CN117106081B

CN117106081B - Anti-NFL protein capture antibody

Info

Publication number: CN117106081B
Application number: CN202311267371.1A
Authority: CN
Inventors: 马咏翔; 庞晓静; 吴俊�
Original assignee: Beijing Kaixiang Hongkang Biotechnology Co ltd
Current assignee: Beijing Kaixiang Hongkang Biotechnology Co ltd
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-05-07
Anticipated expiration: 2043-09-28
Also published as: CN117106081A

Abstract

The invention discloses a capture antibody of an anti-NFL protein. The capture antibody of the anti-NFL protein provided by the invention has stronger binding specificity to the NFL protein, has the value of developing a diagnostic kit for the NFL protein, has the potential value of developing a medicament for preventing or treating the related diseases of the NFL protein, and has wide application prospect.

Description

Anti-NFL protein capture antibody

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an anti-NFL protein capture antibody.

Background

Neurofilament (neurofilament, NF) is a protein specifically expressed in neurons, they are protein polymers that together with microtubules and microfilaments form the cytoskeleton of neurons. Nerve filaments are the main cytoskeletal component of nerve cells, and are important for keeping the caliber and shape of axons intact, and influence the rate and accuracy of nerve transmission. Neurofilament chains can be classified into neurofilament light chains (NFL), neurofilament medium chains (NFM), and neurofilament heavy chains (NFH) according to size. Among them, NFL is closely related to various neurological diseases. Therefore, the detection of the neurofilament protein antibody has important guiding significance for diagnosis, treatment and prognosis judgment of diseases related to the nervous system.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a capture antibody of an anti-NFL protein.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a capture antibody against NFL protein, the antibody comprising:

the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region have amino acid sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequences shown in SEQ ID NOS 1, 2,3, respectively,

The complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region have amino acid sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequences shown in SEQ ID NOS 9, 10, 11, respectively.

Furthermore, the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region have amino acid sequences shown in SEQ ID NO. 1,2 and 3, respectively,

The complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region have the amino acid sequences shown in SEQ ID NO. 9, 10 and 11, respectively.

Further, the antibody further comprises:

The framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region have amino acid sequences that have at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% sequence identity, respectively, to the amino acid sequences shown in SEQ ID NOS.4, 5, 6 and 7,

The framework regions FR1, FR2, FR3 and FR4 of the light chain variable region have amino acid sequences that have at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% sequence identity, respectively, to the amino acid sequences shown in SEQ ID NOS.12, 13, 14 and 15.

Further, the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region have the amino acid sequences shown in SEQ ID NO. 4,5, 6 and 7, respectively,

The framework regions FR1, FR2, FR3 and FR4 of the light chain variable region have the amino acid sequences shown in SEQ ID NOS 12, 13, 14 and 15, respectively.

Further, the amino acid sequence of the heavy chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 8,

The amino acid sequence of the light chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16.

Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

Further, the antibody is defucosylated.

Further, the CDR is defined according to Kabat, IMGT, chothia, abM or Contact numbering system.

Further, the CDRs are defined according to the Kabat numbering system.

Further, the heavy chain of the antibody is operably linked to a first signal peptide and the light chain of the antibody is operably linked to a second signal peptide.

Further, the amino acid sequence of the first signal peptide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 33, and the amino acid sequence of the second signal peptide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 34.

Further, the amino acid sequence of the first signal peptide is shown as SEQ ID NO. 33, and the amino acid sequence of the second signal peptide is shown as SEQ ID NO. 34.

In a second aspect the invention provides a polynucleotide encoding an antibody according to the first aspect of the invention.

Further, the nucleotide sequences of the polynucleotides encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region have at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequences shown in SEQ ID NOS 17, 18, 19, respectively,

The nucleotide sequences of the polynucleotides encoding the complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region have at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequences shown in SEQ ID NOS 25, 26, 27, respectively.

Further, the nucleotide sequences of the polynucleotides encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are shown in SEQ ID NOS 17, 18, 19, respectively,

The nucleotide sequences of the polynucleotides encoding the complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region are shown in SEQ ID NOS 25, 26, 27, respectively.

Further, the nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, FR4 of the heavy chain variable region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOS 20, 21, 22, 23, respectively,

The nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, FR4 of the light chain variable region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOS 28, 29, 30, 31, respectively.

Further, the nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are shown as SEQ ID NOs 20, 21, 22 and 23, respectively,

The nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NOS 28, 29, 30 and 31, respectively.

