Kit and method for detecting neurofilament light chain

Abstract

The present application discloses a kit for detecting neurofilament light chain, which comprises: a first antibody targeting neurofilament light chain conjugated with biotin, a second antibody targeting neurofilament light chain labeled with a labeling agent, and magnetic beads, wherein the magnetic beads are streptavidin 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 labeling agent, wherein the magnetic beads are toluenesulfonyl-activated magnetic beads. Also disclosed is the use of the foregoing kit in the preparation of a product for detecting neurofilament light chain and / or for diagnosing nervous system diseases, as well as a method for detecting the content of neurofilament light chain in a sample. The kit of the present application has a short detection time, a lower detection limit, a wider linear range, high detection sensitivity, and is of great significance for the diagnosis of nervous system diseases.

Description

Technical Field

[0001] This application belongs to the technical field of immunoassay, and particularly relates to a kit for detecting neurofilament light chain and a method for detecting neurofilament light chain. Background Art

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

[0003] NF-L is the backbone of NFs. It is the most abundant subunit and also 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 old age. When the body is in a reaction of axonal injury in the central nervous system (CNS) caused by inflammation, neurodegenerative diseases, trauma, or vascular injury, the release of NF-L increases sharply. The concentration of NF-L in the cerebrospinal fluid (CSF) of patients with nervous system diseases is higher than that of the control group, and existing results have confirmed that the serum NF-L level is closely related to the NF-L level in the CSF. The potential diagnostic value of NF-L lies in the degree of injury of CNS diseases, disease progression, and the differentiation between neurodegenerative diseases and non-neurodegenerative lesions. Compared with imaging methods such as PET-CT and MRI, the immunological monitoring of NF-L helps doctors strengthen the dynamic tracking of CNS injuries, improve the diagnosis and treatment effects, optimize the prognosis management, and bring significant social and economic benefits.

[0004] However, the existing detection platforms for NF-L mainly have the following disadvantages:

[0005] (1) The detection time is too long

[0006] Currently, the main detection platforms for NF-L are enzyme-linked immunosorbent assay and chemiluminescence (including tube-type and plate-type chemiluminescence) platforms. For enzyme-linked immunosorbent assay and plate-type chemiluminescence platforms, a relatively long incubation time is often required, generally 0.5 - 2 hours. Existing tube-type chemiluminescence platforms often use a two-step method to improve detection performance, but this will correspondingly increase the detection time. For example, data from Liu Zhimin et al. showed that their direct chemiluminescence assay kit for neurofilament light chain protein content was within 30 minutes, and the kit for quantitative detection of neurofilament light chain protein developed by Qiu Chunming et al. required incubation at 37°C for 15 minutes and 17 minutes respectively. Such detection durations undoubtedly pose difficulties for high-throughput and high-efficiency NF-L detection in clinical laboratories / physical examination departments.

[0007] (2) Narrow linear range

[0008] A wider linear range can ensure that the sample can be detected without dilution, which can undoubtedly reduce the number of detections and eliminate detection biases caused by sample dilution. The linear range of the NF-L plate-type chemiluminescence immunoassay system developed by Tang Yude et al. is 1.6 - 1000 pg / mL, while the linear range of a magnetic particle chemiluminescence detection kit for determining the content of human neurofilament light chain protein developed by Wang Guowu et al. is only 0 - 500 pg / mL. According to data published by Norgren et al. in 2003, the mean concentration of NF-L in cerebrospinal fluid of patients with multiple sclerosis (MS) reached 2500 pg / mL, far higher than the detection upper limit in existing reports. Therefore, technical improvements are still needed to significantly expand the linear range of the kit.

[0009] (3) Limited detection samples

[0010] Gaetani wrote in a systematic review of NF-L published in 2019: Cerebrospinal fluid and blood NF-L can guide clinicians in further diagnosing and examining patients with potential neurodegenerative diseases; the increase in both cerebrospinal fluid and serum NF-L is also one of the most common differential diagnoses for multiple sclerosis. However, in most current relevant patents and literature, the NF-L assay kits involved mainly target serum or cerebrospinal fluid and cannot detect both simultaneously. Summary of the invention

[0011] Based on the above description, there are still many deficiencies in the current NF-L detection system, which limits the application of NF-L immunological detection in the diagnosis, efficacy evaluation, and prognosis evaluation of CNS injuries and other aspects. Aiming at the deficiencies existing in the prior art, the present application provides a kit for detecting neurofilament light chain, and the use of the kit in the preparation of products for detecting neurofilament light chain and / or for diagnosing nervous system diseases. The present application also provides a method for detecting the content of neurofilament light chain in a sample. The kit of the present application uses a pair of recombinant antibodies with high affinity and stable performance as the core raw materials, has a wide linear range, a short detection time, and can simultaneously detect NF-L in serum and cerebrospinal fluid.

[0012] The specific technical solution of the present application is as follows:

[0013] 1. A kit for detecting neurofilament light chain, wherein the kit includes:

[0014] A first antibody targeting neurofilament light chain conjugated with biotin, a second antibody targeting neurofilament light chain labeled with a labeling agent, and magnetic beads, wherein the magnetic beads are streptavidin magnetic beads; or

[0015] Magnetic beads coated with a first antibody targeting neurofilament light chain and a second antibody targeting neurofilament light chain labeled with a labeling agent, wherein the magnetic beads are tosyl-activated magnetic beads.

[0016] 2. The kit according to item 1, wherein the particle size of the magnetic beads is 1-5 μM;

[0017] Preferably, the particle size of the tosyl-activated magnetic beads is 1.5-3 μM;

[0018] Preferably, the particle size of the streptavidin magnetic beads is 1.5-3 μM, and more preferably 1.5-2 μM.

[0019] 3. The kit according to item 1 or 2, wherein the molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3-40), preferably 1:(3-9).

[0020] 4. The kit according to any one of items 1-3, wherein the mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(100-5000), preferably 1:(500-2000);

[0021] Preferably, the molar ratio of the second antibody targeting neurofilament light chain to the labeling agent is 1:(3-40), preferably 1:(3-9);

[0022] 5. The kit according to any one of items 1 to 4, wherein the kit further comprises a neurofilament light chain standard, a substrate, a washing solution, a magnetic bead diluent, a conjugated antibody diluent, a labeled antibody diluent, and a sample diluent; or, the kit further comprises a neurofilament light chain standard, a substrate, a blocking solution, a coating antibody diluent, a labeled antibody diluent, and a sample diluent.

[0023] 6. The kit according to any one of items 1 to 5, wherein

[0024] the first antibody targeting neurofilament light chain comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0025] the amino acid sequence of the CDR-H1 is as shown in SEQ ID No: 1;

[0026] the amino acid sequence of the CDR-H2 is as shown in SEQ ID No: 3;

[0027] the amino acid sequence of the CDR-H3 is as shown in SEQ ID No: 5;

[0028] the amino acid sequence of the CDR-L1 is as shown in SEQ ID No: 7,

[0029] the amino acid sequence of the CDR-L2 is as shown in SEQ ID No: 9;

[0030] the amino acid sequence of the CDR-L3 is as shown in SEQ ID No: 11;

[0031] the second antibody targeting neurofilament light chain comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0032] the amino acid sequence of the CDR-H1 is as shown in SEQ ID No: 2;

[0033] the amino acid sequence of the CDR-H2 is as shown in SEQ ID No: 4;

[0034] the amino acid sequence of the CDR-H3 is as shown in SEQ ID No: 6;

[0035] the amino acid sequence of the CDR-L1 is as shown in SEQ ID No: 8;

[0036] The amino acid sequence of the CDR-L2 is as shown in SEQ ID No: 10;

[0037] The amino acid sequence of the CDR-L3 is as shown in SEQ ID No: 12.

