will directly affect the sensitivity of the detection reagent, while reducing the specificity and anti-interference of the detection, and ultimately affect the reliability of the test results. Summary of the invention

[0009] In order to overcome the deficiencies in the prior art, the present application provides a recombinant antibody targeting neurofilament light chain and a kit for detecting neurofilament light chain comprising the above recombinant antibody. The recombinant antibody of the present application has higher sensitivity and significantly shortened production cycle.

[0010] The specific technical solutions of this application are as follows:

[0011] 1. A recombinant antibody targeting neurofilament light chain, wherein the recombinant antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0012] The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1 or SEQ ID No: 2;

[0013] The amino acid sequence of CDR-H2 is shown in SEQ ID No: 3 or SEQ ID No: 4;

[0014] The amino acid sequence of CDR-H3 is shown in SEQ ID No: 5 or SEQ ID No: 6;

[0015] The amino acid sequence of CDR-L1 is shown in SEQ ID No: 7 or SEQ ID No: 8;

[0016] The amino acid sequence of CDR-L2 is shown in SEQ ID No: 9 or SEQ ID No: 10;

[0017] The amino acid sequence of CDR-L3 is shown in SEQ ID No:11 or SEQ ID No:12.

[0018] 2. The recombinant antibody according to item 1, wherein

[0019] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1;

[0020] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3;

[0021] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5;

[0022] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7,

[0023] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9;

[0024] The amino acid sequence of the CDR-L3 is shown in SEQ ID No:11.

[0025] 3. The recombinant antibody according to item 1, wherein

[0026] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2;

[0027] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4;

[0028] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6;

[0029] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8;

[0030] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10;

[0031] The amino acid sequence of the CDR-L3 is shown in SEQ ID No:12.

[0032] 4. The recombinant antibody according to any one of items 1 to 3, wherein the recombinant antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0033] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 or SEQ ID No: 14, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 14;

[0034] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:15 or SEQ ID No:16, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:15 or SEQ ID No:16.

[0035] 5. The recombinant antibody according to item 4, wherein

[0036] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 13;

[0037] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:15, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:15.

[0038] 6. The recombinant antibody according to item 4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 14;

[0039] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:16, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:16.

[0040] 7. A nucleic acid molecule encoding the recombinant antibody according to any one of items 1 to 6.

[0041] 8. A kit for detecting neurofilament light chain, wherein the kit comprises:

[0042] A biotin-coupled primary antibody targeting neurofilament light chain, a marker-labeled secondary antibody targeting neurofilament light chain, and magnetic beads; or

[0043] Magnetic beads coated with a first antibody targeting neurofilament light chain and a second antibody targeting neurofilament light chain labeled with a marker;

[0044] The first antibody and the second antibody targeting neurofilament light chain are the recombinant antibodies described in any one of Items 1 to 6.

[0045] 9. The kit according to item 8, wherein it is used for diagnosing a neurological disease;

[0046] Preferably, the nervous system disease is a central nervous system disease;

[0047] Preferably, the nervous system disease is a neurodegenerative disease.

[0048] 10. Use of the recombinant antibody according to any one of items 1 to 6, or the nucleic acid molecule according to item 7, in the preparation of a kit for detecting neurofilament light chain and / or diagnosing a nervous system disease;

[0049] Preferably, the nervous system disease is a central nervous system disease;

[0050] Preferably, the nervous system disease is a neurodegenerative disease.

[0051] Effects of the Invention

[0052] The present application provides a new recombinant antibody targeting neurofilament light chain, a nucleic acid molecule encoding the recombinant antibody, and a kit containing the recombinant antibody. The recombinant antibody of the present application has excellent and stable performance, high affinity, and high sensitivity; at the same time, the present application does not require animal immunization again, and it only takes about 5-6 days from cell recovery to obtaining the target recombinant antibody, and the production cycle is significantly shortened; in addition, the recombinant antibody technology used in the present application can obtain g-level recombinant antibodies in a single batch. Therefore, the NF-L recombinant antibody of the present application is more suitable as a core raw material for application in the field of in vitro diagnostic reagents, which can bring significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the consistency analysis result of the NF-L content in samples detected by the NF-L kit of the present application and by the imported high-sensitivity detection platform product. DETAILED DESCRIPTION

[0054] The present application is further described in detail below in conjunction with specific implementation methods. The implementation methods given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to technicians in the field.

