ATG16L1 monoclonal antibody and its use

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an ATG16L1 monoclonal antibody and its use. The present invention discloses an antibody or antigen-binding fragment thereof that specifically binds to ATG16L1, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 3, 4, and 5, respectively, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 6, 7, and 8, respectively. The antibodies disclosed in the present invention have high sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an ATG16L1 monoclonal antibody and uses thereof. Background Art

[0002] Key proteins in the autophagy signaling pathway have become new candidate targets for disease intervention and tumor therapy, attracting increasing attention. However, independently developed monoclonal antibodies are lacking, and the only commercially available products are rabbit-derived polyclonal antibodies. Many antigens used to immunize mice are native proteins. Because natural proteins are modified and structurally complex (containing both linear and conformational epitopes), they are high-quality antigens. However, obtaining high-purity natural proteins is often difficult. Most secondary antibodies are screened using natural proteins as antigens.

[0003] Chemically synthesized peptide antigens are small molecules that lack strong antigenicity on their own and can only induce a weak immune response in animals. Therefore, crosslinking with a carrier protein is crucial. Carrier proteins contain numerous antigenic determinants, capable of stimulating T-helper cells and, in turn, inducing B-cell responses. A variety of carrier proteins are used for peptide crosslinking, with keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), and bovine thyroglobulin (THY) being the most commonly used carriers. KLH, with its higher antigenicity, is a popular carrier for peptide crosslinking. BSA is also commonly used as a peptide carrier, but its frequent use as a blocking agent in detection assays limits the application of antibodies produced using this method.

[0004] A synthetic peptide is just a linear sequence, and it's uncertain whether an antibody against it will recognize the natural protein. Therefore, peptide design is crucial before antibody preparation. When protein antigens are difficult to obtain, or when homologous proteins exist but with only minor sequence differences, or when preparing modified antibodies at specific sites, peptide antigens can be selected that target proteins with high homology to family members. Specific peptides can then be used to prepare antibodies that distinguish between family members. Summary of the Invention

[0005] The primary purpose of the present invention is to provide an ATG16L1 monoclonal antibody and its use. By designing an ATG16L1 polypeptide sequence, mice can be immunized with the artificially synthesized polypeptide to screen for the desired specific antibody.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ATG16L1, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 3, 4 and 5, respectively, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 6, 7 and 8, respectively.

[0008] As a preferred embodiment of the present invention, the VL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, and the VH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2.

[0009] As a preferred embodiment of the present invention, its light chain constant region CL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9, and the heavy chain constant region CH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10.

[0010] As a preferred embodiment of the present invention, the antigen-binding fragment is selected from a double-chain antibody; the antibody is a murine monoclonal antibody, the light chain of which comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and the heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 12.

[0011] The second aspect of the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to ATG16L1 as described above.

[0012] The third aspect of the present invention provides a vector comprising the nucleic acid as described above.

[0013] The fourth aspect of the present invention provides a host cell comprising the nucleic acid or vector as described above.

[0014] As a preferred embodiment of the present invention, the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

[0015] The fifth aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, comprising: culturing the above-mentioned host cell under conditions that allow the expression of the antibody or antigen-binding fragment thereof.

[0016] The sixth aspect of the present invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 as described above,

[0017] The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes ATG16L1.

[0018] A seventh aspect of the present invention provides a use of the aforementioned antibody or antigen-binding fragment thereof that specifically binds to ATG16L1, or a detection reagent, in the preparation of a product for detecting the content of ATG16L1 in a sample.

[0019] An eighth aspect of the present invention provides a use of the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector, and host cell that specifically binds to ATG16L1 in the preparation of a product for detecting ATG16L1.

[0020] By means of the above technical solution, the present invention has at least the following advantages:

[0021] The present invention provides a monoclonal antibody against ATG16L1 that avoids the high background caused by HAMA and FCR reactions in natural samples. The antibody or antigen-binding fragment thereof provided by the present invention has high affinity and high specificity. This invention addresses the current lack of available antibody raw materials for ATG16L1 detection in the market. The disclosed antibody has high sensitivity and specificity.

