ATG16L1 monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an ATG16L1 monoclonal antibody and application thereof. The invention discloses an antibody specifically bound with ATG16L1 or an antigen binding fragment thereof, amino acid sequences of LCDR1-3 of a light chain variable region VL of the antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO: 3, 4 and 5, and amino acid sequences of HCDR1-3 of a heavy chain variable region VH of the antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO: 6, 7 and 8. The antibody disclosed by the invention has relatively 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] At present, the key proteins of the autophagy signaling pathway have become new candidate targets for disease intervention and tumor treatment, and have attracted the attention of more and more scholars, but there is a lack of independently developed monoclonal antibodies, and the only commercially available products are rabbit-derived polyclonal antibodies. Many antigens for immunizing mice are natural proteins. Since natural proteins are modified and have a relatively complex structure (in addition to linear epitopes, there are also conformational epitopes), natural proteins are high-quality antigens, but high-purity natural proteins are often difficult to obtain. Most secondary antibodies are obtained by screening natural proteins as antigens.

[0003] Chemically synthesized peptide antigens are small molecules that are difficult to have good antigenicity by themselves and can only induce weak immune responses in animals. Therefore, cross-linking with carrier proteins is very important. Carrier proteins contain many antigenic determinants that can stimulate T-helper cells and then induce B-cell responses. There are many carrier proteins used for cross-linking with peptides, among which keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) and bovine thyroglobulin (THY) are commonly used. KLH has higher antigenicity and is a commonly used peptide cross-linking carrier. BSA is also commonly used as a peptide carrier, but because BSA is often used as a blocking agent in detection tests, the antibodies produced by this method have certain limitations in application.

[0004] An artificially synthesized peptide is just a linear sequence. It is uncertain whether the antibody against this peptide can recognize the natural protein. Therefore, the design of the peptide is very important before preparing the antibody. When it is difficult to obtain protein antigens or there are homologous proteins but only a few sequence differences exist, or when preparing modified antibodies at specific sites, peptide antigens can be selected to target proteins with high homology to family members. Specific peptides can be used to prepare specific antibodies that distinguish 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, and then immunizing mice with artificially synthesized polypeptides to screen for the required specific antibodies.

[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 the antigen-binding fragment thereof are shown in SEQID 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 the antigen-binding fragment thereof are shown in SEQID 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, whose light chain 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, and the mammalian cell includes but is not limited to 293F cells and CHO cells.

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

[0016] The sixth aspect of the present invention provides a detection reagent, which comprises the antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 as described above, The antibody or antigen-binding fragment thereof that specifically binds to ATG16L1 is fluorescently labeled and used as a component of a flow fluorescence detection reagent that specifically recognizes ATG16L1.

[0017] The seventh aspect of the present invention provides a use of an antibody or antigen-binding fragment thereof that specifically binds to ATG16L1, or a detection reagent as described above, in the preparation of a product for detecting the content of ATG16L1 in a sample.

[0018] An eighth aspect of the present invention provides a use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, and a host cell that specifically binds to ATG16L1 as described above in the preparation of a product for detecting ATG16L1.

[0019] By means of the above technical solution, the present invention has at least the following advantages: The present invention provides a monoclonal antibody against ATG16L1, which avoids the problem of high background caused by HAMA reaction and FCR reaction in natural samples. The antibody or antigen-binding fragment thereof provided by the present invention has the characteristics of high affinity and high specificity. The present invention solves the problem that there is no antibody raw material for ATG16L1 detection project on the market. The antibody disclosed by the present invention has high sensitivity and specificity.

[0020] When screening antibodies, the present invention selects a variety of natural cell antigens to compare antibody specificity detection. After multiple rounds of screening, antibodies with good specificity for ATG16L1 are obtained. In the animal immunization process, multiple subcutaneous and multi-point injections are used to increase the titer of the final animal serum and improve the positive clone rate.

[0021] 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 preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the amino acid antigenicity prediction map; Figure 2 This is a WB image of the antibody that specifically binds to ATG16L1 obtained by screening according to the present invention; 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

[0023] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination 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 creative work are within the scope of protection of the present invention.

[0024] 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.

[0025] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0026] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including 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.