[0038] 7. The kit according to any one of items 1 to 6, wherein

[0039] The first antibody targeting neurofilament light chain 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 is an amino acid sequence having 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, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15;

[0040] The second antibody targeting neurofilament light chain 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: 14, or is an amino acid sequence having 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, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 16.

[0041] 8. The kit according to any one of items 1 to 7, wherein it is used for diagnosing nervous system diseases;

[0042] Preferably, the nervous system disease is a central nervous system disease;

[0043] Preferably, the nervous system disease is a neurodegenerative disease.

[0044] 9. Use of the kit according to any one of items 1 to 8 in the preparation of a product for detecting neurofilament light chain and / or for diagnosing nervous system diseases;

[0045] Preferably, the nervous system disease is a central nervous system disease;

[0046] Preferably, the nervous system disease is a neurodegenerative disease.

[0047] 10. A method for detecting the content of neurofilament light chain in a sample, wherein the method comprises the following steps:

[0048] Reaction: Mix the sample to be tested, a biotin-conjugated first antibody targeting neurofilament light chain, and a second antibody targeting neurofilament light chain labeled with a label, incubate, add magnetic beads after incubation, and then incubate again;

[0049] Measure the luminescence value: Add a substrate after washing, and measure the luminescence value of the sample to be tested;

[0050] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested by using the luminescence value of the neurofilament light chain standard product;

[0051] Wherein, the magnetic beads are streptavidin magnetic beads.

[0052] 11. A method for detecting the content of neurofilament light chain in a sample, wherein the method comprises the following steps:

[0053] Reaction: Mix the sample to be tested, a biotin-conjugated first antibody targeting neurofilament light chain, a second antibody targeting neurofilament light chain labeled with a label, and magnetic beads, and incubate;

[0054] Measure the luminescence value: Add a substrate after washing, and measure the luminescence value of the sample to be tested;

[0055] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested by using the luminescence value of the neurofilament light chain standard product;

[0056] Wherein, the magnetic beads are streptavidin magnetic beads.

[0057] 12. A method for detecting the content of neurofilament light chain in a sample, wherein the method comprises the following steps:

[0058] Reaction: Mix the sample to be tested, magnetic beads coated with a first antibody targeting neurofilament light chain, and a second antibody targeting neurofilament light chain labeled with a label, incubate, and then perform magnetic separation;

[0059] Measure the luminescence value: Add a substrate after washing, and measure the luminescence value of the sample to be tested;

[0060] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested by using the luminescence value of the neurofilament light chain standard product;

[0061] Wherein, the magnetic beads are tosyl-activated magnetic beads.

[0062] 13. The method according to any one of items 10 to 12, wherein the particle size of the magnetic beads is 1 to 5 μM, preferably 1.5 to 3 μM.

[0063] 14. The method according to item 10 or 11, wherein the particle size of the streptavidin magnetic beads is 1.5 to 2 μM.

[0064] 15. The method according to item 10 or 11, wherein the molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3 - 40), preferably 1:(3 - 9).

[0065] 16. The method according to any one of items 10 - 12, wherein

[0066] the mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(100 - 5000), preferably 1:(500 - 2000);

[0067] Preferably, the molar ratio of the second antibody targeting neurofilament light chain to the label is 1:(3 - 40), preferably 1:(3 - 9).

[0068] 17. The method according to any one of items 10 - 16, wherein the test sample is serum or cerebrospinal fluid;

[0069] Preferably, the test sample is at least 50 μL, preferably at least 100 μL;

[0070] More preferably, for each microliter of the test sample, 0.1 - 0.5 ng of the first antibody targeting neurofilament light chain and 0.05 - 0.25 ng of the second antibody targeting neurofilament light chain are required.

[0071] 18. The method according to any one of items 10 - 17, wherein the first antibody targeting neurofilament light chain and the second antibody targeting neurofilament light chain are the first antibody targeting neurofilament light chain and the second antibody targeting neurofilament light chain as described in item 6 or 7.

[0072] Effects of the Invention

[0073] The kit for detecting NF-L of the present application uses a pair of recombinant antibodies with excellent and stable performance, high affinity, high sensitivity, and short production cycle as the core raw materials, and uses specific magnetic beads, which is more suitable for the field of in vitro diagnostic reagents. Among them, the high-affinity antibody adopted in the present application has a stronger ability to recognize antigen epitopes and a higher degree of binding to antigens, thus having a stronger ability to capture low-abundance target antigens, providing a lower detection limit for the kit. At the same time, the high-affinity antibody can enhance the antigen capture ability of a unit mass of magnetic beads or other media, and then significantly improve the linear range of the kit on the premise of not generating the Hook effect. The magnetic beads used in the present application are specific toluenesulfonyl-activated magnetic beads or streptavidin magnetic beads, with a lower detection limit value, a wider linear range, and high detection sensitivity. Further, the kit of the present application has the advantages of simple operation, high automation degree, and homogeneous reaction system, and the detection time can be further shortened by means of a one-step reaction system. The kit of the present application can detect the NF-L level in both serum and cerebrospinal fluid. Brief Description of the Drawings

[0074] Figure 1 It is the consistency analysis result of the NF-L content in cerebrospinal fluid samples detected by using the NF-L kit of the present application and the products of imported high-sensitivity detection platforms.

[0075] Figure 2 It is the consistency analysis result of the NF-L content in blood samples detected by using the NF-L kit of the present application and the products of imported high-sensitivity detection platforms. Detailed Description of the Embodiments

[0076] The following further describes the present application in detail in combination with specific embodiments. The embodiments given are for the purpose of being able to understand the present application more thoroughly and being able to convey the scope of the present application completely to those skilled in the art. The technical terms mentioned in this specification have the same meaning as those generally understood by those skilled in the art. In case of conflict, the definitions in this specification shall prevail.

[0077] In this article, "neurofilament protein" is an intermediate filament protein that forms the axonal cytoskeleton of nerve cells. It is located in the axons of nerve cells and participates in the composition of the axonal fiber system as one of the cytoskeletal components. Its function is to stabilize the morphology of axons, maintain their diameter and stable conduction velocity, and play a role in processes such as nerve cell differentiation, 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 in the central nervous system are damaged due to inflammation, neurodegeneration, trauma, or vascular injury, the release of NF-L increases sharply.

[0078] In this article, the term "recombinant antigen" refers to an antigen obtained by in vitro recombinant expression and purification after ligating the antigen gene to a vector and then transferring it into prokaryotic or eukaryotic cells. In one embodiment, the recombinant antigen used in this application is a commercial NF-L recombinant antigen.