Further, the nucleotide sequence of the polynucleotide encoding the heavy chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence shown in SEQ ID NO. 24,

The nucleotide sequence of the polynucleotide encoding the light chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 32.

Further, the nucleotide sequence of the polynucleotide encoding the heavy chain variable region is shown as SEQ ID NO. 24, and the nucleotide sequence of the polynucleotide encoding the light chain variable region is shown as SEQ ID NO. 32.

Further, the heavy chain of the polynucleotide is linked to a polynucleotide encoding a first signal peptide and the light chain is linked to a polynucleotide encoding a second signal peptide.

Further, the nucleotide sequence of the polynucleotide encoding the first signal peptide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence shown in SEQ ID NO. 35,

The nucleotide sequence of the polynucleotide encoding the second signal peptide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 36.

Further, the nucleotide sequence of the polynucleotide encoding the first signal peptide is shown as SEQ ID NO. 35, and the nucleotide sequence of the polynucleotide encoding the second signal peptide is shown as SEQ ID NO. 36.

In a third aspect the invention provides an antibody conjugate, the antibody conjugate being the antibody of the first aspect of the invention conjugated to a label comprising one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a radiolabel.

In a fourth aspect, the invention provides a biological material comprising a polynucleotide according to the second aspect of the invention, the biological material comprising an expression cassette, vector or host cell.

Further, the vector comprises a viral vector, an expression vector and a recombinant expression vector.

Further, the host cells include prokaryotic cells, fungal cells.

The fifth aspect of the invention provides the use of any one of the antibodies of the first aspect of the invention, the polynucleotides of the second aspect of the invention, the antibody conjugates of the third aspect of the invention or the biological material of the fourth aspect of the invention, as follows:

(1) Use in detecting the presence or level of NFL protein in a sample;

(2) Use in the preparation of a product for detecting NFL protein;

(3) Use in the preparation of a pharmaceutical composition for the prevention and/or treatment of NFL protein related diseases.

Further, NFL protein related diseases include alzheimer's disease, parkinson's disease, motor neuron disease, demyelinating diseases, traumatic brain injury.

In a sixth aspect, the invention provides a method for detecting NFL protein in a sample, the method comprising contacting the sample with an antibody according to the first aspect of the invention, and detecting the level of NFL protein in the sample.

Further, the method is a method for non-diagnostic purposes.

In a seventh aspect, the invention provides a test kit comprising an antibody according to the first aspect of the invention.

An eighth aspect of the invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, an antibody conjugate according to the third aspect of the invention or a biomaterial according to the fourth aspect of the invention.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier, diluent or excipient.

The invention has the advantages and beneficial effects that:

the capture antibody of the anti-NFL protein provided by the invention has stronger binding specificity to the NFL protein, has the value of developing a diagnostic kit for the NFL protein, has the potential value of developing a medicament for preventing or treating the related diseases of the NFL protein, and has wide application prospect.

Drawings

FIG. 1 is a graph of Western results of monoclonal antibody cell culture supernatant recognizing NFL protein in samples;

FIG. 2 is a diagram of the specificity of binding of purified antibodies to antigens.

Detailed Description

The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention provides a capture antibody against NFL protein, the antibody comprising:

The complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region have amino acid sequences shown in SEQ ID NO. 1,2 and 3,

In the present invention, the term antibody refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (or domain) (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The variable regions of the heavy and light chains of antibodies contain binding domains that interact with the antigen. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q (the first component of the classical pathway of complement activation).

The heavy chain of an immunoglobulin can be divided into three functional regions: fd region, hinge region and Fc region (crystallizable fragment). The Fd region comprises VH and CH1 domains and binds to the light chain to form Fab (antigen binding fragment). The Fc fragment is responsible for immunoglobulin effector functions including, for example, complement fixation and binding to cognate Fc receptors of effector cells. The hinge region found in the IgG, igA and IgD immunoglobulin classes acts as a flexible spacer region, allowing the Fab portion to move freely in space relative to the Fc region. Hinge domains are structurally diverse, with sequence and length varying between immunoglobulin classes and subclasses.