[0055] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0056] In this article, "neurofilament" is an intermediate filament protein that constitutes the skeleton of the axon of a nerve cell. It is located in the axon of a nerve cell and participates in the fiber system of the axon as one of the skeleton components. Its function is to stabilize the morphology of the axon, maintain its diameter and maintain its stable conduction velocity, and play a role in the differentiation of nerve cells, axon growth and regeneration. Neurofilament protein includes three substructures: neurofilament light chain (NF-L), neurofilament medium chain (NF-M), and neurofilament heavy chain (NF-H). Among them, although NF-L has the smallest molecular weight, its function is the most important, because NF-L is the only neurofilament protein among the three substructure proteins that can complete the self-assembly of functional fibers. NF-L is a cylindrical protein specifically located in the cytoplasm of neurons. Under normal circumstances, low levels of NF-L are continuously released from axons, and higher levels of NF-L are released in old age. However, when the axons of the central nervous system are damaged by inflammation, neurodegeneration, trauma or vascular injury, the release of NF-L increases sharply.

[0057] In this article, the term "recombinant antigen" refers to an antigen that is purified by in vitro recombinant expression after the antigen gene is connected to a vector and transferred into prokaryotic or eukaryotic cells. In one embodiment, the recombinant antigen used in this application is a commercial NF-L recombinant antigen.

[0058] In this article, the term "recombinant antibody" refers to an antibody obtained by cloning the immunospecific heavy chain and light chain antibody genes into a high-efficiency expression vector, and then introducing these vectors into an expression host (such as bacteria, yeast or mammals) for antibody expression. The antibody of the present application can be a fully humanized antibody, a humanized antibody, a chimeric antibody, etc.

[0059] In some embodiments, the NF-L recombinant antibody of the present application is an antibody obtained by immunizing an animal with a NF-L recombinant antigen.

[0060] In some embodiments, the recombinant antibody of the present application is a full-length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is generally a polypeptide that consists of a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), an antibody heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.

[0061] In the present application, the light chain constant region and the heavy chain constant region can be any light chain constant region and heavy chain constant region. The light chain constant region (CL) can be κ (kappa) or λ (lambda). The heavy chain constant region can be any one of the heavy chain constant regions of IgG, IgM, IgA, IgE, and IgD.

[0062] It is well known to those skilled in the art that each heavy chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs). The complementary determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, the heavy chain variable region and the light chain variable region both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. There are two common definitions of the CDR sequence of the heavy chain variable region or the light chain variable region, namely the Kabat definition and the Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR sequence in the heavy chain variable region or the light chain variable region sequence can be determined according to the Kabat definition or the Chothia definition. In the embodiment of the present application, the Chothia definition is used to determine the CDR sequence.

[0063] As used herein, the term "identity" may be defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. As used herein, an amino acid sequence that is referenced to have a percentage identity with an amino acid sequence refers to a sequence that has that percentage identity over the entire length of the referenced amino acid sequence.

[0064] In this article, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0065] In this article, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers in single-stranded or double-stranded form thereof. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to reference nucleic acids (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants modified in a conservative manner thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0066] As used herein, the term "EC50 value" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that can induce 50% of the maximal effect.

[0067] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be determined by common methods known in the art.

[0068] In this article, the term "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method.

[0069] Herein, the term "recombinant antibody targeting neurofilament light chain" refers to a recombinant antibody that can bind to neurofilament light chain with sufficient affinity and can be used as a diagnostic agent and / or therapeutic agent targeting neurofilament light chain.

[0070] The recombinant antibody targeting neurofilament light chain of the present application does not bind to proteins unrelated to the target. Here, "unrelated proteins" refer to proteins other than the neurofilament light chain as the target; here, "unbinding" means: when the binding ability of the recombinant antibody targeting neurofilament light chain of the present application to the neurofilament light chain as its target is taken as 100%, the binding ability of the recombinant antibody targeting neurofilament light chain of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0071] Neurofilament light chains suitable for producing antibodies can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Other forms of β-amyloid protein may also include neurofilament light chain expressing cells, preparations or cell extracts or fractions containing neurofilament light chains, and partially purified neurofilament light chains.