[0022] When screening antibodies, the present invention selected multiple natural cell antigens and compared antibody specificity. After multiple rounds of screening, antibodies with good specificity for ATG16L1 were obtained. During the animal immunization process, multiple subcutaneous injections at multiple sites were used, which increased the titer of the final animal serum and the positive clone rate.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the amino acid antigenicity prediction map;

[0025] Figure 2 This is a WB image of the antibody that specifically binds to ATG16L1 obtained by screening according to the present invention;

[0026] Figure 3 This is a sensitivity detection diagram of the antibody that specifically binds to ATG16L1 obtained by screening in the present invention. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0029] Unless otherwise specified, percentage concentrations referred to in the present invention refer to final concentrations, which refer to the percentage of an added component in the system after the addition of the component.

[0030] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically comprise a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains comprise a binding domain that interacts with the antigen. The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

[0034] As used herein, the term "antibody analog" refers to derivatives produced by biological or chemical methods, derived from the antibody structure by deletion, addition, or modification of chemical groups (e.g., amino acids). These derivatives still contain structures similar to those of the antibody variable region (or CDR region within the antibody variable region) and are capable of undergoing reactions similar to antigen-antibody binding through these structures.

[0035] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. In certain embodiments, an antibody that specifically binds to an antigen refers to an antibody that binds to the antigen with an affinity corresponding to a KD of less than about 10-5M, for example, less than about 10-6M, 10-7M, 10-8M, 10-9M or 10-10M or less. As used herein, "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the KD, the higher the binding affinity between the antibody and the antigen.

[0036] As used herein, the term "antibody variable region" refers to a domain within an antibody's heavy and light chains. It includes the light chain variable region (VL) and the heavy chain variable region (VH). In nature, the variable region is encoded by the V, D (for the heavy chain only), and J segments of immunoglobulin (heavy and light chain) genes, spliced ​​together through genetic recombination. The amino acid sequences of the variable region vary significantly between different antibodies (while the amino acid sequences of other regions of the antibody are relatively highly identical) and are responsible for recognizing and binding to specific antigenic determinants. Within the antibody variable region, both the VL and VH domains, from the amino-terminus to the carboxyl-terminus, comprise framework regions (FRs) and CDRs (comlementarity determining regions). A typical antibody variable region has three framework regions and three CDRs, arranged interspersed with each other: FR1, CDR1, FR2, CDR2, FR3, and CDR3. The framework regions (FRs) primarily serve to form the protein domain framework, while the CDRs primarily contribute to specific antigen-antibody recognition and binding. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0037] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410). et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0038] Without substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of the present invention to obtain sequence variants of the antibody or its functional fragment. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties may be substituted in the variable region. The variant sequences of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software, for example, using the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0039] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid residue that is biologically, chemically, or structurally similar. Biologically similar means that the replacement does not destroy the biological activity of the ATG16L1 antibody or the ATG16L1 antigen. Structurally similar means that the amino acids have side chains of similar lengths, such as alanine, glycine, or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine, or methionine are substituted for each other. Or polar amino acids are used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, and so on.

[0040] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide for use in the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0041] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be divided into lambda (λ) chain and kappa (κ) chain. The antibodies disclosed herein can be any of the above classes or subtypes.

[0042] As used herein, the term "antigen-binding fragment" also refers to "antibody fragment." Antibody fragments generally refer to antigen-binding antibody fragments that can include a portion of an intact antibody, generally the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementarity-determining region, single-chain antibody, double-chain antibody, or single-domain antibody molecules. A "Fab fragment" consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain of the Fab fragment; F(ab')2 fragments, which are bivalent fragments comprising two Fab' fragments linked by a disulfide bond at the hinge region; Fd fragments having VH and CH1 domains; Fv fragments having VL and VH domains in a single arm of an antibody; dAb fragments consisting of either a VH domain or a VL domain; isolated CDR regions; and modified forms of any of the above fragments that retain antigen-binding activity.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, except for a small number of naturally occurring mutations, each antibody comprising the population is identical. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.

[0044] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. Another type of vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of the gene operably connected thereto. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. In some embodiments, the vector includes but is not limited to: (1) a plasmid; (2) a phagemid; (3) a cosmid; (4) an artificial chromosome, such as a yeast artificial chromosome, a bacterial artificial chromosome, or an artificial chromosome derived from P1; (5) a bacteriophage, such as lambda phage or M13 phage; (6) an animal virus, such as a retrovirus, adenovirus, adeno-associated virus, sporovirus, poxvirus, or baculovirus.

[0045] When the nucleic acid disclosed in the present invention is connected to a vector, the nucleic acid can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the polynucleotide and the control elements can be operably connected. Herein, "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected function of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the heavy and light chains of the antibody can be independently inserted into different vectors, and it is common to insert them into the same vector.