The light chain variable region (VL) or heavy chain variable region (VH) consists of framework regions separated by three complementarity determining regions or CDRs. The framework regions are used to align CDRs that specifically bind to an epitope. CDRs include amino acid residues in antibodies that are primarily responsible for antigen binding. The VL domain and VH domain both comprise the following Framework (FR) and CDR regions from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDR1, CDR2, and CDR3 of the VL domain may also be referred to as VLCDR1, VLCDR2, and VLCDR3; CDR1, CDR2, and CDR3 of a VH domain may also be referred to as VHCDR1, VHCDR2, and VHCDR3.

The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include Kabat definitions (Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,MD,1987 and 1991), chothia definitions (Chothia and Lesk, j. Mol. Biol.196:901-917,1987; chothia et al, nature 342:878-883,1989); chothia Kabat CDR, wherein CDR-H1 is a complex of a Chothia CDR and a Kabat CDR; abM definition used by Oxford Molecular antibody modeling software; and Martin et al Contact definition (world wide web bioinfo. Org. Uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same number. The present invention may use CDRs defined according to any of these numbering systems, but preferred embodiments use Kabat defined CDRs.

The antibodies disclosed herein may be derived from any animal source, including birds and mammals. Preferably, the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody.

The antibody or antigen binding fragment thereof is defucosylated.

In the present invention, fucosylation refers to the presence of fucose residues within oligosaccharides attached to the peptide backbone of an antibody. Specifically, the fucosylated antibody comprises an alpha (l, 6) linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the Fc region of the antibody, e.g., position Asn297 of the human IgG1 Fc domain (EU numbering of the Fc region residues). Asn297 may also be located about +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in immunoglobulins.

Antibodies that are defucosylated or nonfucosylated or fucose deficient include glycosylated antibody variants of an Fc region in which the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, antibodies with reduced fucose or lacking fucose have improved ADCC function. The defucosylated or nonfucosylated or fucose deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in the cell line.

The present invention provides polynucleotides encoding the antibodies described above.

In the present invention, polynucleotide and nucleic acid are used interchangeably and include single-stranded and double-stranded nucleotide polymers. The polynucleotide or nucleic acid may be a ribonucleotide or deoxyribonucleotide or a modified form of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3' -dideoxyribose, internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenos, phosphorodiselenos, phosphorophenylthioates, phosphoroanilide, and phosphoramidates.

The present invention provides a biological material comprising the polynucleotide described above, the biological material comprising an expression cassette, a vector or a host cell.

In the present invention, any carrier may be suitable for use in the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector comprises a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR、pBABE-puro、pBABE-neo largeTcDNA、pBABE-hygro-hTERT、pMKO.1GFP、MSCV-IRES-GFP、pMSCV PIG(Puro IRES GFP empty plasmids), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, ttRMPVIR, pMSCV-IRES-MCHERRY FP, pRetroX GFP T A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The expression vector may be any suitable recombinant expression vector. Suitable vectors include vectors designed for proliferation and amplification or for expression or both. For example, the vector may be selected from the group consisting of pUC series (FERMENTAS LIFE SCIENCES, glen Burnie, md.), pBluescript series (Stratagene, laJolla, calif.), pET series (Novagen, madison, wis.), pGEX series (PHARMACIA BIOTECH, uppsala, sweden) and pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, zapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present invention include pcDNA, pEUK-Cl, pMAM and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al ,Molecular Cloning:A Laboratory Manual,3rd ed.,Cold Spring Harbor Press,Cold Spring Harbor,N.Y.2001; and Ausubel et al ,Current Protocols in Molecular Biology,Greene Publishing Associates and John Wiley&Sons,NY,1994. Circular or linear expression vector constructs can be prepared to contain the replication system functions in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, COLEL, 2 μ plasmids, λ, SV40, bovine papilloma virus, and the like.

In the present invention, the host cell may be a prokaryotic cell, such as e.coli, bacillus subtilis (Bacillus subtilis), streptomyces sp, pseudomonas sp, proteus mirabilis Proteus mirabilis, or Staphylococcus sp. Also, the host cell may be a fungal cell, such as aspergillus (aspergillus sp.), yeast cells such as saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces pombe (Schizosaccharomyces sp.) and neurospora crassa (Neurospora crassa), lower eukaryotic cells and higher eukaryotic cells such as insect cells. Also, the host cell may be from a plant and/or mammal. Preferred examples of host cells include, but are not limited to, PER.C6 cells, monkey kidney cells 7 (COS 7, particularly simian COS cells), NSO cells, SP2/0, chinese Hamster Ovary (CHO) cells, W138, baby Hamster Kidney (BHK) cells, madin-Darby canine kidney (MDCK) cells, myeloma cell lines, huT78 cells, 293T cells, 293F cells, and other mammalian host cells that produce antibody proteins according to the invention.