[0079] In this article, the term "recombinant antibody" refers to an antibody obtained by cloning immunologically specific heavy and light chain antibody genes into a high-expression vector and then introducing these vectors into an expression host (such as bacteria, yeast, or mammals) for antibody expression. The antibodies of this application can be fully humanized antibodies, humanized antibodies, chimeric antibodies, etc.

[0080] In some embodiments, the NF-L recombinant antibody of this application is an antibody obtained by immunizing an animal with an NF-L recombinant antigen.

[0081] In some embodiments, the recombinant antibody of this application is a full-length antibody. A full-length antibody typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is usually 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 usually 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.

[0082] 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 kappa (κ) or lambda (λ). The heavy chain constant region can be the heavy chain constant region of any one of IgG, IgM, IgA, IgE, and IgD.

[0083] As is well known to those skilled in the art, each heavy chain variable region can be composed of three complementarity-determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementarity-determining regions (CDRs) and four framework regions (FRs). The complementarity-determining regions (CDRs, usually having CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, both the heavy chain variable region and the light chain variable region contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. There are two common definitions for the CDR sequences 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 sequences 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 embodiments of the present application, the Chothia definition is used to determine the CDR sequences.

[0084] As used herein, the term “identity” is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. As used herein, an amino acid sequence having a percentage identity to an amino acid sequence refers to a sequence having the percentage identity over the entire length of the amino acid sequence mentioned.

[0085] In this text, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host and transfers the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple 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. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product.

[0086] In this text, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in its single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have binding properties similar to those of a reference nucleic acid (e.g., showing sequence information) and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, the sequence of a nucleic acid may include variants modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequences.

[0087] In this text, the term "EC50 value" refers to the concentration for 50% of maximal effect (EC50), which is the concentration that can cause 50% of the maximal effect.

[0088] In this text, "affinity" represents 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, the "binding affinity" used in this specification represents the intrinsic binding affinity reflecting the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K D ). The affinity can be determined by common methods known in the art.

[0089] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homologous, i.e., 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 generated during the production of monoclonal antibody preparations), and such variants are typically present in trace amounts. Different from polyclonal antibody preparations that usually include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation binds to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homologous population of antibodies and should not be construed as requiring the antibody to be produced by any specific method.

[0090] As used 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.

[0091] The recombinant antibody targeting neurofilament light chain of the present application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to other proteins other than neurofilament light chain as the target; here, "does not bind" means that: when the binding ability of the recombinant antibody targeting neurofilament light chain of the present application to 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%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0092] Neurofilament light chain suitable for antibody production can be generated 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, articles or cell extracts or fractions containing neurofilament light chain, and partially purified neurofilament light chain.

[0093] In some embodiments, the present application uses a magnetic particle chemiluminescence immunoassay to determine the level of neurofilament light chain in a sample. The specific principle is: directly label an antibody with a chemiluminescent agent (chemiluminescent label), react with the corresponding antigen in the sample to be tested and the antibody on magnetic particles (magnetic beads), separate the chemiluminescent label-labeled antibody in the bound state (precipitation part) and the free state by a magnetic field, and then add a luminescence promoter (luminescence substrate) for a luminescence reaction, and perform quantitative or qualitative detection by detecting the luminescence value.

[0094] In some embodiments, the present application can also use platforms such as POCT, ELISA, SERS, mass spectrometry, microfluidics, and photochemistry to determine the level of neurofilament light chain in a sample.

[0095] In this article, ELISA (enzyme linked immune sorbent assay) is a detection method that utilizes the specific binding property between antibody molecules and antigen molecules to bind free heterologous proteins and target proteins bound to a solid-phase carrier, and qualitatively or quantitatively analyze them using special markers. Its principle is that antigens or antibodies can be physically adsorbed on the surface of the solid-phase carrier while maintaining their immunoreactivity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immunoreactivity or enzyme activity; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of the immune reaction can be determined by adding a substrate color reaction, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen.

[0096] In a first aspect, the present application provides a kit for detecting neurofilament light chain, wherein the kit includes: a first antibody targeting neurofilament light chain conjugated with biotin, a second antibody targeting neurofilament light chain labeled with a marker, and magnetic beads; wherein the magnetic beads are streptavidin magnetic beads.

[0097] 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 liver in the 1930s during the study of yeast growth factors and the growth and respiration promoting factors of rhizobia, and is a factor that can prevent and treat hair loss and skin damage in rats induced by feeding raw egg white. Biotin is a member of the water-soluble vitamin B group. It is abundant in the liver, kidneys, yeast, and milk, and is an important factor for organisms to fix carbon dioxide. It easily binds to a protein in egg white, and excessive consumption of raw egg white can hinder the absorption of biotin, leading to biotin deficiency, such as hair loss, weight loss, dermatitis, etc. Biotin plays an important role in biochemical reaction pathways such as fatty acid synthesis and gluconeogenesis.

[0098] In this article, the term "tosyl-activated magnetic beads (Tosyl beads)" refers to magnetic beads with tosyl-activated groups, and the tosyl-activated groups on the magnetic beads can react with sulfhydryl groups and amino groups. At a pH value of around 7.0 to 8.0, the sulfhydryl reaction occurs. At a higher pH value (such as 8.5 to 9.5), it can react with amino groups. Compared with traditional magnetic beads, tosyl-activated magnetic beads have faster magnetic responsiveness while maintaining good dispersion of the microspheres, extremely low non-specific adsorption, and pre-activated reaction sites, etc., and can be directly used to bind with a variety of biological ligands (proteins, polypeptides, oligonucleotides, drug molecules, etc.) with high loading to achieve the purpose of covalent coupling on the surface of the magnetic beads. The magnetic beads have a long hydrophilic spacer arm and relatively mild reaction conditions, making them particularly suitable for the immobilization of bioactive macromolecules.

[0099] In this article, the term "streptavidin magnetic beads (SA magnetic beads)" refers to streptavidin immobilized on polymer magnetic microspheres. With the high-affinity binding property of streptavidin-biotin, a fixed layer is formed with biotinylated proteins, antibodies, and nucleic acids (collectively referred to as biotinylated molecules), etc., and then the target molecules that interact with the biotinylated molecules are separated. It can be used in various applications such as affinity purification, cell sorting, protein-protein interaction, DNA-protein interaction, probe capture, and mRNA separation, which is conducive to conveniently and quickly capturing target molecules and realizing magnetic separation.

[0100] In some embodiments, the molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3 - 40), preferably 1:(3 - 9). For example, it can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 3:20, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:20, 1:20.5, 1:21, 1:21.5, 1:22, 1:22.5, 1:23, 1:23.5, 1:24, 1:24.5, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40 or any range therebetween.

