CN118406145A - An anti-phosphorylated ASC_Tyr 144 antibody and its application - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-phosphorylation ASC_Tyr144 antibody and application thereof. The invention discloses a monoclonal antibody 9S26 and/or a single-chain antibody scFV9S26 aiming at phosphorylated ASC_Tyr144, and the antibody provided by the invention has the characteristics of high affinity and specificity, and simultaneously provides a method for detecting human peripheral THP-1 cell phosphorylated ASC_Tyr144 protein by a flow method based on the antibody, thereby greatly improving the accuracy and convenience of detection. The invention solves the problem of blank raw materials of human peripheral THP-1 cell phosphorylation ASC_Tyr 144 detection project antibodies in the current market. The antibody provided by the invention can be used as an auxiliary diagnosis for the occurrence and progress of Alzheimer's disease, multiple sclerosis and parkinsonism through detecting the phosphorylated ASC_Tyr144 protein of human peripheral THP-1 cells.

Description

An anti-phosphorylated ASC_Tyr 144 antibody and its application

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-phosphorylated ASC_Tyr 144 antibody and an application thereof.

Background technique

In 1999, Masumoto et al. discovered ASC (Apoptosis-associated speck-like protein containing a CARD, TMS1) molecules in leukemia cells, with a molecular weight of 22kDa. ASC is constitutively expressed at a high level in a variety of macrophages. In unstimulated cells, ASC exists as a soluble protein in the cytoplasm and nucleus. During apoptosis, ASC aggregates into hollow spots in the cytoplasm, so it is called apoptosis-associated speck-like protein. Subsequent studies have shown that ASC protein is an important adaptor protein of inflammasomes. On the one hand, it recruits pro-caspase-1 to the inflammasome complex, and on the other hand, ASC aggregates to form a caspase-1 activation platform, the ASC speck. When the inflammasome is activated, almost all ASC molecules gather together to form this cytoplasmic supramolecular polymer. This process is strictly regulated and the aggregation is very rapid. In most cases, only one ASC spot can be formed in one cell. The latest study used single-cell intracellular polymer three-dimensional imaging technology to find that caspase-1 forms dimers wrapped around the periphery of spherical ASC spots. The process of ASC spots recruiting pro-caspase-1 and inducing its self-hydrolysis and activation has been studied relatively clearly, but the specific mechanism of ASC spot formation during inflammasome activation is still unclear. A possible explanation is that ASC is recruited by oligomerized NLRs or the effector region of PYHIN receptors, causing close contact between ASC molecules, thereby inducing the initial ASC aggregation, which forms ASC spots with this as the core.

ASC specks are oligomers of ASC dimers, and ASC may bind through CARD-CARD or PYD-PYD homologous protein interactions. When overexpressed in COS-7 cells, ASC mutants lacking the PYD or CARD domains aggregated to form filamentous polymers, suggesting that both the PYD and CARD domains are involved in the formation of ASC specks. A point mutation (K26A) in the PYD domain can prevent the formation of ASC specks, confirming that the PYD domain plays an important role in the formation of ASC specks. The PYD domain of ASC has positively and negatively charged surfaces, and the two binding sites are located at the two ends of the PYD domain, which is very important for binding and interaction with other PYD molecules. However, another study showed that the CARD domain is a key region for the formation of ASC specks, because the CARD domain of overexpressed ASC can form ASC speck-like polymers with the participation of the CARD domain of caspase-1, and the researchers also confirmed the amino acid residues necessary for the formation of specks on the CARD domain of ASC (especially Glu130, Asp134 and Glu144). It has been reported that phosphorylation of Tyr144 on the CARD domain plays a key role in the formation of ASC specks. Studies have shown that the PYD domain of the ASC molecule is phosphorylated under the stimulation of tumor necrosis factor (TNF). In the activation of NLRP3 and AIM2 inflammasomes, phosphorylation of ASC occurs downstream of Syk and JNK. Inhibitors of Syk or JNK can attenuate IL-18 secretion, caspase-1 activation and ASC speck formation induced by NLRP3 and AIM2 agonists without affecting the interaction between ASC and NLRP3. Therefore, the phosphorylation of ASC in inflammasome activation requires the participation of Syk and JNK, and Tyr144 on the CARD domain of the ASC molecule has been confirmed to be an important phosphorylation site for spot formation. In summary, downstream of Syk and JNK, phosphorylation of the ASC molecule (Tyr144) plays an important role in regulating ASC spot formation and inflammasome-mediated immune responses.