[0030] As used herein, the term "antibody analogs" refers to derivatives produced by biological or chemical methods, which are obtained by deleting, adding, or modifying chemical groups (such as amino acids) based on the antibody structure. These derivatives still contain structures similar to the antibody variable region (or the CDR region in the antibody variable region), and can undergo reactions similar to antigen-antibody binding through these structures.

[0031] 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, such as 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, which 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.

[0032] As used herein, the term "antibody variable region" refers to a domain in the heavy chain and light chain of an antibody. It includes a light chain variable region (VL) and a heavy chain variable region (VH). In nature, the antibody variable region is encoded by V, D (only applicable to heavy chain), and J fragments in the immunoglobulin (heavy chain and light chain) gene through gene recombination splicing. The amino acid sequences of the variable regions between different antibodies have a high degree of difference (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. In the antibody variable region, the VL domain and the VH domain contain a framework region (FR) and a CDR region (comlementarity determining region) from the amino terminus to the carboxyl terminus. A typical antibody variable region has 3 framework regions and 3 CDR regions, which are interspersed with each other: FR1, CDR1, FR2, CDR2, FR3 and CDR3. The framework region FR mainly plays a role in the formation of the protein domain framework, while the CDR region mainly plays a role in the specific recognition and binding of antigen-antibody. 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.

[0033] 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 similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and 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.

[0034] Without substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, 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 variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence 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, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0035] 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 length, 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 replace each other. Or with polar amino acids, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, and so on.

[0036] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes 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 a variant or fragment thereof. For example, the antigen-binding polypeptide used in the present disclosure may include: 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, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. In addition, the antibody used 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 so that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0037] Based on the amino acid sequence of the constant region of the heavy chain of the antibody, 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.

[0038] As used herein, the term "antigen-binding fragment" is also referred to as "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementary determining region, single-chain antibody, double-chain antibody, or single-domain antibody molecule, etc. Among them, the "Fab fragment" is composed of a light chain and a heavy chain CH1 and variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. Fab' fragment, which has one or more cysteine ​​residues at the C-terminus of the CH1 domain of the Fab fragment; F(ab')2 fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region; Fd fragment, which has VH and CH1 domains; Fv fragment, which has VL and VH domains in a single arm of the antibody; dAb fragment, which consists of a VH domain or a VL domain; isolated CDR regions; and modified forms of any of the above fragments that retain antigen-binding activity.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, except for a small amount of mutations that may occur naturally, each antibody constituting 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.

[0040] 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 (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free 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 guide the expression of the gene operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, the vector includes but is not limited to: (1) plasmid; (2) phagemid; (3) cosmid; (4) artificial chromosome, such as yeast artificial chromosome, bacterial artificial chromosome or artificial chromosome derived from P1; (5) bacteriophage, such as λ phage or M13 phage; (6) animal virus, such as retrovirus, adenovirus, adeno-associated virus, sporovirus, poxvirus, baculovirus.

[0041] 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, they are not from the vector itself. Of course, the polynucleotide can be operably connected to the control elements. In this article, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended 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.

[0042] 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, a recombinant cell can be constructed, and then the antibody or antigen-binding fragment provided by the present invention can be expressed using these recombinant cells. The corresponding antibody can be obtained by culturing the recombinant cell. In some embodiments, the host cell includes, for example, a CHO cell, such as a CHOS cell and a CHO-K1 cell, or a HEK293 cell, such as HEK293A, HEK293T, and HEK293F.

[0043] As used herein, the term "hybridoma cell" refers to a cell formed by the fusion of myeloma cells and B lymphocytes in the process of preparing monoclonal antibodies. In the classic monoclonal antibody preparation method, a suitable antigen is first required and used to immunize animals. In order to prepare antibodies (or antibody analogs) against ATG16L1, the suitable antigen must contain ATG16L1. This antigen can be isolated and purified from natural human tissues or blood, or expressed in prokaryotic or eukaryotic cells and purified by artificial recombinant protein expression. Antigens are used for animal immunization and antibody screening detection.