[0072] In this article, the term "biotin" is one of the B vitamins, also known as vitamin H, vitamin B7, coenzyme R, etc. It was discovered in the 1930s when studying yeast growth factor and rhizobium growth and respiration promoting factor from the liver. It is a factor that can prevent and treat hair loss and skin damage in rats induced by feeding raw egg protein. Biotin is a member of the water-soluble B vitamins. It is present in large amounts in the liver, kidney, yeast, and milk, and is an important factor in the fixation of carbon dioxide in organisms. It is easy to bind to a protein in egg white. Eating a large amount of raw protein can hinder the absorption of biotin and lead to biotin deficiency, such as hair loss, weight loss, dermatitis, etc. Biotin plays an important role in biochemical reaction pathways such as fat synthesis and gluconeogenesis.

[0073] In some embodiments, the present application uses a magnetic particle chemiluminescent immunoassay to determine the level of neurofilament light chain in a sample. The specific principle is: directly label the antibody with a chemiluminescent agent (chemiluminescent marker), react with the corresponding antigen in the sample and the antibody of the magnetic particle (magnetic bead), separate the bound state (precipitation part) and the free state of the antibody labeled with the chemiluminescent marker by the magnetic field, and then add a luminescent promoter (luminescent substrate) to carry out a luminescent reaction, and perform quantitative or qualitative detection by detecting the luminescent value.

[0074] In some embodiments, the present application can also use POCT, ELISA, SERS, mass spectrometry, microfluidics, photochemistry and other platforms to determine the level of neurofilament light chain in the sample.

[0075] In this article, ELISA (enzyme linked immune sorbent assay) refers to a detection method that uses the characteristics of antibody molecules that can specifically bind to antigen molecules to bind free impurities and target proteins bound to solid phase carriers, and uses special markers to conduct qualitative or quantitative analysis. The principle is that antigens or antibodies can be physically adsorbed on the surface of solid phase carriers and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of immune reactions can be determined by the color reaction of adding substrates, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen.

[0076] In the first aspect, the present application provides a recombinant antibody targeting neurofilament light chain, which comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID No: 1 or SEQ ID No: 2; the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 3 or SEQ ID No: 4; the amino acid sequence of CDR-H3 is as shown in SEQ ID No: 5 or SEQ ID No: 6; the amino acid sequence of CDR-L1 is as shown in SEQ ID No: 7 or SEQ ID No: 8; the amino acid sequence of CDR-L2 is as shown in SEQ ID No: 9 or SEQ ID No: 10; the amino acid sequence of CDR-L3 is as shown in SEQ ID No: 11 or SEQ ID No: 12.

[0077] In some embodiments, the amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1 (GFSLTGM); the amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3 (YWDED); the amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5 (RPFHRYYVEALDY); the amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7 (RSSQSIVHSNGATSLE), the amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9 (KVSWRFS); the amino acid sequence of the CDR-L3 is shown in SEQ ID No: 11 (FNGSRVPLT).

[0078] In some embodiments, the amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2 (GFIFSIR); the amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4 (APSGN); the amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6 (APDGNYGPFFY); the amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8 (RASETITNSLH); the amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10 (YATNSIS); the amino acid sequence of the CDR-L3 is shown in SEQ ID No: 12 (QQSDSWPLT).

[0079] In some embodiments, the recombinant antibody comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 or SEQ ID No: 14, or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15 or SEQ ID No: 16, or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 14. No:16 has an amino acid sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0080] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 (QVSLRESGPGILQPSNTLSLTCSFSGFSLTGMGVSWIRQPSGKGIEWI AHIYWDEDKTYNPSLKSHLSITRDTSRNQVFLHVTNVDTPDSVTYYQAR RPFHRYYVEALDYWGQGLAVTVDS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15 (EIMTQTPLSLPVSLGEQASISCRSSQSIVHSNGATSLEWYLQKPRQH PKLLIYKVSWRFSGVPDRFSGSGSGTDFTLHISRVEADELGVYYCFNGSRV PLTFGAGTKLELKKA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No:15.