At present, there are no diagnostic reagent raw materials specific for phosphorylated ASC_Tyr 144 in the industry. Since phosphorylated ASC_Tyr 144 is an intracellular protein, it only exists in cells and its content in peripheral blood and body fluids is extremely low. Traditional immunodiagnostic methods, such as chemiluminescence assay (CLIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), latex enhanced turbidimetry, etc., are suitable for the detection of secretory proteins in peripheral blood and other body fluids, but not for the detection of intracellular proteins. Immunohistochemistry methods have the advantages of long detection time, complex operation, poor repeatability and low sensitivity. Flow cytometry can detect natural cell intracellular proteins, so it is more suitable for the analysis and detection of phosphorylated ASC_Tyr 144.

At present, there is an urgent need to develop a phosphorylated ASC_Tyr 144 antibody with strong specificity and high sensitivity for the development of ASC_Tyr144 kits.

Summary of the invention

The purpose of the present invention is to overcome the problems existing in the prior art and provide an anti-phosphorylated ASC_TYR 144 antibody and its application. The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylated ASC_TYR 144, which can be in the form of a monoclonal antibody or a single-chain antibody. The antibody provided by the present invention has the characteristics of high affinity and specificity.

The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO:3-5, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO:6-8, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto.

In some embodiments of the invention, the amino acid sequence of the VL is as shown in SEQ ID NO:1 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO:2 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

In some embodiments of the present invention, the amino acid sequence of VL comprises LCDR1, LCDR2 and LCDR3 sequences having a light chain variable region as shown in SEQ ID NO: 1, or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof; the amino acid sequence of VH comprises HCDR1, HCDR2 and HCDR3 sequences having a heavy chain variable region as shown in SEQ ID NO: 2, or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof.

In some embodiments of the present invention, it further comprises a light chain constant region CL and/or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:14.

In some embodiments of the invention, the antibody is of an isotype selected from IgG.

In some embodiments of the present invention, the antibody is of the IgG1 subtype.

In some embodiments of the present invention, the antigen-binding fragment is selected from a single-chain antibody.

In some embodiments of the present invention, the antibody is a murine monoclonal antibody or a single-chain antibody.

In some embodiments of the present invention, the antibody is a monoclonal antibody, wherein the antibody is a monoclonal antibody, clone number 9S26, which further comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:15, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:16.

In some embodiments of the present invention, the antibody is a single-chain antibody, clone number is scFV9S26, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ IDNO:17.

The second aspect of the present invention provides a nucleic acid comprising a nucleic acid encoding any of the above-mentioned antibodies or antigen-binding fragments thereof that specifically bind to phosphorylated ASC_TYR 144.

In some embodiments of the invention, the nucleic acid is DNA, such as cDNA or recombinant DNA.

The third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

The fourth aspect of the present invention provides a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector.

In some embodiments of the present invention, the host cell is a eukaryotic cell.

In some embodiments of the present invention, the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

The fifth aspect of the present invention provides a hybridoma cell, which can produce any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to phosphorylated ASC_Tyr 144.

The sixth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in any one of the above items, comprising: culturing the host cell or hybridoma cell described in any one of the above items under conditions that allow the antibody or antigen-binding fragment thereof to be expressed.

A seventh aspect of the present invention provides a composition comprising:

(i) an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or hybridoma cell that specifically binds to ITGAX as described in any one of the above;

(ii) a pharmaceutically acceptable carrier or adjuvant.

The eighth aspect of the present invention provides an antibody-conjugate, which comprises the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr144 as described in any one of the above items.