[0044] In the classic monoclonal antibody preparation method, first of all, a suitable antigen is needed to be available and used to immunize animals. In order to prepare anti-ATG16L1 antibodies (or antibody analogs), the suitable antigen must contain ATG16L1. This antigen can be obtained by separation and purification from natural human tissue or blood, chemical synthesis, or a combination of these methods. After that, the antigen peptide needs to be coupled to KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) carrier proteins to prepare the whole antigen for animal immunization and antibody screening detection.

[0045] In the classic monoclonal antibody preparation method, animals are first immunized with KLH / BSA-coupled polypeptide antigens, and blood is collected at intervals to verify whether the animals have produced antibody responses to the KLH / BSA-coupled polypeptide antigens. B cells are then isolated from the spleen of animals with antibody responses and fused with immortal myeloma cells in vitro to obtain hybridoma cells. These hybridoma cells are then diluted to the limit in culture plates and regrown (monoclonal hybridoma cell lines), and the culture supernatants of these hybridoma cell lines are taken to detect whether they contain specific antibodies against the antigen. Based on the antibody yield, quality and cell line growth characteristics, the best monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

[0046] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the above animals can be isolated and then incubated with labeled antigens. Since antibody-producing B cells usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and can be sorted by a fluorescent flow cytometer. The mRNA of these sorted B cells can be isolated, and a library of antibody variable region cDNA can be obtained 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 expression in mammalian cells, or a phage expression plasmid suitable for expression in bacterial cells) and expressed in a host cell suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method described above, or the phage plaque plating method). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

[0047] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0048] Example 1: ATG16L1 polypeptide design and coupling First, according to the ATG16L1 sequence published on NCBI, the amino acid sequence of human species 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, so as to express 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: Sequence 1: NQMQRKDREMQMNE; Sequence 2: LQKELAEAAKEPLP; Sequence 3: ALQITFTALEGKLR; The above three peptides were synthesized by a third-party company using a peptide synthesizer, and the purity was greater than 95% as detected by HPLC.

[0049] 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.

[0050] Example 2: Immunization of BALB / c mice with three coupled polypeptides Fifteen healthy and active BALB / C female mice aged 6-8 weeks were selected and numbered. Five mice were immunized with each coupled polypeptide, and the following immunization procedure was performed: 100 μg of antigen was used for each mouse. According to the number of mice, the total volume was calculated, and the antigen was mixed with an equal volume of Freund's adjuvant and emulsified for 15 minutes to obtain an emulsion. First immunization: 0.2 ml of emulsion (0.2 ml containing 100 ug antigen) was injected subcutaneously into the back of each mouse with a 1 ml syringe, and the injection time and number of subcutaneous injection points were recorded. 14 days after the first immunization, 0.2 ml of emulsion (0.2 ml containing 100 ug antigen) was injected subcutaneously into the back of each mouse with a 1 ml syringe for the second immunization, and the injection time and number of subcutaneous injection points were recorded. 14 days after the second immunization, 0.2 ml of emulsion (0.2 ml containing 100 ug antigen) was injected subcutaneously into the back of each mouse with a 1 ml syringe for the third immunization, and the injection time and number of subcutaneous injection points were recorded. 7 days after the third immunization (±1 day), collect the blood from the mouse jaw and separate the serum for Elisa test. The specific process is as follows: prepare 1× coating solution (0.05M carbonate buffer), dilute the BSA-coupled antigen to 1ug / ml with the coating solution (the antigen concentration has no effect on the experiment and can be converted to unit concentration), add 100ul to each well, add to the ELISA plate, seal the film and place it at 4℃ overnight; discard the sample in the ELISA plate and pat the coating solution on the paper (to prevent cross contamination when discarding the solution), wash the plate 3 times with PBST (PBS pH 7.4, 0.05% TWEN20), 3min each time, pat the buffer dry for the last time, add blocking solution, 200ul per well, incubate at 37℃ for 1.5h; discard the blocking solution in the ELISA plate, wash the plate 3 times with PBST, 3min each time, and pat the buffer dry for the last time. If the primary antibody is not incubated in time, place it at 4℃ for use.