[0081] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14 (DVKLVESGCNLVHPGGSPRLSCTATGFIFSIRAMSWVYCTPDHRLE WIASIAPSGNTHYPDYVKGRPTISRADATNVLYLQMSTLKSEDTAMYYCT GAPDGNYGPFFYWGQGTLVTVTA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16 (EVVLTQSPVSLTVTPGDSVSLSCRASETITNSLHWYQHKSHESPRLL IKYATNSISGIPSRFSASGTGTDFTLTIDTVETEDFGVYPCQQSDSWPLTFSA GTKLNLKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No:16.

[0082] In a second aspect, the present application provides a nucleic acid molecule encoding any of the aforementioned recombinant antibodies.

[0083] In a third aspect, the present application provides a kit for detecting neurofilament light chain, the kit comprising: a biotin-coupled first antibody targeting neurofilament light chain, a second antibody targeting neurofilament light chain labeled with a marker, and magnetic beads; wherein the first antibody and the second antibody targeting neurofilament light chain are respectively the recombinant antibodies described in any one of the aforementioned first aspects.

[0084] In the above embodiment, the kit also includes a sample standard to be tested, a substrate, a washing solution, a magnetic bead diluent, a coupled antibody diluent, a labeled antibody diluent and a sample diluent, etc. In some embodiments, the substrate includes a luminescent excitation liquid A and a luminescent excitation liquid B, wherein the luminescent excitation liquid A is an aqueous solution of nitric acid and hydrogen peroxide, and the luminescent excitation liquid B is an aqueous solution of sodium hydroxide. In some embodiments, the magnetic bead diluent is a phosphate buffer containing BSA and Proclin300. In some embodiments, the coupled antibody diluent and the labeled antibody diluent are TBS-T buffer. In some embodiments, the washing solution is PBS and Tween-20. In some embodiments, the sample diluent is a PBS buffer. In some embodiments, the sample diluent is a PBS buffer containing BSA and Proclin-300.

[0085] In a fourth aspect, the present application provides a kit for detecting neurofilament light chain, the kit comprising: magnetic beads coated with a first antibody targeting neurofilament light chain and a second antibody targeting neurofilament light chain labeled with a marker; wherein the first antibody and the second antibody targeting neurofilament light chain are respectively the recombinant antibodies described in any one of the aforementioned first aspects.

[0086] In the above-mentioned embodiment, the kit also includes a sample standard to be tested, a substrate, a washing solution, a blocking solution, a coated antibody diluent, a labeled antibody diluent and a sample diluent, etc. In some embodiments, the substrate, the washing solution, and the sample diluent are as described in the aforementioned third aspect. In some embodiments, the coated antibody diluent and the labeled antibody diluent are TBS-T buffer. The blocking solution is used to block the uncoupled vacancy to prevent non-specific binding in the later stage. In some embodiments, the blocking solution is a BSA blocking solution.

[0087] In some embodiments, the kits described in the third and fourth aspects further include a storage buffer and a quenching buffer, wherein the storage buffer is used to provide a stable pH environment, reduce antibody degradation, and preserve, etc.; the quenching buffer is used to quench the markers that are not bound to the antibody. In some embodiments, the storage buffer is TBS-T buffer, and the quenching buffer is 5% DL-lysine.

[0088] In some embodiments, the magnetic beads described in the third and fourth aspects may be SA magnetic beads, tosyl-activated magnetic beads, and the like.

[0089] In some embodiments, the magnetic beads described in the third aspect are SA magnetic beads.

[0090] In some embodiments, the magnetic beads described in the fourth aspect are tosyl-activated magnetic beads.

[0091] In some embodiments, the label described in the third and fourth aspects may be a chemiluminescent label, an electrochemiluminescent label, etc. Preferably, the chemiluminescent label is an acridinium ester.

[0092] The kit of the present application can be used to diagnose nervous system diseases, such as brain atrophy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, anterior temporal lobe dementia, Huntington's disease, brain injury-related diseases, etc. The brain injury-related diseases can be, for example, primary craniocerebral injury or secondary craniocerebral injury.