The present invention provides a pharmaceutical composition comprising the above antibody, the above polynucleotide, the above antibody conjugate, or the above biological material.

In the present invention, the pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. For example, the pharmaceutical composition may be lyophilized, such as by freeze-drying, spray-cooling, or by using particles from supercritical particle formation.

The pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier, diluent or excipient.

In the present invention, the term pharmaceutically acceptable is intended to mean a non-toxic material that does not reduce the effectiveness of the binding activity of the antibodies or antigen binding fragments of the invention. Pharmaceutically acceptable buffers, carriers, diluents or excipients are for example well known in the art.

Wherein the buffer means an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Examples of buffers are Trizma, bicine, tricine, MOPS, MOPSO, MOBS, tris, hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, dimethylarsinate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

Diluents means aqueous or non-aqueous solutions with the aim of diluting the antibody or antigen binding fragment in the pharmaceutical formulation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or an oil such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil.

An adjuvant means any compound that is added to a formulation to increase the biological effect of an antibody or antigen binding fragment of the invention. The adjuvant may be one or more salts with zinc, copper or silver salts of different anions such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetate of different acyl compositions. Adjuvants may also be cationic polymers (such as cationic cellulose ethers, cationic cellulose esters), deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers (such as poly (vinylimidazole)), and cationic polypeptides (such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids).

The excipient may be one or more of a carbohydrate, a polymer, a lipid, and a mineral. Examples of carbohydrates include lactose, glucose, sucrose, mannitol and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate of varying degrees of hydrolysis, and polyvinylpyrrolidone, all of which have different molecular weights, which are added to the composition, for example, for viscosity control, for achieving bioadhesion, or for protecting lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids (all of which have different acyl chain lengths and saturations), egg lecithin, soy lecithin, hydrogenated egg lecithin and soy lecithin (which are added to the composition for reasons similar to polymers). Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or advantageous pigment properties.

The invention provides application of the antibody, the polynucleotide, the antibody conjugate or the biological material in preparing a pharmaceutical composition for preventing and/or treating NFL protein related diseases.

In the present invention, NFL protein related diseases include, but are not limited to, alzheimer's disease, parkinson's disease, motor neuron diseases (e.g., amyotrophic lateral sclerosis), demyelinating diseases (e.g., multiple sclerosis), traumatic brain injury (e.g., diffuse axonal injury).

The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.

Examples

1. Immunogen treatment: the immunogen is recombinant NFL protein, the purity and molecular weight of the protein are identified by SDS-PAGE, and the immunogenicity is enhanced by ImmunoPlus technology treatment.

2. Animal immunization: BALB/c mice were selected and immunized by conventional methods. After three immunizations, the antiserum titers were tested by indirect ELISA, and the mice with high titers were selected for subsequent experiments, and the antiserum was tested for recognition of recombinant antigens using Western.

3. Spleen cell preparation: the mice were sacrificed by cervical guide, spleens were removed under aseptic conditions and placed in sterilized 90-100 mesh stainless steel mesh. 3ml of serum-free culture medium was injected into the spleen by syringe, and the cells were obtained by repeated aspiration several times, and then cell suspension was prepared. The cell suspension is poured into a 50ml centrifuge tube, 10-20 ml of culture solution is added, gently beaten for several times, and the mixture is kept stand for 5 minutes at room temperature. Centrifuging (800-1000 rpm) and counting for standby.

4. Cell fusion: mouse myeloma cells and mouse spleen cells were prepared at a ratio of 1: mix at 5 ratio, centrifuge discard supernatant and blot excess supernatant with sterile filter paper. 1ml of 40% PEG solution was added dropwise to the cell pellet over 60 seconds while the centrifuge tube was constantly gently turned. In a continuously rotating centrifuge tube, 1ml of serum-free medium was added dropwise over 60 seconds. Then 20ml of serum free medium was slowly added over 5 minutes. Centrifugation (800 rpm, 8 min), removal of supernatant, suspension with 10ml of complete medium, and gentle mixing. The cell suspension was added to a 96-well plate at 50 microliters per well. After culturing in a CO ₂ incubator at 37 ℃ for 24 hours, the culture medium is replaced by HAT selective culture medium.