[0051] 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 multiple dilution until the seventh well. After mixing the seventh well, discard 50ul, and keep the eighth well as the negative control of antibody diluent. Seal the coated plate and incubate it in a 37℃ constant temperature box for 1.5h; discard the primary antibody in the ELISA plate, wash the plate 3 times with PBST, 3min each time, and tap the buffer dry for the last time. If the secondary antibody is not incubated in time, place it at 4℃ for later use; dilute HRP-labeled goat anti-mouse IgG (H+L) with antibody diluent PBST at 1:10000 (select the dilution multiple according to the titer provided by the manufacturer), then add 100ul / well to the ELISA plate, and then incubate it in a 37℃ constant temperature box for 1h; discard the secondary antibody in the ELISA plate, wash the plate 3 times with PBST, 3min each time, and tap the buffer dry for the last time; add 100ul of TMB substrate colorimetric solution to each well, and incubate it in a 37℃ constant temperature box for 4min; add 50ul of stop solution to each well to stop the color development reaction; read the OD450nm measurement value with an ELISA reader and record the data.

[0052] Elisa results are shown in Tables 1-3.

[0053] Table 1 KLH-1 polypeptide serum titer test in mice Table 2 KLH-2 polypeptide serum titer detection in mice Table 3 KLH-3 polypeptide serum titer detection in mice The test results of three typical immunized mouse sera are shown in Tables 1 to 3. As shown in Tables 1 to 3, it can be seen from the above results that the titers of the mouse sera immunized with the three peptides (KLH-1, KLH-2, and KLH-3) are 1:81000, 1:9000, and 1:9000, respectively. Therefore, the titer of the mouse sera immunized with KLH-1 is the highest. Since the detection antigen is a BSA-coupled peptide, the antibody of the mouse that recognizes KLH in the immunogen can be excluded. The antibody in the serum determined by the analytical experiment specifically recognizes ATG16L1.

[0054] Example 3: Cell fusion It should be noted that the antibody titer should be tested one week after the third immunization, and two mice with the highest titer (and the titer is at least 80,000) should be selected as 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.

[0055] 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. Use DMEM culture medium containing 15% fetal bovine serum to subculture for one week, 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 1000rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in serum-free culture medium, count, and adjust to 2×10 7 / ml for standby use.

[0056] 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 with the abdomen facing up and fixed on the dissecting board; use ophthalmic forceps to pick up the mouse 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 peritoneal cavity, remove the syringe, use tweezers to gently press the mouse abdomen several times, 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 many times. 3 to 5×10 cells can be obtained from the peritoneal fluid of 1 mouse. 6 cells.

[0057] Preparation of spleen cells: Select one mouse with the best immune effect, collect blood from the orbits, and kill it by dislocation. Place it in 75% alcohol for 5 minutes for disinfection. Collect blood before the killing process, which is used as positive serum. After the mouse is disinfected, fix it in the clean bench, remove the spleen, and use scissors to remove the connective tissue and adipose tissue adhering to the spleen cells; rinse the spleen with serum-free DMEM culture medium, transfer it to a 40um cell sieve (placed in a 9cm sterile culture dish in advance, add serum-free DMEM culture medium), crush the spleen with the inner core of the syringe, and gently rinse the sieve with serum-free DMEM culture medium, grind and rinse repeatedly to make a spleen cell suspension, and transfer it to a 50ml centrifuge tube; centrifuge at 1000rpm for 5 minutes, discard the supernatant, and wash the precipitate twice with serum-free DMEM culture medium, count and use.

[0058] Cell fusion: Mix SP2 / 0 cells and spleen cells in a 50ml centrifuge tube at a ratio of 1:5, and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, tap the bottom of the centrifuge tube to loosen the cell pellet; slowly drip 1ml of 50% PEG solution preheated to 37℃ within 1min; drip slowly first and then quickly, and shake the centrifuge tube continuously during the dripping process to fully mix the cells and PEG solution; let it stand for 1min; then add 30ml of 37℃ preheated PEG solution to the cell mixture. DMEM medium terminates the reaction, first drip slowly and then drip quickly, add 1ml in 1 minute, then drip a little faster, still add one drop and stir well, then slowly add the remaining medium; tighten the tube cap and gently place it in the incubator for 10 minutes; then take out the cells, centrifuge at 1000rpm for 10 minutes, discard the supernatant after centrifugation, and try to dry the fusion agent (note that it cannot be violently shaken); gently flick the cells evenly, slowly add a small amount of DMEM culture solution (2ml), add the prepared complete culture medium, including feeder cells, HAT (1× final concentration), serum (15% final concentration), double antibody (1× final concentration), resuspend the cells, gently mix it, and add it to 5 96-well cell culture plates. Use a spray gun to drip 100 microliters (10ml / plate), culture and observe in a CO2 incubator at 37℃; starting from the first day after cell fusion, carefully observe the cells, record the cell growth status, the number of hybrid cell tumors in each well, and whether the culture medium is contaminated. After 5-7 days of culture, change the medium completely and replace it with HT culture medium.