[0101] In some embodiments, the kit further includes a neurofilament light chain standard, a substrate, a washing solution, a magnetic bead diluent, a conjugated antibody diluent, a labeled antibody diluent, and a sample diluent. In some embodiments, the substrate includes a luminescence excitation solution A and a luminescence excitation solution B, wherein the luminescence excitation solution A is an aqueous solution of nitric acid and hydrogen peroxide, and the luminescence excitation solution B is an aqueous solution of sodium hydroxide. In some embodiments, the magnetic bead diluent is a phosphate buffer solution containing BSA and Proclin 300. In some embodiments, the conjugated antibody diluent and the labeled antibody diluent are TBS-T buffer solutions. In some embodiments, the washing solution is PBS and Tween-20. In some embodiments, the sample diluent is a PBS buffer solution. In some embodiments, the sample diluent is a PBS buffer solution containing BSA and Proclin-300. In some embodiments, the kit further includes a preservation buffer and a quenching buffer, wherein the preservation buffer is used to provide a stable pH environment, reduce antibody degradation, prevent corrosion, etc.; the quenching buffer is used to quench the label that has not bound to the antibody. In some embodiments, the preservation buffer is a TBS-T buffer solution, and the quenching buffer is 5% DL-lysine.

[0102] In some embodiments, the mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(100 - 5000), preferably 1:(500 - 2000), and for example, it can be 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000 or any range therebetween.

[0103] In some embodiments, the molar ratio of the second antibody targeting neurofilament light chain to the label is 1:(3-40), preferably 1:(3-9). For example, it can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 3:20, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:20, 1:20.5, 1:21, 1:21.5, 1:22, 1:22.5, 1:23, 1:23.5, 1:24, 1:24.5, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40 or any range therebetween.

[0104] In some embodiments, the particle size of the streptavidin magnetic beads is 1-5 μM, preferably 1.5-3 μM, and further preferably 1.5-2 μM. For example, it can be 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM or any range therebetween. Compared with Tosyl magnetic beads, SA magnetic beads have a better signal-to-noise ratio. Further, using magnetic beads with a smaller particle size can further effectively improve the signal-to-noise ratio and effectively improve the linear range of the kit.

[0105] In some embodiments, the label can be a chemiluminescent label, an electrochemiluminescent label, etc. Preferably, the label is a chemiluminescent label. Further preferably, the chemiluminescent label is acridinium ester.

[0106] In some embodiments, the first antibody targeting neurofilament light chain comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID No: 1 (GFSLTGM); the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 3 (YWDED); the amino acid sequence of CDR-H3 is as shown in SEQ ID No: 5 (RPFHRYYVEALDY); the amino acid sequence of CDR-L1 is as shown in SEQ ID No: 7 (RSSQSIVHSNGATSLE); the amino acid sequence of CDR-L2 is as shown in SEQ ID No: 9 (KVSWRFS); the amino acid sequence of CDR-L3 is as shown in SEQ ID No: 11 (FNGSRVPLT); the second antibody targeting neurofilament light chain comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID No: 2 (GFIFSIR); the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 4 (APSGN); the amino acid sequence of CDR-H3 is as shown in SEQ ID No: 6 (APDGNYGPFFY); the amino acid sequence of CDR-L1 is as shown in SEQ ID No: 8 (RASETITNSLH); the amino acid sequence of CDR-L2 is as shown in SEQ ID No: 10 (YATNSIS); the amino acid sequence of CDR-L3 is as shown in SEQ ID No: 12 (QQSDSWPLT).

[0107] In some embodiments, the first antibody targeting neurofilament light chain 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 (QVSLRESGPGILQPSNTLSLTCSFSGFSLTGMGVSWIRQPSGKGIEWI AHIYWDEDKTYNPSLKSHLSITRDTSRNQVFLHVTNVDTPDSVTYYQARRPFHRYYVEALDYWGQGLAVTVDS), or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to 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 is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No: 15; the second antibody targeting neurofilament light chain 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: 14 (DVKLVESGCNLVHPGGSPRLSCTATGFIFSIRAMSWVYCTPDHRLE WIASIAPSGNTHYPDYVKGRPTISRADATNVLYLQMSTLKSEDTAMYYCT GAPDGNYGPFFYWGQGTLVTVTA), or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to 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 is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No: 16.

[0108] In a second aspect, the present application provides another kit for detecting neurofilament light chain, wherein the kit includes: magnetic beads coated with a first antibody targeting neurofilament light chain and a second antibody targeting neurofilament light chain labeled with a labeling agent; wherein the magnetic beads are tosyl-activated magnetic beads.

[0109] In some embodiments, the kit further includes a neurofilament light chain standard, a substrate, a blocking solution, a coated antibody diluent, a labeled antibody diluent, and a sample diluent. In some embodiments, the substrate, washing solution, and sample diluent are as described in the first aspect above. In some embodiments, the coated antibody diluent and the labeled antibody diluent are TBS-T buffer solutions. The blocking solution is used to block uncoupled vacancies to prevent non-specific binding in the later stage. In some embodiments, the blocking solution is a BSA blocking solution. In some embodiments, the kit further includes a storage buffer and a quenching buffer. Among them, the storage buffer is used to provide a stable pH environment, reduce antibody degradation, prevent corrosion, etc.; the quenching buffer is used to quench the labeling agent that has not bound to the antibody. In some embodiments, the storage buffer is a TBS-T buffer solution, and the quenching buffer is 5% DL-lysine.

[0110] In some embodiments, the particle size of the tosyl-activated magnetic beads is 1-5 μM, preferably 1.5-3 μM, and can be, for example, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, or any range therebetween.

[0111] In some embodiments, the mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads, the molar ratio of the second antibody targeting neurofilament light chain to the labeling agent, the labeling agent, the first antibody targeting neurofilament light chain, and the second antibody targeting neurofilament light chain are all as described in the first aspect.

[0112] In some embodiments, any of the kits in the first and second aspects above can 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, a photochemistry kit, etc., and preferably a magnetic particle chemiluminescence kit.

[0113] In some embodiments, the kit for detecting NF-L according to any one of the foregoing first and second aspects is used for diagnosing nervous system diseases. In some embodiments, the nervous system disease is a central nervous system disease. In some embodiments, the nervous system disease is a neurodegenerative disease. The nervous system disease may be, for example, brain atrophy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, brain injury-related diseases, etc. The brain injury-related diseases may be, for example, primary craniocerebral injury or secondary craniocerebral injury.

[0114] Any one of the foregoing first and second aspect kits of the present application can also be used to distinguish between neurodegenerative diseases and non-neurodegenerative lesions.

[0115] In a third aspect, the present application provides the use of any one of the foregoing first and second aspect kits in the preparation of a product for detecting neurofilament light chain and / or for diagnosing a nervous system disease. In some embodiments, the nervous system disease is a central nervous system disease. In some embodiments, the nervous system disease is a neurodegenerative disease. The nervous system disease may be, for example, brain atrophy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, brain injury-related diseases, etc. The brain injury-related diseases may be, for example, primary craniocerebral injury or secondary craniocerebral injury.

[0116] In a fourth aspect, the present application further provides a method for detecting the content of neurofilament light chain in a sample, which includes the following steps:

[0117] Reaction: Mix the sample to be tested, a biotin-conjugated first antibody targeting neurofilament light chain, and a second antibody targeting neurofilament light chain labeled with a label, incubate, add magnetic beads after incubation, and then incubate again;

[0118] Measure the luminescence value: Add a substrate after washing and measure the luminescence value of the sample to be tested;

[0119] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested using the luminescence value of the neurofilament light chain standard product;

[0120] Wherein, the magnetic beads are streptavidin magnetic beads.