[0093] The kit of the present application can be used to distinguish neurodegenerative diseases from non-neurodegenerative lesions.

[0094] In a fifth aspect, the present application also provides a use of any of the aforementioned recombinant antibodies or any of the aforementioned nucleic acid molecules in the preparation of a kit for diagnosing nervous system diseases.

[0095] In some embodiments, the kit may be a magnetic particle chemiluminescence kit, a magnetic particle electrochemiluminescence kit, an electrochemiluminescence kit, an ELISA kit, a POCT detection kit, a SERS detection kit, a mass spectrometry kit, a microfluidics kit and a photochemical kit, etc., preferably a magnetic particle chemiluminescence kit.

[0096] Example

[0097] The present application content will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the embodiments.

[0098] Example 1 Preparation of recombinant antibodies rNF-LA and rNF-LB

[0099] The preparation method of the recombinant antibody in this embodiment is: immunize mice with commercial NF-L recombinant antigen. After obtaining the mouse monoclonal antibody cell line combination with the highest sensitivity, the antibody variable region sequence is sequenced. According to the variable region sequence, the chimeric vector is constructed and transfected into the CHO cell line for recombinant antibody expression. Among them, the light chain vector used is pFUSE2ss-CLIg-mk, and the heavy chain vector is pFUSEss-CHIg-mG1. The above two vectors are commercial vectors with constant region sequences and no additional processing is required; after the recombinant antibody is expressed in the CHO cell line, the purified recombinant antibodies rNF-LA and rNF-LB are obtained through Protein A filler. Among them, the amino acid sequence of the rNF-LA heavy chain variable region is shown in SEQ ID No: 13, the amino acid sequence of the rNF-LA light chain variable region is shown in SEQ ID No: 15, the amino acid sequence of the rNF-LB heavy chain variable region is shown in SEQ ID No: 14, and the amino acid sequence of the rNF-LB light chain variable region is shown in SEQ ID No: 16.

[0100] Example 2 Performance Verification of Recombinant Antibodies rNF-LA and rNF-LB

[0101] (1) Determination of recombinant antibody concentration

[0102] The protein concentration of multiple batches of different recombinant antibodies was tested by BCA (Bicinchoninic Acid Assay) technology. The concentration statistical results are shown in Table 1 below. Table 1 shows the concentration of the purified recombinant antibody. The BCA protein concentration determination kit was used. The specific operation was carried out according to the kit instructions.

[0103] Table 1

[0104] Concentration (mg / mL) 20230301 20230302 rNF-LA 8.9 7.3 rNF-LB 7.0 8.2

[0105] (2) Elisa test results

[0106] Elisa technology was used to test the affinity of multiple batches of recombinant antibodies. Each well of the ELISA plate was coated with 100 ng of NF-L recombinant antigen, and the recombinant antibody diluted in multiples was added and incubated at 37°C for 1 hour. After the incubation, a goat anti-human secondary antibody labeled with HRP was added, and finally TMB was added for color development. 2M sulfuric acid was added to terminate the reaction, and the wavelength of the ELISA reader was set to 450nm for detection. The test results are shown in Tables 2 to 6 below.

[0107] Batch 20230301 rNF-LA:

[0108] Table 2

[0109]

[0110]

[0111] Batch 20230302 rNF-LA:

[0112] Table 3

[0113]

[0114] Batch 20230301 rNF-LB:

[0115] Table 4

[0116]

[0117] Batch 20230302 rNF-LB:

[0118] Table 5

[0119]

[0120] The EC50 of each batch of recombinant antibodies was calculated using SigmaPlot 12.5 software as the K D The data are summarized in Table 6 below. The results in Table 6 show that the above recombinant antibodies have a higher affinity. D The K value of the recombinant antibody of the present application D The value can reach pM level, which is about one thousandth of the prior art. The affinity of the recombinant antibody of the present application is much higher than that of the antibody in the prior art.

[0121] Table 6

[0122]

[0123] Example 3 Preparation of a kit for detecting neurofilament light chain and detection process

[0124] (1) Preparation of magnetic bead working solution

[0125] Select SA magnetic beads (MS160), vortex mix, ultrasonicate for 5 min and fully resuspend. After magnetic separation to remove the liquid, add magnetic bead diluent (0.1M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA and 0.1% Proclin300) to make the final concentration 0.25 mg / mL, and store at 4°C as M working solution for later use.