The ninth aspect of the present invention provides a detection reagent, which comprises the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 as described in any one of the above items.

In some embodiments of the present invention, the antibody or antigen-binding fragment thereof against phosphorylated ASC_Tyr 144 is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes phosphorylated ASC_Tyr 144.

In some embodiments of the present invention, substances used for fluorescent labeling include but are not limited to PE, PerCP, FITC, and APC.

The tenth aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr144, or a detection reagent as described in any of the above items, for use in preparing a reagent or product for detecting the content of phosphorylated ASC_Tyr 144 in a sample, a reagent or product for diagnosing a disease related to phosphorylated ASC_Tyr 144, or a drug for preventing and/or treating a disease related to phosphorylated ASC_Tyr 144.

In some embodiments of the present invention, the phosphorylated ASC_Tyr 144-related disease is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson's disease.

The eleventh aspect of the present invention provides an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, hybridoma cell, composition and/or antibody-conjugate specifically binding to phosphorylated ASC_Tyr144 as described in any one of the above items for use in preparing any of the following products:

(1) Detection of phosphorylated ASC_Tyr 144 products;

(2) Products that stimulate or enhance immune response;

(3) Products for preventing and/or treating diseases related to phosphorylated ASC_Tyr 144.

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

The present invention provides a monoclonal antibody against phosphorylated ASC_Tyr 144, and scFV phosphorylated ASC_Tyr 144 after modification by single-chain antibody, 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 specificity. The present invention solves the problem that there is no antibody raw material for phosphorylated ASC_Tyr 144 detection project on the market. The antibody disclosed in the present invention has high sensitivity and specificity, and can be used as an auxiliary diagnostic tool for the occurrence and progression of neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis and Parkinson's disease.

When screening antibodies, the present invention uses a variety of natural cell antigens to compare antibody specificity detection. After multiple rounds of screening, 9S26 was obtained as an antibody with good specificity for phosphorylated ASC_Tyr 144. In the animal immunization process, multiple subcutaneous and multi-point injections were used to increase the titer of the final animal serum and the positive clone rate. The addition of HAT screening reagents was postponed from the day of fusion to the second day, which significantly improved the cell positive rate.

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

Fig. 1 is a HPLC chromatogram of three coupled polypeptides;

FIG2 is a comparison of the antigenic activity of two pASC144s coupled to carrier proteins;

FIG3 is a gel image showing subtype identification of antibodies expressed by hybridoma cell line 9S26 and cloned antibody variable regions according to an embodiment of the present invention, and the lane sequence is shown in Table 6;

FIG4 is a plasmid map of a single-chain antibody expression plasmid pCDNA3.1-scFV9S26 constructed using antibody 9S26 according to an embodiment of the present invention;

FIG5 shows the application of monoclonal antibody 9S26 and single-chain antibody scFV9S26 in detecting phosphorylated ASC_Tyr 144 in human THP-1 cells by flow cytometry according to an embodiment of the present invention.

Detailed ways

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.

In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

As used herein, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

As used herein, the terms “comprise” or “include” are open expressions, namely including the contents specified in the present invention but not excluding other contents.

As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO:3-5, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto, respectively, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO:6-8, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto, respectively.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to phosphorylated ASC_Tyr 144 disclosed herein, the amino acid sequence of the VL is as shown in SEQ ID NO:1, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO:2, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to phosphorylated ASC_Tyr 144 disclosed herein, the amino acid sequence of the VL comprises LCDR1, LCDR2 and LCDR3 sequences of a light chain variable region as shown in SEQ ID NO:1, or having one or more conservative amino acid substitutions, deletions or insertions, or any combination thereof; the amino acid sequence of the VH comprises HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region as shown in SEQ ID NO:2, or having one or more conservative amino acid substitutions, deletions or insertions, or any combination thereof.

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, 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.

As used herein, the term "antibody analogs" refers to derivatives produced by biological or chemical methods, based on the antibody structure, by deletion, addition, or modification of chemical groups (such as amino acids). 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.

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 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.