[0121] In a fifth aspect, the present application further provides a method for detecting the content of neurofilament light chain in a sample, which includes the following steps:

[0122] Reaction: Mix the sample to be tested, a biotin-conjugated first antibody targeting neurofilament light chain, a second antibody targeting neurofilament light chain labeled with a label, and magnetic beads, and incubate;

[0123] Measure the luminescence value: After washing, add the substrate and measure the luminescence value of the sample to be tested.

[0124] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested by using the luminescence value of the neurofilament light chain standard.

[0125] Wherein, the magnetic beads are streptavidin magnetic beads.

[0126] In a sixth aspect, the present application further provides a method for detecting the content of neurofilament light chain in a sample, which includes the following steps:

[0127] Reaction: Mix the sample to be tested, magnetic beads coated with a first antibody targeting neurofilament light chain, and a second antibody targeting neurofilament light chain labeled with a labeling agent, and perform magnetic separation after incubation.

[0128] Measure the luminescence value: After washing, add the substrate and measure the luminescence value of the sample to be tested.

[0129] Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested by using the luminescence value of the neurofilament light chain standard.

[0130] Wherein, the magnetic beads are tosyl-activated magnetic beads.

[0131] In some embodiments of the fourth to sixth aspects, the mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(100 - 5000), preferably 1:(500 - 2000), for example, it can be 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000 or any range therebetween.

[0132] In some embodiments of the fourth and fifth aspects, the particle size of the streptavidin magnetic beads is 1 - 5 μM, preferably 1.5 - 3 μM, more preferably 1.5 - 2 μM, for example, it can be 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM or any range therebetween. Compared with Tosyl magnetic beads, SA magnetic beads have a better signal-to-noise ratio. Further, using magnetic beads with a smaller particle size can further effectively improve the signal-to-noise ratio and effectively improve the linear range of the kit. Compared with Tosyl magnetic beads, SA magnetic beads have a better signal-to-noise ratio. Using magnetic beads with a smaller particle size can effectively improve the signal-to-noise ratio and effectively improve the linear range of the kit.

[0133] In some embodiments of the sixth aspect, the particle size of the tosyl-activated magnetic beads is 1 to 5 μM, preferably 1.5 to 3 μM, and may be, for example, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM or any range therebetween.

[0134] In some embodiments of the fourth to sixth aspects, the label may be a chemiluminescent label, an electrochemiluminescent label, etc. Preferably, the label is a chemiluminescent label, and more preferably, the chemiluminescent label is acridinium ester.

[0135] In some embodiments of the fourth and fifth aspects, the molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3 to 40), preferably 1:(3 to 9), and may be, for example, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 3:20, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:20, 1:20.5, 1:21, 1:21.5, 1:22, 1:22.5, 1:23, 1:23.5, 1:24, 1:24.5, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40 or any range therebetween.

[0136] In some embodiments of the fourth to sixth aspects, the molar ratio of the second antibody targeting neurofilament light chain to the label is 1:(3 to 40), preferably 1:(3 to 9). For example, it can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 3:20, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:20, 1:20.5, 1:21, 1:21.5, 1:22, 1:22.5, 1:23, 1:23.5, 1:24, 1:24.5, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40 or any range therebetween.

[0137] In some embodiments of the fourth to sixth aspects, the sample to be tested is serum or cerebrospinal fluid. That is, the kit of the present application can detect the NF-L level in both serum and cerebrospinal fluid. The blood NF-L concentration is approximately 1 / 40 of that in CSF. Since the kit of the present application has a large linear range, it can simultaneously detect the NF-L concentration in serum and cerebrospinal fluid samples. The blood NF-L concentration is relatively low. Since the kit of the present application has better sensitivity, it can ensure stable and accurate measurement values. The main proteins contained in serum and cerebrospinal fluid are different, which will cause different detection interferences. Since the recombinant antibody used in the kit of the present application has sufficient affinity, it can ensure accurate detection of the substance to be detected.

[0138] In some embodiments of the fourth to sixth aspects, the sample to be tested is at least 50 μL. For example, it can be 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL or any range therebetween, preferably at least 100 μL.

[0139] In some embodiments of the fourth to sixth aspects, the sample to be tested is serum or cerebrospinal fluid. The sample to be tested is at least 50 μL, preferably at least 100 μL. For each microliter of the sample to be tested, 0.1 - 0.5 ng of the first antibody targeting neurofilament light chain and 0.05 - 0.25 ng of the second antibody targeting neurofilament light chain are required. The amount of the first antibody targeting neurofilament light chain required for each microliter of the sample to be tested can be, for example, 0.1 ng, 0.15 ng, 0.2 ng, 0.25 ng, 0.3 ng, 0.35 ng, 0.4 ng, 0.45 ng, 0.5 ng or any range therebetween. The amount of the second antibody targeting neurofilament light chain required for each microliter of the sample to be tested can be, for example, 0.05 ng, 0.07 ng, 0.09 ng, 0.1 ng, 0.12 ng, 0.14 ng, 0.16 ng, 0.18 ng, 0.2 ng, 0.21 ng, 0.23 ng, 0.25 ng or any range therebetween.

[0140] In some embodiments of the fourth to sixth aspects, the first antibody targeting neurofilament light chain and the second antibody targeting neurofilament light chain are the first antibody targeting neurofilament light chain and the second antibody targeting neurofilament light chain as described in the first aspect above.

[0141] Example

[0142] The following will illustrate the content of the present application 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 conventional reagents and instruments in the art and can be obtained through commercial purchase. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described solutions and obtain corresponding results according to the content of the examples.

[0143] Example 1

[0144] Preparation of recombinant antibodies rNF-L-A and rNF-L-B

[0145] In this embodiment, the preparation method of the recombinant antibody is as follows: Mice are immunized with a commercial NF-L recombinant antigen. After obtaining the mouse-derived monoclonal antibody cell line combination with the highest sensitivity, antibody variable region sequence sequencing is performed. According to the variable region sequence, a chimeric vector is constructed and transfected into a 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. Both of the above two vectors are commercial vectors and carry constant region sequences without additional treatment. After the recombinant antibody is expressed in the CHO cell line, the purified recombinant antibodies rNF-L-A and rNF-L-B are obtained through Protein A packing material. Among them, the amino acid sequence of the heavy chain variable region of rNF-L-A is shown in SEQ ID No: 13, the amino acid sequence of the light chain variable region of rNF-L-A is shown in SEQ ID No: 15, the amino acid sequence of the heavy chain variable region of rNF-L-B is shown in SEQ ID No: 14, and the amino acid sequence of the light chain variable region of rNF-L-B is shown in SEQ ID No: 16.

[0146] Example 2

[0147] Affinity detection of recombinant antibodies rNF-L-A and rNF-L-B

[0148] The affinity of multiple batches of recombinant antibodies is detected by means of the Elisa technique. 100 ng of NF-L recombinant antigen is coated on each well of the enzyme-linked immunosorbent assay (ELISA) plate, and serially diluted recombinant antibodies are added and incubated at 37 °C for 1 hour. After incubation, a goat anti-human secondary antibody labeled with HRP is added. Finally, TMB is added for color development, 2 M sulfuric acid is added to terminate the reaction, and the absorbance is measured at a wavelength of 450 nm using an ELISA reader. The EC50 values of each batch of recombinant antibodies are calculated according to SigmaPlot 12.5 software and used as the K D value. The data are summarized in Table 1 below. The results in Table 1 show that the above-mentioned recombinant antibodies have a relatively high affinity. Compared with the nM-level K D value in the prior art, the K D value of the recombinant antibodies in this application can reach the pM level, which is about one-thousandth of that in the prior art. The affinity of the recombinant antibodies in this application is far higher than that of the antibodies in the prior art.