[0046] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into a host cell to construct a recombinant cell, which is then used to express the antibody or antigen-binding fragment provided by the present invention. By culturing the recombinant cell, the corresponding antibody can be obtained. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293F.

[0047] As used herein, the term "hybridoma" refers to cells formed by the fusion of myeloma cells and B lymphocytes during the production of monoclonal antibodies. In the classic monoclonal antibody production method, a suitable antigen must be obtained and used to immunize an animal. To produce anti-ATG16L1 antibodies (or antibody analogs), the appropriate antigen must contain ATG16L1. This antigen can be isolated and purified from natural human tissue or blood, or expressed and purified using prokaryotic or eukaryotic cells through artificial recombinant protein expression. The antigen is used for animal immunization and antibody screening and detection.

[0048] The classic monoclonal antibody production method first requires a suitable antigen to be prepared and used to immunize animals. To generate anti-ATG16L1 antibodies (or antibody analogs), a suitable antigen must contain ATG16L1. This antigen can be obtained by purification from natural human tissue or blood, chemical synthesis, or a combination of these methods. The antigen peptide is then conjugated to the carrier proteins KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) to prepare the complete antigen for animal immunization and antibody screening.

[0049] In the classic monoclonal antibody production method, animals are first immunized with a KLH / BSA-conjugated peptide antigen. Blood samples are collected at intervals to verify whether the animals have produced an antibody response to the KLH / BSA-conjugated peptide antigen. B cells are then isolated from the spleens of animals that have produced an antibody response and fused in vitro with immortalized myeloma cells to produce hybridoma cells. These hybridoma cells are then diluted to a minimum in culture plates and regrown (monoclonal hybridoma cell lines). The culture supernatant of these hybridoma cell lines is then collected and tested for the presence of specific antibodies against the antigen. Based on antibody yield, quality, and cell line growth characteristics, the optimal monoclonal antibody-producing cell line can be selected for subsequent monoclonal antibody production.

[0050] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the aforementioned animals can be isolated and incubated with labeled antigen. Since antibody-producing B cells typically display antibody molecules on their cell membranes, these cells will be bound by the labeled antigen (stained), allowing them to be sorted using a fluorescent flow cytometer. The mRNA from these sorted B cells can be isolated, and a library of antibody variable region cDNAs can be generated through in vitro reverse transcription and specific PCR reactions. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for mammalian cell expression or a phage expression plasmid suitable for bacterial cell expression) and expressed in host cells compatible with the expression plasmid. These host cells (or phage) can be isolated and purified (cloned) using various methods (such as the limiting dilution cell culture method or phage plaque plating method described above). These cell lines or phage clones can then be used to produce antibodies and analyze and characterize the antibodies.

[0051] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0052] Example 1: ATG16L1 polypeptide design and coupling

[0053] First, based on the ATG16L1 sequence published on NCBI, the human amino acid sequence was downloaded. The amino acid antigenicity of ATG16L1 was predicted using DNASTAR7.0 software, and the antigenic epitope region of the ATG16L1 structural protein was analyzed, thereby expressing it in stages. Figure 1 , the following three short peptide sequences that specifically bind to ATG16L1 were screened out, and their amino acid sequences are as follows:

[0054] Sequence 1: NQMQRKDREMQMNE;

[0055] Sequence 2: LQKELAEAAKEPLP;

[0056] Sequence 3: ALQITFTALEGKLR;

[0057] The above three peptides were synthesized by a third-party company using a peptide synthesizer, and the purity was greater than 95% after HPLC detection.

[0058] The immune antigens synthesized and coupled with KLH were named KLH-1, KLH-2, and KLH-3, and the specific synthesis process was completed by Wuhan Chentai Biotechnology Co., Ltd.