[0126] (2) Biotin conjugation of antibodies

[0127] 0.5 mg of rNF-LA recombinant antibody was taken, mixed thoroughly with 3.33 μL 10 mM Sulfo-NHS-LC-LC-Biotin, and then allowed to stand at room temperature for 60 min. Biotin that was not coupled to the recombinant antibody was removed by ultrafiltration tube, and PBS (pH 7.4) was used for 3 rinses during ultrafiltration, and then an equal volume of glycerol was added and stored at -20°C for use. In this embodiment, the molar ratio of the recombinant antibody to biotin is 1:10. The mother liquor after coupling needs to be further diluted 1000 times with buffer TBS-T to be configured as R1 working solution for use.

[0128] (3) Antibody labeling

[0129] Pipette 0.5 mg of rNF-LB recombinant antibody and mix it thoroughly with 8.25 μL 4mM NSP-SA-NHS (dissolved in DMF) solution for 2 hours. Add 200 μl 5% DL-lysine to the solution and continue mixing for 30 minutes. Use Sephadex G-25 for desalination, buffer TBS-T (0.1M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin300). After adding an equal volume of glycerol and mixing thoroughly, store at -20°C for use. In this example, the molar ratio of the recombinant antibody to NSP-SA-NHS is 1:10. The labeled mother solution needs to be diluted 2000 times with TBS-T buffer to be configured as R2 working solution for use.

[0130] (4) Sample traceability: Trace the NF-L antigen on an imported high-sensitivity detection platform.

[0131] (5) Packaging of calibrators and quality controls: NF-L calibrators and quality controls are prepared from sample diluents. The concentrations of NF-L calibrators are 1000pg / ml and 3000pg / ml; the concentrations of NF-L quality controls are 1000pg / ml and 3000pg / ml. The sample diluent is PBS buffer at pH 7.4, and each liter of sample diluent also contains 1% BSA and 0.1% Proclin-300.

[0132] (6) Kit assembly: The conjugated antibodies and labeled antibodies produced in three batches of pilot tests were packaged in the reagent compartment and assembled into a kit together with the calibrators and quality control products. The specific information is shown in Table 7 below:

[0133] Table 7

[0134]

[0135] (7) On-machine testing

[0136] Testing process: Use the fully automatic chemiluminescence immunoassay analyzer as the testing tool, check and replenish consumables, and complete self-test after starting up. Manually or by scanning the code, enter the parameters and reagent batch number required for the NF-L test, and place the reagent in the corresponding reagent position. Use the NF-L calibrator to calibrate the master curve, and then use the NF-L quality control product to perform quality control on the detection system. After the quality control is qualified, add the samples to be tested in the sample position in turn for testing. After the test is completed, the data is output and analyzed.

[0137] Reaction system: The machine automatically aspirates 100 μL of the sample and mixes it thoroughly with 50 μL of R1 working solution and 50 μL of R2 working solution, and incubates it at 37°C for 10 min. Then add the M working solution, mix it thoroughly and continue to incubate it at 37°C for 10 min. After discarding the supernatant, add the immunoassay instrument's supporting washing solution (PBS and Tween-20), and repeat the washing 4 times. Then send the reaction solution into the darkroom, and add 100 μL of the immunoassay instrument's supporting luminescence excitation solution A (PBS and Tween-20) and luminescence excitation solution B (sodium hydroxide aqueous solution) for luminescence reaction, and record the luminescence value. Combined with the entered and calibrated calculation curve, the corresponding sample concentration value is calculated.

[0138] Example 4 Kit Performance Evaluation

[0139] The performance of the kit was evaluated using the method described in Example 3:

[0140] (1) Precision

[0141] According to the EP5-A2 protocol of the National Committee for Clinical Laboratory Standards (NCCLS / CLSI), the intra-batch precision and inter-laboratory precision were tested using a multi-factor integrated nested design. Different operators, different equipment, and different locations were tested once in the morning and afternoon each day. Each sample was tested twice for 20 consecutive days. A total of 160 data results were collected for each batch of test kits to calculate their precision. At the same time, the traced NF-L antigen was configured into 1000pg / mL and 3000pg / mL solutions, and the test was repeated 3 times and the data was analyzed. The test results are shown in Table 8 below:

[0142] Table 8

[0143]

[0144]

[0145] In summary, the results of repeated testing of low and high concentration samples by the above-mentioned kit showed that the intra-batch precision and inter-batch precision CV were both less than 10%, indicating that the above-mentioned kit meets the requirements for precision performance evaluation.