[0059] Example 4: Screening and subcloning of positive hybridoma cell lines After the fusion cells grow for about 10 days, indirect Elisa detection is performed. The antigen is coated on the ELISA plate at 100 ng / well, overnight at 4°C, washed three times with PBST, 200 ml of blocking solution is added, incubated in a 37°C incubator for 1.5 hours, washed three times with PBST, and then the hybridoma cell culture supernatant is added, incubated in a 37°C incubator for 1.5 hours, washed three times with PBST, 1:5000 diluted HRP-labeled goat anti-mouse IgG is added, incubated at 37°C for 1 hour, washed three times with PBST, TMB substrate colorimetric solution is added, color is developed at 37°C in the dark for 10 minutes, stop solution is added to terminate the color development reaction, and the absorbance value (OD value) is immediately measured at a wavelength of 450 nm on the ELISA reader, as shown in Table 4. In addition, normal mouse serum before immunization is used as a negative control, and mouse serum after immunization is used as a positive control. If the absorbance value is greater than 2.1 times of the negative control group, it is judged to be positive. Select the hybridoma cell well subclone with specific antibody secretion in the cell growth well, single clone growth and 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.

[0060] Table 4 Positive clone screening results After resuspending the hybrid cells in the positive wells, count them, prepare a 15% FBS-DMEM-HT medium containing feeder cells, use the medium to make a cell suspension of the hybridoma at a density of 12 cells / ml, and prepare 5ml; each clone is spread on 0.5 96-well plates, and 0.1ml / well (i.e. 1.2 / well) is added to the culture plate for culture. The remaining cells of each clone can be cultured in a 24-well dish first; about 7 days, single clone wells are selected under a microscope, marked and recorded, and ELISA is used to detect antibodies, see Table 5. Normal mouse serum before immunization is used as a negative control, and mouse serum after immunization is used as a positive control. If the absorbance value is greater than 2.1 times the negative control group, it is judged to be positive. Select the hybridoma cell well subclone that has specific antibody secretion in the cell growth well, grows as a single clone and has good morphology. As shown in Table 5, the highest OD value is 2.805, and the cell clone number 1G4 with the highest OD value (OD450=2.805) is selected for antibody production.

[0061] Table 5 Positive clone screening results The positive wells were replaced with medium, and after the 96-well cell plate was full, the cells were cultured from the 96-well plate to the 24-well plate. After the cells were full of 24 wells, the cell supernatant was retained and continued to the next step of protein immunoblotting (Western Blot) detection. Then the cells were cultured from the 24-well plate to the 6-well plate and the hybridoma cells were frozen.

[0062] The mouse tissue protein sample was added to the loading buffer and boiled. After the protein was denatured, it was cooled. The protein sample was added to the corresponding channel at 20 μg / well, 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 .

[0063] like Figure 2 As shown, 1 is mouse colon tissue, 2 is mouse liver tissue, and 3 is mouse testicular 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 testicular tissue, which is consistent with the expression of mouse ATG16L1 in different tissues provided by NCBI.

[0064] Example 5: Mass preparation of monoclonal antibodies and determination of antibody titer Large-scale preparation of monoclonal antibodies: Liquid paraffin is inoculated intraperitoneally, 0.3-0.5 ml per Balb / c mouse. After 7-10 days, hybridoma cells diluted with PBS are inoculated intraperitoneally, 1-1.5×106 / ml per mouse. After one week, observe the ascites production of the mice every day. If the abdomen is obviously swollen and the skin feels tense when touched, the ascites can be collected with a sterile syringe, once every one to two days, and repeatedly collected until the mouse dies naturally. The collected ascites is centrifuged at 5000 rpm for 10 minutes, the supernatant is collected and aliquoted, and finally stored in a -20℃ refrigerator.