[0059] Example 2: Immunization of BALB / c mice with three coupled polypeptides

[0060] Fifteen healthy, active 6-8 week-old BALB / C female mice were selected and numbered. Five mice were immunized with each conjugated peptide using the following immunization protocol: 100 μg of antigen was administered to each mouse. The total volume was calculated based on the number of mice. The antigen was mixed with an equal volume of Freund's adjuvant and emulsified for 15 minutes to prepare an emulsion. For the first immunization, 0.2 ml of the emulsion (100 μg antigen) was injected subcutaneously into the back of each mouse using a 1 ml syringe. The injection time and number of subcutaneous injection sites were recorded. Fourteen days after the first immunization, a second immunization was performed with a 1 ml syringe subcutaneously into the back of each mouse using a 1 ml syringe. The injection time and number of subcutaneous injection sites were recorded. Fourteen days after the second immunization, a third immunization was performed with a 1 ml syringe subcutaneously into the back of each mouse using a 1 ml syringe. The injection time and number of subcutaneous injection sites were recorded. Seven days (± 1 day) after the third immunization, submandibular blood was collected from mice and serum was isolated for ELISA analysis. The specific procedure was as follows: 1× coating buffer (0.05 M carbonate buffer) was prepared. BSA-conjugated antigen was diluted to 1 μg / ml using the coating buffer (the antigen concentration did not affect the assay and could be converted to unit concentration). 100 μl of the solution was added to each well of the ELISA plate, which was sealed and incubated at 4°C overnight. The sample was discarded from the plate and the coating buffer was tapped off on paper (to prevent cross-contamination). The plate was washed three times with PBST (pH 7.4, 0.05% TWEN20) for 3 minutes each, tapping the buffer dry for the final time. Blocking buffer was added, and 200 μl of the solution was added to each well. The plate was incubated at 37°C for 1.5 hours. The blocking buffer was discarded and the plate was washed three times with PBST for 3 minutes each, tapping the buffer dry for the final time. If the primary antibody incubation was not immediately initiated, the plate was incubated at 4°C until use.

[0061] After diluting the serum at 1:1000 with antibody diluent PBST, add 150ul of diluted serum to the first well of the ELISA plate, and then add 100ul of PBST diluent to each well. Pipette 50ul from the first well to the second well and mix, then aspirate 50ul from the second well to the third well and mix, and so on for serial dilution until the seventh well. After mixing the seventh well, discard 50ul and keep the eighth well as the negative control of antibody dilution. Seal the coated plate and incubate it in a 37°C incubator for 1.5h; discard the primary antibody in the ELISA plate, wash the plate 3 times with PBST, 3 minutes each time, and pat dry the buffer for the last time. If the secondary antibody is not incubated in time, place it at 4°C until needed; dilute HRP-labeled goat anti-mouse IgG (H+L) with antibody diluent PBST at 1:10,000 (select the dilution multiple based on the titer provided by the manufacturer), then add 100 μl / well to the ELISA plate and incubate in a 37°C incubator for 1 hour; discard the secondary antibody from the ELISA plate, wash the plate three times with PBST for 3 minutes each, and tap dry the buffer the last time; add 100 μl of TMB substrate colorimetric solution to each well and incubate in a 37°C incubator for 4 minutes; add 50 μl of stop solution to each well to terminate the color development reaction; read the OD450nm measurement value with a microplate reader and record the data.

[0062] Elisa results are shown in Tables 1-3.

[0063] Table 1 KLH-1 polypeptide titer detection in mouse serum

[0064] Table 2 KLH-2 polypeptide titer detection in mouse serum

[0065]

[0066] Table 3 KLH-3 polypeptide titer detection in mouse serum

[0067] The test results for three typical immunized mouse sera are shown in Tables 1 through 3. As shown in Tables 1 through 3, the titers of sera from mice immunized with the three peptides (KLH-1, KLH-2, and KLH-3) were 1:81,000, 1:9,000, and 1:9,000, respectively. Therefore, the highest titer was observed in sera from mice immunized with KLH-1. Because the test antigen was a BSA-coupled peptide, the presence of antibodies in the mice that recognized KLH in the immunogen could be ruled out. The antibodies in the serum determined by the analytical experiments specifically recognized ATG16L1.

[0068] Example 3: Cell fusion

[0069] It should be noted that the antibody titer should be tested one week after the third immunization, and the two mice with the highest titer (and the titer is at least 80,000) should be selected as the mice for later fusion. If the titer of the mouse is below 80,000, the fourth immunization (boosting immunization) should be continued until the expected result is achieved.

[0070] Preparation of mouse myeloma cells: One week before fusion, SP2 / 0 cells stored in liquid nitrogen were revived and cultured in 25 cm 2 Cell culture flask. Subculture for one week in DMEM containing 15% fetal bovine serum. Select SP2 / 0 cells in the logarithmic growth phase, discard the culture medium, and wash twice with PBS. Add an appropriate amount of serum-free DMEM culture medium, gently resuspend the cells, transfer to a 50ml centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in serum-free culture medium, count, and adjust to 2×10 7 / ml for future use.