As used herein, the term "antibody variable region" refers to a domain in the heavy and light chains 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 chains), and J fragments in immunoglobulin (heavy and light chain) genes through gene recombination splicing. The amino acid sequences of the variable regions between different antibodies have a high degree of diversity (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 both 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.

The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDR. Conventional definitions include Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), Chothia definition (Chothia and Lesk, J. Mol. Biol. 196: 901 917, 1987; Chothia et al., Nature 342: 878 883, 1989); a composite of Chothia Kabat CDRs, wherein CDR H1 is a composite of Chothia CDRs and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (World Wide Web bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to either of these numbering systems, but preferred embodiments use CDRs defined according to Kabat or Chothia.

As used herein, the term "anti-phosphorylated ASC_Tyr 144 antibody (or antibody analog)" exists in a single (or monoclonal) form, and a single antibody (or antibody analog) contains a single structure (such as a single amino acid sequence).

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 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. 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. 85:24 ... l.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., 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.

Under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can 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 a manner from N-terminus to C-terminus.

As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid by 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 phosphorylated ASC_Tyr 144 antibody or the phosphorylated ASC_Tyr144 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 replace each other. Or with polar amino acids, for example, arginine replaces lysine, glutamic acid replaces aspartic acid, glutamine replaces asparagine, serine replaces threonine, and the like.

Therefore, the antibodies of the present invention can also be directly synthesized according to the antibody variable region amino acid sequence provided by the present invention, and can be derived into different antibody analogs through further chemical modification.

In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 disclosed herein, it further comprises a light chain constant region CL and/or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14.

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 for use 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 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 so that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

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.

In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein that specifically bind to phosphorylated ASC_Tyr 144, the antibodies have an isotype selected from IgG. In some embodiments, the antibodies have a subtype selected from IgG1.

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 the CH1 and variable region of a heavy chain. 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.

In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein that specifically binds to phosphorylated ASC_Tyr 144, the antigen-binding fragment is selected from a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, or a small molecule antibody. For example, the antibody is a single-chain antibody. For example, the small molecule antibody mentioned includes at least one of a Fab antibody, a Fv antibody, a single-chain antibody, and a minimum recognition unit.

In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein that specifically binds to phosphorylated ASC_Tyr 144, the antibody is a murine monoclonal antibody or a single chain antibody.

In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 disclosed herein, the antibody is a monoclonal antibody with clone number 9S26, which further comprises a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:15, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:16.

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.

In some embodiments of the antibody or its antigen-binding fragment that specifically binds to phosphorylated ASC_Tyr 144 disclosed herein, the antibody is a single-chain antibody, clone number scFV9S26, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:17.

As used herein, the term "single-chain antibody" scFv refers to an antibody formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide (linker) of 15 to 20 amino acids. scFv can better retain its affinity activity for antigens and has the characteristics of small molecular weight, strong penetration and weak antigenicity. scFv contains antibody fragments of the VH and VL domains of antibodies, wherein these domains are present in a single polypeptide chain. In general, the Fv polypeptide additionally contains a polypeptide linker between the VH and VL domains, which enables scFv to form the desired structure for antigen binding. It should be noted that the term "single-chain antibody" herein is not limited to antibodies produced by the techniques described herein, nor should it be interpreted as requiring antibodies produced by any particular method.

In yet another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylated ASC_TYR 144 disclosed herein.

In yet another aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein.

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.

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.

In another aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

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, and then the recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. 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.

In some embodiments of the host cells disclosed herein, the host cell is a eukaryotic cell.

In some embodiments of the host cells disclosed herein, the host cells are mammalian cells, including but not limited to 293F cells and CHO cells.

In another aspect, the present disclosure provides hybridoma cells that can produce antibodies or antigen-binding fragments that specifically bind to phosphorylated ASC_Tyr 144 disclosed in the present invention.

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 phosphorylated ASC_Tyr 144, the suitable antigen must contain phosphorylated ASC_Tyr 144. This antigen can be isolated and purified from natural human tissue 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.