[0149] Table 1

[0150]

[0151] Example 3

[0152] Preparation and detection process of the SA magnetic bead system kit

[0153] (1) Preparation of magnetic bead working solution

[0154] Select SA magnetic beads (M-270), vortex mix and then sonicate for 5 min and resuspend thoroughly. After magnetic separation to remove the liquid, add magnetic bead diluent (0.1 M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA and 0.1% Proclin 300) to make the final concentration 0.5 mg / mL, and store as Working solution M at 4 °C for later use.

[0155] (2) Biotinylation of antibodies

[0156] Pipette 0.5 mg of rNF-L-A recombinant antibody, mix well with 3.33 μL of 10 mM Sulfo-NHS-LC-LC-Biotin, and let stand at room temperature for 60 min. Remove the uncoupled biotin with an ultrafiltration tube, and perform 3 rinses with PBS (pH 7.4) during the ultrafiltration process. Then add an equal volume of glycerol and store at -20 °C for later use. The molar ratio of rNF-L-A recombinant antibody to biotin is 1:20, 1:10, 1:6.7. Dilute the coupled mother liquor 1000-fold with TBS-T buffer to prepare Working solution R1 for use.

[0157] (3) Labeling of antibodies

[0158] Pipette 0.5 mg of rNF-L-B recombinant antibody, mix well with 8.25 μL of 4 mM NSP-SA-NHS (dissolved in DMF) solution for 2 h. Add 200 μl of 5% DL-lysine to the solution and continue to mix for 30 min. Use Sephadex G-25 for desalting, with buffer TBS-T (0.1 M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin 300). After adding an equal volume of glycerol and mixing well,

[0159] store at -20 °C for later use. The molar ratio of rNF-L-B recombinant antibody to NSP-SA-NHS is 1:20, 1:10, 1:6.7. Dilute the labeled mother liquor 2000-fold with TBS-T buffer to prepare Working solution R2 for use.

[0160] (4) Sample traceability

[0161] Perform traceability of NF-L antigen on an imported high-sensitivity detection platform.

[0162] (5) Sub-packaging of calibrators and quality control products

[0163] The calibrator and quality control product of NF-L are both prepared with sample diluent. Among them, the concentrations of NF-L calibrator are 1000 pg / ml and 3000 pg / ml; the concentrations of NF-L quality control product are 1000 pg / ml and 3000 pg / ml. The sample diluent is PBS buffer solution with pH 7.4, and each liter of sample diluent also contains 1% BSA and 0.1% Proclin-300.

[0164] (6) Kit assembly

[0165] The conjugated antibody and labeled antibody produced in small-scale production of 3 batches are aliquoted into the reagent bin and assembled into a kit together with the calibrator and quality control product. The specific information is shown in Table 2 below:

[0166] Table 2

[0167]

[0168] (7) Detection on the machine

[0169] Detection process: Using an automatic chemiluminescence immunoassay analyzer as the detection tool, check and supplement the consumables, and complete the self-check after starting up. Manually or by scanning the code, input the parameters required for NF-L test and the reagent batch number, and place the reagent in the corresponding reagent position. Calibrate the main curve with the NF-L calibrator, 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 sequence at the sample position for detection. After the detection is completed, output and analyze and sort out the data.

[0170] Two-step reaction: 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 at 37 °C for 10 min. Immediately add 50 μL of M working solution, mix thoroughly and continue to incubate at 37 °C for 10 min. Discard the supernatant and add the washing solution (PBS and Tween-20) supporting the immunoassay analyzer, and repeat the washing 4 times. Then send the reaction solution into the dark room, and add 100 μL each of the luminescence excitation solution A (PBS and Tween-20) and luminescence excitation solution B (sodium hydroxide aqueous solution) supporting the immunoassay analyzer for luminescence reaction, and record the luminescence value. Calculate the corresponding concentration value of the sample by combining with the calculated curve that has been entered and calibrated.

[0171] Example 4

[0172] Preparation and detection process of the toluenesulfonyl-activated magnetic bead system kit

[0173] (1) Preparation of magnetic bead working solution

[0174] Select toluenesulfonyl-activated magnetic beads (M-280) with an original concentration of 100 mg / mL. After vortex mixing, sonicate for 5 min and resuspend thoroughly. Pipette 100 μL of the thoroughly resuspended magnetic bead mother liquor (about 10 mg of magnetic beads), and perform magnetic separation for 5 min to remove the liquid. Add 165 μL of 0.1 M borate buffer (pH 9.5) and 200 μg of rNF-L-A recombinant antibody, mix well and incubate overnight at 37 °C. After incubation, perform magnetic separation for 5 min to remove the liquid. Add 10% BSA and block at 37 °C for 3 h. After blocking, perform magnetic separation for 5 min to remove the liquid. Finally, add buffer TBS-T (0.1 M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin 300) and store at 4 °C for later use. The mass ratio of rNF-L-A recombinant antibody to magnetic beads is 1:50. The coated mother liquor needs to be further diluted 20-fold to prepare the M working solution for use.

[0175] (2) Labeling of antibodies

[0176] Respectively pipette 0.25 mg of rNF-L-B recombinant antibody and mix well with 8.25 μL of 4 mM ASDFGH (dissolved in DMF) solution for 2 h. Add 200 μl of 5% DL-lysine to the solution and continue to mix for 30 min. Use Sephadex G-25 for desalting with buffer TBS-T (0.1 M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin 300). Add an equal volume of glycerol, mix well, and store at -20 °C for later use. The molar ratio of rNF-L-B recombinant antibody to NSP-SA-NHS is 1:20. The labeled mother liquor needs to be further diluted 2000-fold to prepare the R1 working solution for use.

[0177] (3) Sample traceability: Refer to Example 3.

[0178] (4) Sub-packaging of calibrators and quality control products: Refer to Example 3.

[0179] (5) Kit assembly: Refer to Example 3.

[0180] (6) Instrument detection

[0181] Detection procedure: Refer to Example 3.

[0182] One-step reaction: The machine automatically aspirates 150 μL of the sample and mixes it thoroughly with 50 μL of the M working solution and 50 μL of the R1 working solution, incubates it at 37 °C for 20 min, and then performs magnetic separation. After discarding the supernatant, the immunological analyzer's supporting washing solution (PBS and Tween-20) is added, and the washing is repeated 3 times. Subsequently, the reaction is sent to a dark room, and 100 μL each of the immunological analyzer's supporting luminescence excitation solution A (aqueous solution of nitric acid and hydrogen peroxide) and luminescence excitation solution B (aqueous solution of sodium hydroxide) are added for luminescence reaction, and the luminescence value is recorded. The corresponding concentration value of the sample is calculated by combining the calculated curve that has been entered and calibrated.