[0071] Preparation of feeder cells: Balb / c mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, and then placed in a clean bench and fixed on the dissecting board with the abdomen facing up; use ophthalmic forceps to pick up the mouse's abdominal skin and use scissors to make a small cut. Be careful not to cut the peritoneum to prevent the outflow of peritoneal fluid. Then use scissors to perform blunt separation on the upper and lower sides to fully expose the peritoneum. Use alcohol cotton balls to wipe the peritoneum for disinfection; use a syringe to draw 5ml of DMEM basal culture medium and inject it into the mouse's peritoneal cavity. Remove the syringe and use tweezers to gently press the mouse's abdomen several times to cut the peritoneum. Use a pipette to draw peritoneal cells into a centrifuge tube for use, and draw 5ml of culture medium again. This step can be repeated multiple times. 3 to 5×10 cells can be obtained from the peritoneal fluid of 1 mouse. 6 cells.

[0072] Preparation of splenocytes: Select one mouse with the best immune response, draw blood from its orbital cavity, and sacrifice by dislocation. Disinfect the mouse by soaking it in 75% alcohol for 5 minutes. Collect blood before sacrifice, which will serve as the positive serum. After disinfection, secure the mouse in a laminar flow hood. Remove the spleen, and use scissors to remove any connective and adipose tissue adhering to the splenocytes. Rinse the spleen with serum-free DMEM and transfer it to a 40 μm cell sieve (previously placed in a 9 cm sterile dish and filled with serum-free DMEM). Crush the spleen with a syringe plunger and gently rinse the sieve with serum-free DMEM. Repeated grinding and rinsing to prepare a splenocyte suspension is then transferred to a 50 ml centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash the pellet twice with serum-free DMEM. Count the pellet and count it for later use.

[0073] Cell fusion: Mix SP2 / 0 cells and spleen cells at a ratio of 1:5 in a 50ml centrifuge tube and centrifuge at 1000rpm for 5 minutes. Discard the supernatant and gently tap the bottom of the centrifuge tube to loosen the cell pellet. Slowly add 1ml of 50% PEG solution pre-warmed to 37℃ over 1 minute, starting with a slow drip and then a fast drip. During the addition process, shake the centrifuge tube continuously to thoroughly mix the cells and PEG solution. Let it stand for 1 minute. Then, add 30ml of 37℃ pre-warmed PEG solution to the cell mixture. Stop the reaction with DMEM medium, adding 1 ml dropwise at first, then more quickly, over 1 minute. Add a little more quickly, still adding a drop and stirring thoroughly, then slowly add the remaining medium. Tighten the cap and gently place in the incubator for 10 minutes. Remove the cells and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant and aspirate as much of the fusion agent as possible (be careful not to shake violently). Gently flick the cells to mix well and slowly add a small amount of DMEM medium (2 ml). Add the prepared complete medium containing feeder cells, HAT (1x final concentration), serum (15% final concentration), and double-antibody (1x final concentration). Resuspend the cells, gently mix, and add to five 96-well cell culture plates. Add 100 μl dropwise (10 ml / plate) using a pipette. Incubate at 37°C in a CO2 incubator and observe. Starting from the first day after cell fusion, carefully observe the cells and record cell growth, the number of hybridomas per well, and any contamination of the culture medium. Perform a complete medium change with HT medium every 5-7 days.

[0074] Example 4: Screening and subcloning of positive hybridoma cell lines

[0075] After the fusion cells were grown for approximately 10 days, indirect ELISA was performed. Antigen was coated onto the microplate at 100 ng / well and incubated overnight at 4°C. The plates were washed three times with PBST, then 200 ml of blocking buffer was added and incubated at 37°C for 1.5 hours. The plates were then washed three times with PBST. Hybridoma cell culture supernatant was then added and incubated at 37°C for 1.5 hours. The plates were washed three times with PBST, and a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG was added. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and TMB substrate was added for color development at 37°C in the dark for 10 minutes. The color reaction was terminated with stop solution, and the absorbance (OD) was immediately measured at 450 nm on a microplate reader (see Table 4). Pre-immune normal mouse serum was used as a negative control, and post-immune mouse serum was used as a positive control. Positive results were considered if the absorbance was 2.1 times greater than the negative control. Hybridoma subclones were selected from wells that secreted specific antibodies, exhibited single clonal growth, and had good morphology. As shown in Table 4, the highest OD value was 2.859. Therefore, the cell clone number 1A3 with the highest OD value (OD450 = 2.859) was selected for subsequent subcloning.