In the classic monoclonal antibody preparation method, first of all, a suitable antigen is required and animals are immunized with it. In order to prepare antibodies (or antibody analogs) against phosphorylated ASC_Tyr 144, the suitable antigen must contain phosphorylated ASC_Tyr144. 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 pASC144 polypeptide 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.

In the classic monoclonal antibody preparation method, animals are first immunized with KLH-coupled pASC144 polypeptide antigens, and blood is collected at intervals to verify whether the animals have produced antibody responses to BSA-coupled pASC144 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.

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 (such as fluorescein-labeled phosphorylated ASC_Tyr 144). 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 cDNAs can be obtained by 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.

In another aspect, the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof disclosed in the present invention, comprising: culturing the host cell or hybridoma cell disclosed in the present invention under conditions that allow the antibody or antigen-binding fragment thereof to be expressed.

In another aspect, the present disclosure provides a composition comprising: (i) an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or hybridoma cell disclosed in the present invention that specifically binds to ITGAX; and (ii) a pharmaceutically acceptable carrier or adjuvant.

As used herein, the term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not substantially toxic to the recipient thereof, and/or such carrier or adjuvant is approved or available for inclusion in pharmaceutical compositions for parenteral administration to humans.

In some embodiments, the carrier or adjuvant used with the compositions disclosed herein include, but are not limited to, sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier can be water. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W.Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, together with a suitable carrier, to provide a dosage form suitable for the patient. The preparation should be suitable for the mode of administration. The preparation can be packaged in an ampoule, a disposable syringe or a multi-dose vial made of glass or plastic.

In yet another aspect, the present disclosure provides an antibody-conjugate comprising the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 disclosed in the present invention.

In another aspect, the present invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 disclosed in the present invention.

In some embodiments of the detection reagent disclosed herein, the antibody or antigen-binding fragment thereof against phosphorylated ASC_Tyr 144 is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes phosphorylated ASC_Tyr 144.

In some embodiments of the detection reagents disclosed herein, substances used for fluorescent labeling include but are not limited to PE, PerCP, FITC, and APC.

On the other hand, the present disclosure provides the use of the antibody or its antigen-binding fragment that specifically binds to phosphorylated ASC_Tyr 144 disclosed in the present invention, or the detection reagent in the preparation of a reagent or product for detecting the content of phosphorylated ASC_Tyr 144 in a sample, a reagent or product for diagnosing a disease related to phosphorylated ASC_Tyr144, or a drug for preventing and/or treating a disease related to phosphorylated ASC_Tyr 144.

In some embodiments of the uses disclosed herein, the phosphorylated ASC_Tyr 144-related disease is selected from: Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease and other nervous system-related inflammatory diseases.

In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, hybridoma cell, composition and/or antibody-conjugate disclosed in the present invention that specifically binds to phosphorylated ASC_Tyr 144 in the preparation of any of the following products:

(1) Detection of products phosphorylated at ASC_Tyr 144;

(2) Products that stimulate or enhance immune response;

(3) Products for preventing and/or treating diseases related to phosphorylated ASC_Tyr 144.

The anti-phosphorylated ASC_Tyr 144 antibody of the present invention can be used to detect phosphorylated ASC_Tyr144 on the surface of human peripheral blood THP-1 cells, or substances containing phosphorylated ASC_Tyr 144, and substances that can specifically bind to phosphorylated ASC_Tyr 144 (such as anti-phosphorylated ASC_Tyr 144 antibodies). The principle of detection is mainly based on the specific recognition and binding of the phosphorylated ASC_Tyr 144 antibody of the present invention to phosphorylated ASC_Tyr144, and detection is performed using chemical labeling technology (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection technology (such as light scattering technology, plasma resonance technology, etc.).

The present invention provides a monoclonal antibody 9S26 against phosphorylated ASC_Tyr 144, which has the characteristics of high affinity and specificity, and also provides a flow detection method based on the above antibody. The present invention solves the problem of relative shortage of antibody raw materials for phosphorylated ASC_Tyr 144 detection project in the current market, can be used as an auxiliary diagnosis for related neurological diseases, greatly improves the accuracy and convenience of detection, and fills the gap in the domestic related market.