[0183] The detection results of the two different magnetic bead system kits in Examples 3 and 4 are shown in Table 3 below.

[0184] Table 3

[0185]

[0186] The above results show that compared with Tosyl magnetic beads, SA magnetic beads have a better signal-to-noise ratio. The kit using SA magnetic beads can obtain a relatively better linear range.

[0187] Example 5

[0188] The difference between this example and Example 3 is only that the M-270 magnetic beads are replaced with MS160 magnetic beads, and the detection results are shown in Table 4 below.

[0189] Table 4

[0190]

[0191]

[0192] The above results show that compared with M-270 magnetic beads, although the RLU decreases after using the smaller particle size MS160, the signal-to-noise ratio is effectively improved, thereby effectively improving the linear range of the kit.

[0193] Examples 6-1 to 6-2

[0194] The difference between Example 6-1 and Example 5 is only that the usage amount of the rNF-L-A recombinant antibody is 0.25 mg.

[0195] The difference between Example 6-2 and Example 5 is only that the usage amount of the rNF-L-A recombinant antibody is 0.75 mg.

[0196] The detection results of Example 5 and Examples 6-1 to 6-2 are shown in Table 5 below.

[0197] Table 5

[0198]

[0199] The above results show that in the process of conjugating rNF-L-A recombinant antibody with biotin, increasing the antibody usage to 0.75 mg can effectively detect antigens at lower concentrations and effectively improve the detection sensitivity.

[0200] Examples 7-1 to 7-3

[0201] The difference between Example 7-1 and Example 5 is only that the usage of rNF-L-A recombinant antibody is 0.75 mg and the usage of rNF-L-B recombinant antibody is 0.25 mg;

[0202] The difference between Example 7-2 and Example 5 is only that the usage of rNF-L-A recombinant antibody is 0.75 mg and the usage of rNF-L-B recombinant antibody is 0.5 mg;

[0203] The difference between Example 7-3 and Example 5 is only that the usage of rNF-L-A recombinant antibody is 0.75 mg and the usage of rNF-L-B recombinant antibody is 0.75 mg.

[0204] The detection results of Examples 7-1 to 7-3 are shown in Table 6 below.

[0205] Table 6

[0206]

[0207] The above results show that in the rNF-L-B labeling process, increasing the usage of rNF-L-B antibody to 0.75 mg can further improve the detection sensitivity.

[0208] Example 8

[0209] The difference between this example and Example 7-3 is that the reaction in the step of detecting on the machine is a one-step reaction: the machine automatically aspirates 100 μL of the sample and mixes it thoroughly with 50 μL of M working solution, 50 μL of R1 working solution, and 50 μL of R2 working solution, and incubates at 37 °C for 10 min. After discarding the supernatant, the immunology analyzer's supporting washing solution (PBS and Tween-20) is added and washed 4 times repeatedly. Subsequently, the reaction is sent to a dark room, and 100 μL each of the immunology analyzer's supporting luminescence excitation solution A (nitric acid and hydrogen peroxide aqueous solution) and luminescence excitation solution B (sodium hydroxide aqueous solution) are added for luminescence reaction, and the luminescence value is recorded. The corresponding concentration value of the sample is calculated by combining the calculation curve that has been entered and calibrated. The detection results of Example 7-3 and Example 8 are summarized in Table 7 below.

[0210] Table 7

[0211]

[0212]

[0213] The above results show that after changing the reaction procedure to a one-step method and shortening the detection time, the detection performance is similar to that of the two-step method, and the overall linear range is not affected.

[0214] The preparation process parameters, detection method parameters, etc. of the kits in Examples 3 to 8 are listed in Table 8 below.

[0215] Table 8

[0216] Magnetic bead model Dosage of rNF-L-A Dosage of rNF-L-B Reaction on the machine Example 3 SA-M-270 0.5 mg 0.5 mg Two-step method Example 4 Tosyl-M-280 200 μg 0.25 mg One-step method Example 5 SA-MS160 0.25 mg 0.5 mg Two-step method Example 6-1 SA-MS160 0.5 mg 0.5 mg Two-step method Example 6-2 SA-MS160 0.75 mg 0.5 mg Two-step method Example 7-1 SA-MS160 0.75 mg 0.25 mg Two-step method Example 7-2 SA-MS160 0.75 mg 0.5 mg Two-step method Example 7-3 SA-MS160 0.75 mg 0.75 mg Two-step method Example 8 SA-MS160 0.75 mg 0.75 mg One-step method

[0217] Performance Evaluation of the Kit in Example 9

[0218] (1) Accuracy

[0219] The NF-L antigen after traceability was respectively configured into a 1500 pg / mL solution, detected 3 times repeatedly, and data analysis was carried out. The accuracy results are shown in Table 9 below (the concentration unit is pg / mL).

[0220] Table 9

[0221] Kit batch Test result 1 Test result 2 Test result 3 Mean value Labeled value Deviation 20230401 1460.458 1578.402 1444.221 1494.3603 1500 99.62％ 20230401 1596.086 1451.047 1578.828 1541.987 1500 102.80％ 20230401 1589.392 1556.941 1495.532 1547.2883 1500 103.15％

[0222] In summary, the relative deviation of the measurement results is within the range of ±10%, and the results of 3 times all meet the requirements. The accuracy of the above 3 batches of kits is qualified.

[0223] (2) Precision

[0224] According to the NCCLS / CLSI document EP5-A2 protocol in the United States, the within-batch precision and between-laboratory precision were experimentally carried out using a nested design with multi-factor integration. Different operators, different equipment, different locations, were detected once in the morning and afternoon every day, each sample was detected 2 times repeatedly, continuously detected for 20 days, and a total of 160 data results were collected for each batch of kits to calculate their precision. At the same time, the NF-L antigen after traceability was configured into 1000 pg / mL and 3000 pg / mL solutions, detected 3 times repeatedly, and data analysis was carried out. The detection results are shown in Table 10 below.

[0225] Table 10

[0226]

[0227] In summary, the repeated detection results of the above kits for low- and high-concentration samples show that the within-batch precision and between-batch precision CV are both less than 5%, indicating that the above kits meet the requirements of precision performance evaluation.

[0228] (3) Linear Range

[0229] One sample of cerebrospinal fluid supplemented with NF-L antigen was selected, and 12 concentration levels were diluted with diluent. Each diluted sample was tested 3 times, and the mean was calculated. According to the results, the concentration points were gradually reduced to calculate the corresponding linear relationship, and the widest linear range of the kit was determined. The test results are shown in Table 11 below (concentration units are pg / mL).

[0230] Table 11

[0231]

[0232]

[0233] The above Table 10 shows that the correlation coefficient r of the NF-L kit is not less than 0.9900 in the range of [1 to 5000] pg / mL and there is no outlier, and the linear range of the NF-L kit is [1 to 5000] pg / mL.

[0234] (4) Sensitivity

[0235] The blank sample was tested with the above kit and repeated 20 times to determine the sensitivity. The test results are shown in Table 12 below (concentration unit is pg / mL):

[0236] Table 12

[0237] Item Parameter Mean 424.45 SD 26.912 Mean + 2SD 478.274 Corresponding concentration value 0.73

[0238] The average luminescence value of the blank samples measured by the NF-L kit was 424.45. Substituting Mean+2SD into the reaction curve, the sensitivity of the NF-L kit was 0.73 pg / mL.