[0076] Table 4 Positive clone screening results

[0077] Resuspend and count the hybridoma cells in the positive wells. Prepare 15% FBS-DMEM-HT medium containing feeder cells. Prepare a 5ml cell suspension in this medium at a density of 12 cells / ml. Plate each clone onto 0.5 96-well plates and add 0.1ml / well (i.e., 1.2 cells / well) to each plate for culture. The remaining cells from each clone can be cultured in a 24-well dish. After approximately 7 days, single clones are identified under a microscope, labeled, and recorded. Antibody detection is performed by ELISA (see Table 5). Pre-immune normal mouse serum serves as a negative control, and post-immune mouse serum serves as a positive control. Positive results are considered if the absorbance is 2.1 times greater than the negative control. Hybridoma subclones are selected from wells that secrete specific antibodies, exhibit single clonal growth, and have good morphology. As shown in Table 5, the highest OD value is 2.805. Clone 1G4, with the highest OD value (OD450 = 2.805), was selected for antibody production.

[0078] Table 5 Positive clone screening results

[0079] The medium in the positive wells was changed. Once the 96-well plate was confluent, the cells were cultured from the 96 wells to the 24 wells. After the cells had confluent all 24 wells, the cell supernatant was retained for Western blot analysis. The cells were then cultured from the 24-well plate to the 6-well plate and the hybridoma cells were frozen.

[0080] The mouse tissue protein sample was added to the loading buffer and boiled. After the protein was denatured, it was cooled and the protein sample was added to the corresponding channel at 20 μg / well. It was then separated by polyacrylamide gel electrophoresis and electrotransferred to the nitrocellulose membrane. After blocking with 5% skim milk powder at room temperature for 1.5 hours, the membrane was incubated in the diluted cell supernatant (1:100) at 4°C overnight. After the membrane was removed and washed three times with TBST, a 1:2000 diluted HRP-labeled goat anti-mouse IgG antibody was added and incubated for 1.5 hours. The membrane was washed three times with TBST. The ECL colorimetric solution was prepared in a 1:1 ratio of A / B solution and exposed using a chemical imaging system. The results are shown in Figure 2 .

[0081] like Figure 2 As shown, 1 is mouse colon tissue, 2 is mouse liver tissue, and 3 is mouse testis tissue. All of them express the target protein with a size of 66~68KDa, and all of them have internal reference GAPDH as a control. It can be seen that the expression of the target antibody in mouse colon tissue and liver tissue is higher than that in mouse testis tissue, which is consistent with the expression of mouse ATG16L1 in different tissues provided by NCBI.

[0082] Example 5: Mass preparation of monoclonal antibodies and determination of antibody titer

[0083] To prepare large quantities of monoclonal antibodies: Intraperitoneally inoculate Balb / c mice with liquid paraffin (0.3-0.5 ml per mouse). 7-10 days later, intraperitoneally inoculate hybridoma cells diluted in PBS at a concentration of 1-1.5 × 10⁶ cells / ml per mouse. After one week, observe the mice daily for ascites production. If the abdomen is noticeably distended and the skin feels tense when touched, collect ascites with a sterile syringe. Repeat this collection every one to two days until the mouse dies naturally. Centrifuge the collected ascites at 5000 rpm for 10 minutes, collect the supernatant, and aliquot and store at -20°C.

[0084] Secure the rProtein G Beads gravity column to a metal stand. Remove the lower and upper stoppers, draining the column's protective buffer. Add 5 column volumes of equilibration buffer (0.15 M NaCl, 20 mM NazHPO4, pH 7.0) to the column tube for equilibration. Load the sample onto the equilibrated gravity column, retaining the sample for at least 2 minutes. Collect the flow-through for SDS-PAGE analysis of protein binding. Wash with 10-15 column volumes of wash buffer to remove non-specifically adsorbed contaminants, and collect the wash buffer. Elute the target protein using 5-10 column volumes of elution buffer (0.1 M glycine, pH 3.0). Collect fractions, collecting each column volume into a separate tube, for analysis. The eluted fractions must be immediately neutralized. It is generally recommended to neutralize with 1 / 10 the volume of the eluted fraction using neutralizing buffer (1 M Trs-HCl, pH 8.5). The packing was equilibrated with 3 column volumes of equilibration buffer and then 5 column volumes of deionized water. The gravity column was stored in an equal volume of 20% ethanol at 2-8°C. Antibody concentration was determined using a BCA kit (Shanghai Bioengineering), and the purified monoclonal antibody concentration was 1.6 mg / mL.