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, those skilled in the art can easily obtain the corresponding nucleic acid sequence, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below are obtained based on the IMGT database. Those skilled in the art should know 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.

Table 1 Sequence information corresponding to different numbers

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.

Example 1: Design and coupling of phosphorylated ASC_Tyr 144 peptide

The peptide phosphorylated ASC_Tyr 144) was designed based on the Uniprot ID: Q9ULZ3-1 (fASC) sequence. Through immunogenicity analysis, the following two peptide sequences were selected for synthesis:

ASC144 polypeptide sequence (Tyr 144 unphosphorylated polypeptide): “WLLDALYGKVLTDYQYQAVR” (Trp131-Arg150);

pASC144 polypeptide sequence (Tyr 144 phosphorylated polypeptide): “WLLDALYGKVLTDY(p)QYQAVR” (Trp131-Arg150);

The above two peptides were synthesized by a third-party company using a peptide synthesizer, and the purity was greater than 95% as detected by HPLC.

The pASC144 polypeptide was coupled to the KLH carrier protein using a two-step glutaraldehyde method, and the ASC144 and pASC144 polypeptides were coupled to BSA. First, 10 mg of KLH or BSA was dissolved in 0.2 mL of glutaraldehyde solution (10 mM PB, 12.5 mM glutaraldehyde, pH 6.8), reacted at room temperature for 18 hours, and then dialyzed with PBS buffer (10 mM PBS, pH 7.2) at 4 ° C overnight to obtain a aldehyded KLH or BSA solution. 2 mg of the polypeptide (ASC144 or pASC144) was dissolved in 1 mL of 150 mM NaCl to obtain a polypeptide solution. Then 0.5 mL of the polypeptide solution was mixed with the aldehyded KLH or BSA solution. Add 0.1mL 1M pH9.6 carbonate buffer solution (adjust pH to 9.0-9.6), react at 4°C for 24 hours, then add 0.1mL 0.2M lysine solution, let stand at 4°C for 2 hours to terminate the reaction. Finally, dialyze with 10mM PBS buffer (10mM PBS, pH7.2) at 4°C overnight, and the dialysate is analyzed by HPLC (High Performance Liquid Chromatography). The results are shown in Figure 1, verifying the A280 absorption peak. The single peak is that no free polypeptide peak is found, and the polypeptide has been fully coupled to the carrier protein. The dialysate collected after verification is the coupled protein.

The coupled proteins obtained through the coupling experiment are: pASC144-KLH (pASC144 polypeptide coupled to KLH carrier protein); pASC144-BSA (pASC144 polypeptide coupled to BSA carrier protein); ASC144-BSA (ASC144 polypeptide coupled to BSA carrier protein).

Example 2: Activity identification of pASC144 coupled protein

The specific operation for the activity identification of the coupled protein of pASC144 is as follows: KLH, BSA, and the polypeptide coupled KLH (pASC144-KLH) and BSA (pASC144-BSA) of phosphorylated ASC_Tyr144 were diluted with a carbonate buffer solution at pH 9.6 and coated on the ELISA plate at 1 μg/mL, and 100 μL/well was coated on a 96-well ELISA plate at 4°C overnight; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: PTB was used to dilute different anti-pASC144 rabbit polyclonal antibodies (abcam, ab180799) in different proportions or concentrations, and 0.1 mL of the diluted sample was placed in the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (without sample), negative control wells and positive control wells were made at the same time. Add 0.1 mL of freshly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody (HRP-labeled goat anti-rabbit antibody, abcam, #ab6728): Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result, use an enzyme reader to measure the absorbance value (A450) at 450 nm, and use the OD450 reading of the enzyme reader to compare the antigen activity of pASC144 after coupling with the carrier protein. The results are shown in Figure 2. As shown in FIG2 , both pASC144-KLH and pASC144-BSA coupled proteins have binding activity, and the highest antigen activity was detected for the protein after pASC144 was coupled to KLH (pASC144-KLH).