[0065] Fix the rProtein G Beads gravity column on the iron stand, remove the lower and upper plugs in turn, and drain the protective liquid of the gravity column. Add 5 column volumes of equilibration solution (0.15 M NaCl, 20 mM NazHPO4, pH7.0) to the column tube for equilibration. Add the sample to the balanced gravity column, and the sample retention time is at least 2 minutes. Collect the effluent for SDS-PAGE analysis of protein binding. Wash with 10-15 column volumes of washing solution to remove non-specifically adsorbed impurities, and collect the washing solution. Use 5-10 column volumes of elution solution (0.1M glycine, pH 3.0) to elute the target protein, collect in sections, collect one tube for each column volume, and detect them separately. The eluted fraction needs to be immediately adjusted to neutrality. It is generally recommended to use 1 / 10 volume of neutralizing solution (1 M Trs-HCl, pH 8.5) for neutralization. The packing was equilibrated with 3 column volumes of equilibration solution and 5 column volumes of deionized water. The gravity column was stored in an equal volume of 20% ethanol at 2-8°C. The antibody concentration was determined using a BCA kit (Shanghai Bioengineering), and the concentration of the purified monoclonal antibody was 1.6 mg / mL.

[0066] Antibody titer determination: The titer of ATG16L1 antibody was detected by ELSA indirect method. The BSA-coupled protein was diluted to 1ug / ml with coating solution, and 100ul per well was coated in a 96-well ELISA plate. After overnight at 4°C, it was washed 3 times with PBST solution, 3 minutes each time, and 300ul of washing solution was added to each well. After washing, pat dry. The ELISA plate was blocked with 5% skimmed milk powder in PBS solution, 200ul per well, and placed at 37°C for 1.5 hours. After blocking, the PBST solution was washed 3 times, 3 minutes each time, and 300ul of washing solution was added to each well, and drained after washing. ATG16L1 antibody diluted to 20ng / ml with PBST was added, placed at 37°C for 1 hour, and pat dry after washing 3 times with PBST. TMB substrate solution was added, 100ul per well was placed at 37°C for 10 minutes, and 50ul / well of 1MH2SO4 was added to terminate the reaction. The OD450nm measurement value was read by the microplate reader, and the results are shown in Table 6 below. As shown in Table 6, the titer of the purified antibody is 1:729000.

[0067] Table 6 Antibody titer Example 6: Sequence Analysis It can be seen from the analysis of a third-party testing agency that the nucleic acid encoding the heavy chain and / or light chain of the antibody of the present invention is within the scope of the present invention. According to the amino acid sequence of the heavy chain and / or light chain, a person skilled in the art can easily obtain the corresponding nucleic acid sequence, as shown in Table 7. It should be noted that the CDR sequences listed in Table 7 below are obtained based on the IMGT database. It should be known to those skilled in the art that the CDR sequences analyzed from different databases may be different, but these changes should be included in the scope of protection of the present invention.

[0068] Table 7 Antibody sequence information Example 7: Antibody sensitivity and specificity detection 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. The results are shown in Figure 2 .like Figure 2 As shown, 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 the antibody is 9,000 times.

[0069] 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 3000-fold dilutions of the present antibody, and Lane 3 is 3000-fold dilutions of the commercial antibody. The specificity of the antibody 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 WB detection specificity.

[0070] Example 8: Application This antibody is used as a research antibody for the study of 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 studying the autophagy process through ATG16L1 protein, and further studying the drug treatment of certain diseases. The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the above-disclosed methods and technical contents without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still belongs to the scope of the technical solution of the present invention.

Claims

1. 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 the 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 the antigen-binding fragment thereof are shown in SEQ ID NOs: 6, 7 and 8, respectively.

2. An 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. An 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. An 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 double-chain antibody; the antibody is a murine monoclonal antibody, whose light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and whose 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 any one of claims 1 to 4 . 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 such that the antibody or antigen-binding fragment thereof is expressed.

10. A detection reagent comprising an antibody or an 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 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 the preparation of a product for detecting ATG16L1.

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