[0085] Antibody titer determination: The titer of the ATG16L1 antibody was determined using an indirect ELISA assay. BSA-conjugated protein was diluted to 1 μg / ml with coating buffer and coated onto a 96-well microtiter plate at 100 μl per well. After overnight at 4°C, the plate was washed three times with PBST for 3 minutes each time. 300 μl of wash buffer was added to each well and the plate was patted dry. The plate was blocked with 5% skim milk powder in PBS (200 μl per well) and incubated at 37°C for 1.5 hours. After blocking, the plate was washed three times with PBST for 3 minutes each time. 300 μl of wash buffer was added to each well and the plate was drained. ATG16L1 antibody diluted to 20 ng / ml in PBST was added and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST and patted dry. TMB substrate solution (100 μl per well) was added and incubated at 37°C for 10 minutes. The reaction was terminated by adding 50 μl / well of 1M H₂SO₄. The OD 450 nm readings were measured using a microtiter plate reader. The results are shown in Table 6. As shown in Table 6, the titer of the purified antibody was 1:729,000.

[0086] Table 6 Antibody titer

[0087] Example 6: Sequence Analysis

[0088] Analysis by a third-party testing agency indicates that the nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 7. It should be noted that the CDR sequences listed in Table 7 below were obtained from the IMGT database. Those skilled in the art will appreciate that the CDR sequences analyzed from different databases may differ, but such variations are intended to be within the scope of protection of the present invention.

[0089] Table 7 Antibody sequence information

[0090]

[0091] Example 7: Antibody sensitivity and specificity detection

[0092] Sensitivity test: Hela cells were used as the sample for the test, and the purified antibodies were diluted 3000 times, 9000 times, 12000 times, and 15000 times, and the sensitivity of the antibodies was tested by Western Blot. Figure 2 .like Figure 2 As shown in the figure, when diluted 12,000 times, the target band is lightly colored and has unclear boundaries, while when diluted 9,000 times, the target band is darkly colored and has clear boundaries, so the sensitivity of this antibody is 9,000 times.

[0093] Specificity detection: Hela cells are used as the sample for detection, and the purified antibodies are compared with commercial antibodies, such as Figure 3 , Lanes 1-2 are the antibodies diluted 3000 times, and Lane 3 is the commercial antibody diluted 3000 times. The specificity of the antibodies was tested by Western Blot. Figure 3 It can be seen that the target band detected by the antibody of the present invention is obviously darker in color than the commercial antibody. Therefore, the antibody of the present invention has better specificity in WB detection.

[0094] Example 8: Application

[0095] This antibody is used as a research antibody for studying the ATG16L1 protein in the autophagy signaling pathway. It is a member of the autophagy-related protein family and plays an important role in regulating the autophagy process. Therefore, more and more researchers are using the ATG16L1 protein to study the autophagy process and further research on drug treatments for certain diseases. The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make slight changes or modifications to the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ATG16L1, wherein the amino acid sequences of LCDR1 and LCDR3 of the light chain variable region VL of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 3 and 5, respectively, the amino acid sequence of LCDR2 is LMS, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 6, 7 and 8, respectively.

2. The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to claim 1, wherein the VL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, and the VH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to claim 1 or 2, wherein the light chain constant region CL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9, and the heavy chain constant region CH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to claim 1 or 2, wherein the antigen-binding fragment is selected from a diabody; the antibody is a murine monoclonal antibody, the light chain of which comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and the heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

12. 5 . A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to claim 1 . A vector comprising the nucleic acid according to claim 5 .

7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. The host cell according to claim 7, wherein the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: The host cell according to claim 7 or 8 is cultured under conditions that allow the expression of the antibody or antigen-binding fragment thereof.

10. A detection reagent comprising an antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to any one of claims 1 to 4, The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes ATG16L1.

11. Use of the antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to any one of claims 1 to 4, or the detection reagent according to claim 10, in the preparation of a product for detecting the content of ATG16L1 in a sample.

12. Use of the antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 according to any one of claims 1 to 4, the nucleic acid according to claim 5, the vector according to claim 6, or the host cell according to claim 7 or 8 in preparing a product for detecting ATG16L1.

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