Example 3: Immunization of BALB/c mice with KLH-coupled polypeptide phosphorylated ASC_Tyr 144 (pASC144-KLH)

BALB/c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg pASC144-KLH protein was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 μg phosphorylated ASC_Tyr 144 protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the second immunization, 12.5 μg pASC144-KLH protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated, and pASC144-BSA protein was coated in a 96-well ELISA plate at 0.2 μg/mL and 100 μL/well. After overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL/well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA in PBS solution, 200 μL/well, at room temperature for 2 hours, and then patted dry; mouse serum diluted in gradient PTB was added, 100 μL/well, and reacted in a 37°C incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL/well each time. Patted dry after washing. HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, and reacted in a 37°C incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL/well each time. Patted dry after washing. TMB substrate solution was added, 100 μL/well, and reacted at room temperature for 10 minutes. 100 μL/well of 1MH ₂ SO ₄ was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 2.

Typical immune mouse serum test results are shown in Table 2. As shown in the results of Table 2, the titer of the mouse antiserum prepared in this example is 1:72900. Since the detection antigen is pASC144-BSA coupled polypeptide, the mouse antibodies that recognize KLH in the immunogen can be excluded, and the antibodies in the serum measured by the analytical experiment specifically recognize the pASC144 polypeptide.

Table 2 Mouse antiserum titer detection

Serum dilution multiple OD450 100 1.256 300 0.954 900 0.682 2700 0.452 8100 0.325 24300 0.258 72900 0.169 Background 0.065

Example 4: Cell fusion

In Example 5, after the third immunization, the mice were boosted with the following specific steps: 12.5 μg pASC144-KLH protein was injected and mixed with PBS to a volume of 200 μL for intraperitoneal injection. Cell fusion was performed 3 days after the booster immunization of the mice. After removing the mouse eyeballs to collect blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied to find the spleen, removed with tweezers, and gently ground into a 200-mesh stainless steel filter membrane, and the cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200g for 10 minutes in a centrifuge at room temperature, and the supernatant was discarded for later use.

When preparing feeder cells, the mice were killed by dislocation and placed in a 70% alcohol bottle for 2 minutes. The mice were then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied, PBS was drawn up with a syringe and gently injected into the subperitoneum, and the liquid containing the feeder cells was washed out from the other side. The mice were then centrifuged at 200g in a centrifuge at room temperature for 10 minutes, and the supernatant was discarded for later use. 2.0×10 ⁷ FO myeloma cells and 2.0×10 ⁸ spleen cells were mixed, centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 mL of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, followed by 20 ml of DMEM culture medium. The mixture was centrifuged at 200 g for 10 minutes, the supernatant was discarded, the washing was repeated, and the mixture was centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the hybridoma cells were plated into 10 96-well culture plates with 150 μL per well. 10,000 cells/well of feeder layer cells were added to the above 10 96-well cell culture plates, 100 μL per well, and the culture plates were marked and placed in a cell culture incubator at 37°C containing 5% CO _2. HAT selection medium (Merk, H0262) was added on the second day, and a large number of tumor cells died within 1-2 days of HAT selection culture. After 3-4 days, the tumor cells disappeared and hybrid cells formed small colonies. After 7-10 days of HAT selection culture medium, HT culture medium (Merk, H0137) should be used, and then maintained for another 2 weeks, and then 20% FBS (ExCell, FSP500) DMEM culture medium was used to continue culture. During the above selection culture period, when the hybridoma cells covered 1/10 of the bottom area of the well, specific antibodies could be detected to screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium was generally replaced every 2-3 days.