5. Cell culture after fusion: half-quantity liquid exchange is carried out on HAT culture solution 7-10 days after fusion, and then half-quantity liquid exchange is carried out every 2-3 days. Hybrid cell colonies appeared after 2-3 weeks. When the colony grows to 1/3 hole, the indirect ELISA method is applied to carry out affinity test on the monoclonal antibody in the culture supernatant of the mouse hybridoma. The NFL recombinant protein and the HIS tag protein are used as antigen coating ELISA plates, and the concentration of the coating antigen is 1 mu g/ml and 100 mu l/hole. The coating buffer was PBS (ph=7.4). The mixture was left overnight at 4 ℃. The next day the PBS was washed 3 times for 5 minutes each. Blocking with 1% BSA was performed with 200. Mu.l of each well. Incubate at 37℃for 2 hours. BSA was discarded and 100 μl of cell culture supernatant containing monoclonal antibody was added per well. Positive antisera from mice were used as positive control and blank culture supernatant as negative control. Incubate at 37℃for 2 hours. The primary antibody was discarded, washed 5 times with a solution of Peroxydase-AffiniPure Goat Anti-Mouse IgG and incubated for 1 hour at 37 ℃. After the substrate is added for color development, the absorbance value is measured by an enzyme-labeled instrument.

The results show that the absorbance of the antibody against the NFL recombinant protein is greater than 2.7 and is far greater than the negative control value of 0.067, and the absorbance of the antibody against the HIS tag protein is equivalent to the negative control value, which indicates that the antibody has good affinity for the NFL recombinant protein antigen (Table 2), and the sequence of the antibody is shown in Table 1.

TABLE 1 3D1F9 antibody sequences

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Table 2 monoclonal antibody cell culture supernatant recognizes recombinant NFL proteins

6. Human neurofilament light chain protein in clinical samples is used as an antigen, and a mouse hybridoma cell culture supernatant containing monoclonal antibodies is used for detection. At the target site, a strong positive band appears, indicating that the antibodies have strong binding to the antigen in the clinical sample (fig. 1).

7. The ELISA plate was coated with 2.5. Mu.g/ml of purified antibody using a double antibody sandwich ELISA method, with PBS (pH=7.4) as the coating solution, and left overnight at 4 ℃. Washing the washing solution for 3 times, adding recombinant protein as antigen, wherein the antigen concentration is 0, 1, 10 and 100ng/ml respectively. Incubate at 37℃for 1 hour. Washing 3 times, adding biotin-labeled detection antibody at a concentration of 1. Mu.g/ml. Incubate at 37℃for 1 hour. Wash 3 times, add HRP-labeled streptavidin, bind to detection antibody, dilute at 1mg/ml,1:10,000, add 100 μl per well. Incubate at 37℃for 30 min. And adding a substrate for color development, and measuring the absorbance value by using an enzyme-labeled instrument.

The results showed that antibody 3D1F9 successfully paired with antibody 18a12C11, with an increase in absorbance with increasing antibody content. At an antigen concentration of 100ng/ml, the absorbance was >2.96, significantly higher than the negative control. This antibody demonstrated strong recognition and capture of antigen (Table 3).

TABLE 3 recognition of antigen by purified antibodies

8. Antibody 3D1F9 was paired with antibody 18a12C11 and recombinant proteins were detected. The antigen concentration was subjected to double dilution and plotted against the absorbance. FIG. 2 shows that as the antigen concentration increases, the absorbance increases, indicating that the antibody binds specifically to the antigen.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. A capture antibody against NFL protein, the antibody comprising:

Complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region having amino acid sequences as shown in SEQ ID NOS 1, 2, 3, respectively, and

2. The antibody of claim 1, wherein the antibody further comprises:

The framework regions FR1, FR2, FR3, FR4 of the heavy chain variable region have an amino acid sequence that has at least 90% sequence identity to the amino acid sequences shown in SEQ ID NOs 4,5, 6, 7, respectively, and

The framework regions FR1, FR2, FR3 and FR4 of the light chain variable region have amino acid sequences that have at least 90% sequence identity to the amino acid sequences shown in SEQ ID NOS 12, 13, 14 and 15, respectively.

3. The antibody according to claim 2, wherein the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region have the amino acid sequences shown in SEQ ID NO. 4, 5, 6 and 7, respectively,

4. The antibody of claim 2, wherein the amino acid sequence of the heavy chain variable region has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8,

The amino acid sequence of the light chain variable region has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO. 16.