[0146] (2) Linear range

[0147] Select one cerebrospinal fluid supplemented with NF-L antigen, dilute it to 12 concentration levels with diluent, repeat the test for each diluted sample 3 times, and calculate the mean. According to the results, gradually reduce the concentration points to calculate the corresponding linear relationship, determine the widest linear range of the kit, and the NF-L kit test results are shown in Table 9 below (concentration unit is pg / mL).

[0148] Table 9

[0149]

[0150] Table 9 shows that the correlation coefficient r of the NF-L kit is not less than 0.9900 in the range of [10-4000] pg / mL and there is no outlier. Therefore, the linear range of the NF-L kit is [10-4000] pg / mL.

[0151] (3) Sensitivity

[0152] The blank sample was tested with the above kit and repeated 20 times to determine the sensitivity. The test results of the NF-L kit are shown in Table 10 below (concentration unit is pg / mL):

[0153] Table 10

[0154] project parameter Mean 443.5 SD 60.82 Mean+2SD 565.14 Corresponding concentration value 9.79pg / mL

[0155] The average luminescence value of the blank sample measured by the NF-L kit was 443.5. Substituting this value into the reaction curve, the sensitivity of the NF-L kit was 9.79 pg / mL.

[0156] (4) Sample test results

[0157] 20 samples assigned by the imported high-sensitivity detection platform were tested to determine the consistency of the kit with the platform products. For the part beyond the linear range of the benchmark reagent, the dilution method was used for testing. The analysis results are as follows Figure 1 As shown (concentration unit is pg / mL), regression analysis data showed that y = 0.9957x + 16.388 and R 2 =0.9443, which proves that this product has good consistency with the commercial product.

[0158] It can be seen from the above embodiments that the NF-L kit of the present application has good stability, high accuracy, high precision, and high sensitivity. The linear range of the kit is [10-4000] pg / mL, which is much wider than the linear range of the prior art. The upper limit of detection of the kit is at least 2 times that of the prior art. At the same time, the kit of the present application has good consistency with the commercial product.

[0159] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A recombinant antibody targeting neurofilament light chain, wherein: The recombinant antibody is a monoclonal antibody, which comprises three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7, The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9; The amino acid sequence of the CDR-L3 is shown in SEQ ID No:

11.

2. The recombinant antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 13; The amino acid sequence of the light chain variable region is as shown in SEQ ID No:15, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:

15.

3. A recombinant antibody targeting neurofilament light chain, wherein: The recombinant antibody is a monoclonal antibody, which comprises three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8; The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10; The amino acid sequence of the CDR-L3 is shown in SEQ ID No:

12.

4. The recombinant antibody according to claim 3, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 14; The amino acid sequence of the light chain variable region is shown in SEQ ID No: 16, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:

16.

5. A nucleic acid molecule encoding the recombinant antibody according to any one of claims 1 to 4.

6. A kit for detecting neurofilament light chain, wherein: The kit comprises: A biotin-coupled primary antibody targeting neurofilament light chain, a marker-labeled secondary antibody targeting neurofilament light chain, and magnetic beads; or Magnetic beads coated with a first antibody targeting neurofilament light chain and a second antibody targeting neurofilament light chain labeled with a marker; Wherein, the first antibody targeting neurofilament light chain is the recombinant antibody according to claim 1 or 2, and the second antibody targeting neurofilament light chain is the recombinant antibody according to claim 3 or 4.

7. The kit according to claim 6, wherein It is used to diagnose central nervous system diseases or neurodegenerative diseases.

8. Use of the recombinant antibody according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, in the preparation of a kit for detecting neurofilament light chain and / or diagnosing central nervous system diseases or neurodegenerative diseases.

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