[0239] (5) Cerebrospinal fluid test results

[0240] 20 cerebrospinal fluid 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 = 1.0036x-21.662 and R 2 =0.926, which proves that this product has good consistency with the commercial product.

[0241] (6) Blood test results

[0242] 40 blood samples assigned by the imported high-sensitivity detection platform were tested to determine the consistency of the kit with the platform products. Figure 2 As shown (concentration unit is pg / mL), regression analysis data showed that y = 1.0227x + 0.7245 and R 2= 0.9505, demonstrating that the product has good consistency with the commercial product.

[0243] As can be seen from the above embodiments, the NF-L kit of the present application has good stability, high accuracy, high precision, high sensitivity, and short detection time. Through the coupling or coating process, labeling process, and optimization of the detection system in the kit preparation process, the present application can further improve the detection limit and linear range of the kit on the original basis, and further shorten the detection time. The linear range of the kit of the present application is [1 - 5000] pg / mL. The linear range of the kit is much wider than that of the prior art, and the upper detection limit of the kit is at least twice the upper detection limit of the prior art. At the same time, the kit of the present application also has good consistency with the commercial product.

[0244] The above is only the preferred embodiment of the present application, and it is not a limitation of the present application in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present application without departing from the technical solution content of the present application still belong to the protection scope of the technical solution of the present application.

Claims

1. A kit for detecting neurofilament light chain, wherein, The kit includes: a first antibody targeting neurofilament light chain conjugated with biotin, a second antibody targeting neurofilament light chain labeled with a labeling agent, and magnetic beads, wherein the magnetic beads are streptavidin 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 labeling agent, wherein the magnetic beads are tosyl-activated magnetic beads; wherein, the first antibody targeting neurofilament light chain comprises three heavy-chain complementarity-determining regions CDR-H1, CDR-H2, CDR-H3 and three light-chain complementarity-determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the amino acid sequence of the CDR-H1 is as shown in SEQ ID No:1; the amino acid sequence of the CDR-H2 is as shown in SEQ ID No:3; the amino acid sequence of the CDR-H3 is as shown in SEQ ID No:5; the amino acid sequence of the CDR-L1 is as shown in SEQ ID No:7, the amino acid sequence of the CDR-L2 is as shown in SEQ ID No:9; the amino acid sequence of the CDR-L3 is as shown in SEQ ID No:11; the second antibody targeting neurofilament light chain comprises three heavy-chain complementarity-determining regions CDR-H1, CDR-H2, CDR-H3 and three light-chain complementarity-determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the amino acid sequence of the CDR-H1 is as shown in SEQ ID No:2; the amino acid sequence of the CDR-H2 is as shown in SEQ ID No:4; the amino acid sequence of the CDR-H3 is as shown in SEQ ID No:6; the amino acid sequence of the CDR-L1 is as shown in SEQ ID No:8; the amino acid sequence of the CDR-L2 is as shown in SEQ ID No:10; the amino acid sequence of the CDR-L3 is as shown in SEQ ID No:

12.

2. The kit according to claim 1, wherein, The particle size of the magnetic beads is 1 - 5 μM.

3. The kit according to claim 1, wherein The particle size of the tosyl-activated magnetic beads is 1.5 - 3 μM.

4. The kit according to claim 1, wherein, The particle size of the streptavidin magnetic beads is 1.5 - 3 μM.

5. The kit according to claim 1, wherein The particle size of the streptavidin magnetic beads is 1.5 - 2 μM.

6. The kit according to any one of claims 1 to 5, wherein, The molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3 - 40).

7. The kit according to any one of claims 1 to 5, wherein The molar ratio of the first antibody targeting neurofilament light chain to biotin is 1:(3 - 9).

8. The kit according to any one of claims 1 to 5, wherein, The mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(100 - 5000).

9. The kit according to any one of claims 1 to 5, wherein, The mass ratio of the first antibody targeting neurofilament light chain to the magnetic beads is 1:(500 - 2000).

10. The kit according to any one of claims 1 to 5, wherein, The molar ratio of the second antibody targeting neurofilament light chain to the labeling agent is 1:(3 - 40).

11. The kit according to any one of claims 1 to 5, wherein, The molar ratio of the second antibody targeting neurofilament light chain to the labeling agent is 1:(3 - 9).

12. The kit according to any one of claims 1 to 5, wherein, The kit further includes a neurofilament light chain standard, a substrate, a washing solution, a magnetic bead diluent, a conjugated antibody diluent, a labeled antibody diluent, and a sample diluent; or, the kit further includes a neurofilament light chain standard, a substrate, a blocking solution, a coating antibody diluent, a labeled antibody diluent, and a sample diluent.

13. The kit according to any one of claims 1 to 5, wherein The first antibody targeting neurofilament light chain 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 is an amino acid sequence having 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, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15; The second antibody targeting neurofilament light chain 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: 14, or is an amino acid sequence having 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, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No:

16.

14. The kit according to any one of claims 1 to 5, wherein, It is used for diagnosing neurodegenerative diseases.

15. The kit according to claim 1, wherein, When detecting neurofilament light chain in a sample using the kit, the following steps are included: Reaction: Mix the sample to be tested, the first antibody targeting neurofilament light chain conjugated with biotin, and the second antibody targeting neurofilament light chain labeled with a label, incubate, add magnetic beads, and then incubate again; or, mix the sample to be tested, magnetic beads coated with the first antibody targeting neurofilament light chain, and the second antibody targeting neurofilament light chain labeled with a label, incubate, and then perform magnetic separation; Measure the luminescence value: After washing, add the substrate and measure the luminescence value of the sample to be tested; Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested using the luminescence value of the neurofilament light chain standard; Wherein, the magnetic beads are tosyl-activated magnetic beads.

16. The kit according to claim 1, wherein, When detecting neurofilament light chain in a sample using the kit, the following steps are included: Reaction: Mix the sample to be tested, magnetic beads coated with the first antibody targeting neurofilament light chain, and the second antibody targeting neurofilament light chain labeled with a label, incubate, and then perform magnetic separation; Measure the luminescence value: After washing, add the substrate and measure the luminescence value of the sample to be tested; Calculate the content: Calculate the content of neurofilament light chain in the sample to be tested using the luminescence value of the neurofilament light chain standard; Wherein, the magnetic beads are tosyl-activated magnetic beads.

17. The kit according to claim 15 or 16, wherein, The sample to be tested is serum or cerebrospinal fluid.

18. The kit according to claim 17, wherein The sample to be tested is at least 50 μL.

19. The kit according to claim 17, wherein, The sample to be tested is at least 100 μL.

20. The kit according to claim 15 or 16, wherein For each microliter of the sample to be tested, 0.1 - 0.5 ng of a first antibody targeting neurofilament light chain and 0.05 - 0.25 ng of a second antibody targeting neurofilament light chain are required.

21. Use of the kit according to any one of claims 1 - 20 in the preparation of a product for detecting neurofilament light chain and / or for diagnosing neurodegenerative diseases.

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