5. The antibody of claim 4, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16.

6. The antibody of claim 1, wherein the antibody is defucosylated.

7. The antibody of claim 1, wherein the CDRs are defined according to Kabat, IMGT, chothia, abM or Contact numbering system.

8. The antibody of claim 7, wherein the CDRs are defined according to the Kabat numbering system.

9. The antibody of claim 8, wherein the heavy chain of the antibody is operably linked to a first signal peptide and the light chain of the antibody is operably linked to a second signal peptide.

10. The antibody of claim 9, wherein the amino acid sequence of the first signal peptide has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID No. 33 and the amino acid sequence of the second signal peptide has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID No. 34.

11. The antibody of claim 10, wherein the amino acid sequence of the first signal peptide is shown in SEQ ID No. 33 and the amino acid sequence of the second signal peptide is shown in SEQ ID No. 34.

12. A polynucleotide encoding the antibody of any one of claims 1-11.

13. The polynucleotide according to claim 12, wherein the nucleotide sequences of the polynucleotides encoding complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region have at least 95% sequence identity to the nucleotide sequences shown in SEQ ID NOS 17, 18, 19, respectively,

The nucleotide sequences of the polynucleotides encoding the complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region have at least 95% sequence identity to the nucleotide sequences shown in SEQ ID NOS 25, 26, 27, respectively.

14. The polynucleotide according to claim 13, wherein the nucleotide sequences of the polynucleotides encoding complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are shown in SEQ ID NOS 17, 18, 19, respectively,

15. The polynucleotide according to claim 12, wherein the nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, FR4 of the heavy chain variable region have at least 90% sequence identity to the nucleotide sequences shown in SEQ ID NOS 20, 21, 22, 23, respectively,

The nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, FR4 of the light chain variable region have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOS 28, 29, 30, 31, respectively.

16. The polynucleotide according to claim 15, wherein the nucleotide sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are shown in SEQ ID NOS 20, 21, 22 and 23, respectively,

17. The polynucleotide according to claim 15, wherein the nucleotide sequence of the polynucleotide encoding the heavy chain variable region has at least 70% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 24,

The nucleotide sequence of the polynucleotide encoding the light chain variable region has at least 70% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 32.

18. The polynucleotide according to claim 17, wherein the polynucleotide encoding the heavy chain variable region has a nucleotide sequence shown in SEQ ID No. 24 and the polynucleotide encoding the light chain variable region has a nucleotide sequence shown in SEQ ID No. 32.

19. The polynucleotide of claim 12, wherein the heavy chain of the polynucleotide is linked to a polynucleotide encoding a first signal peptide and the light chain is linked to a polynucleotide encoding a second signal peptide.

20. The polynucleotide of claim 19, wherein the nucleotide sequence of the polynucleotide encoding the first signal peptide has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 35,

The nucleotide sequence of the polynucleotide encoding the second signal peptide has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 36.

21. The polynucleotide according to claim 20, wherein the polynucleotide encoding the first signal peptide has a nucleotide sequence shown in SEQ ID NO. 35 and the polynucleotide encoding the second signal peptide has a nucleotide sequence shown in SEQ ID NO. 36.

22. An antibody conjugate, wherein the antibody conjugate is obtained by coupling the antibody of any one of claims 1-11 with a label comprising one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

23. A biological material comprising the polynucleotide of any one of claims 12-21, the biological material comprising an expression cassette, vector or host cell.

24. The biomaterial of claim 23, wherein the vector comprises a viral vector, an expression vector.

25. The biomaterial of claim 23, wherein the host cell comprises a prokaryotic cell or a fungal cell.

26. The antibody of any one of claims 1-11, the polynucleotide of any one of claims 12-21, the antibody conjugate of claim 22, or the biological material of any one of claims 23-25 for use as follows:

(1) Use in detecting the presence or level of NFL protein in a sample for non-diagnostic purposes;

(2) Use in the preparation of a product for detecting NFL protein.

27. A method for detecting NFL protein in a sample for non-diagnostic purposes, comprising contacting the sample with the antibody of any one of claims 1-11, and detecting the level of NFL protein in the sample.

28. A test kit comprising the antibody of any one of claims 1-11.

29. A pharmaceutical composition comprising the antibody of any one of claims 1-11, the polynucleotide of any one of claims 12-21, the antibody conjugate of claim 22, or the biological material of claims 23-25.

30. The pharmaceutical composition of claim 29, further comprising a pharmaceutically acceptable buffer or excipient.