Example 5: Screening and subcloning of positive hybridoma cell lines

First, the optimal coating amount of pASC144-BSA as an antigen was determined by the founder experiment. 0.5, 1.0, 2.0, and 4.0 μg of pASC144-BSA protein was coated on a 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Founder titration was performed with positive serum from mice immunized with pASC144-KLH protein at different dilutions, and negative serum from unimmunized mice was used as a negative control. Coat a 96-well ELISA plate with 0.5 μg of purified pASC144-BSA and ASC144-BSA proteins per well at 4°C overnight; wash twice with PBST; add 200 μL of 1% BSA in PBS to each well, block for 2 hours at room temperature, and then pat dry on trifold paper; add samples: add 0.1 mL of the sample to be tested to the reaction wells of the ELISA plate coated with pASC144-BSA and ASC144-BSA proteins in parallel, incubate at 37°C for 1 hour, and then wash, and make blank wells (without adding samples), negative control wells, and positive control wells at the same time. Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 ml of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result, and use a microplate reader to measure the absorbance (A450) at 450 nm. A value 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select the cell wells with pASC144-BSA positive and ASC144-BSA negative to determine that they are hybridoma cell monoclones specific for anti-phosphorylated ASC_Tyr 144.

According to the above method, the positive hybridoma cells screened were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution method and redistributed into 96-well culture plates, and then the morphology and number of cells were observed. The cells were adjusted to 3-10 cells/mL. Take the cell culture plate with feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Culture statically in a 37°C, 5% _CO2 incubator. Change the medium on the 7th day, and change the medium once every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity can be detected in time. Move the cells in the positive wells to a 24-well plate for expanded culture. The binding activity of antibodies and pASC144-BSA in the culture supernatants of different cell lines after 4 days of culture in 24-well plates was compared by indirect ELISA detection. 96-well ELISA plates were coated with 0.2 μg/mL pASC144-BSA and washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: add 0.1 mL of cell culture supernatant samples in 24 wells of different clones to be tested, incubate at 37°C for 1 hour, and then wash, and do negative wells (blank cell culture medium) and positive wells (PTB 100-fold diluted pASC144-BSA positive mouse serum). Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 ml of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result, and use a microplate reader to measure the absorbance value (A450) at 450 nm. A value 2.1 times or more of the negative control OD value is considered positive (calculated after zeroing the blank control well). The results are shown in Table 3. As shown in Table 3, the highest OD value is 2.145. Therefore, the cell line clone number 9S26 with the highest OD value (OD450 = 2.145) was selected for antibody production.

Table 3 Identification of binding activity of antibodies in culture supernatant with pASC144-BSA

Example 6: Mass preparation of monoclonal antibodies and determination of antibody titer

(1) Large-scale preparation of monoclonal antibodies

0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into 8-week-old BALB/C mice. 1×10 ⁶ hybridoma cells were injected intraperitoneally 2 weeks later. Ascites can be produced 7 to 10 days after the cells were inoculated. The health status of the animals and the signs of ascites were closely observed. When the ascites was as much as possible and the mice were on the verge of death, the mice were killed and the ascites was sucked into a test tube with a dropper. 5 to 10 mL of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained was centrifuged at 3000g for 10 minutes, the upper layer of fat and the bottom sediment were discarded, the supernatant was collected and aliquoted at -20℃, and after the ascites supernatant was thawed and equilibrated to room temperature, 1/10 volume of 1M Tris-HCl pH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was balanced with 20 column volumes of 100mM Tris-HCl at pH 8.0, and the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100mM Tris-HCl at pH 8.0, and finally eluting the antibody with 100mM Glycine-HCL pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at room temperature for 20 minutes at 3000×g in a centrifuge (Xiangyi, L550). The solution was centrifuged in batches until the volume of the solution reached 1mL/concentrator tube (2 tubes), 4mL of 10mM PBS pH 7.4 buffer was added, and centrifugation at room temperature for 20 minutes at 3000×g was continued. The centrifugation was repeated 3 times to make the antibody buffer 10mM PBS pH 7.4, and 10mM PBS pH 7.4 was added to a total volume of 10mL. Finally, 2 mL/tube of the concentrated antibody solution was dispensed into centrifuge tubes and stored at -80° C. The antibody concentration was determined using a BCA kit (Solabo, PC0020), and the concentration of the purified monoclonal antibody was 1.1 mg/mL.

(2) Antibody titer determination

The titer of anti-phosphorylated ASC_Tyr 144 antibody 9S26 was detected by indirect ELISA method. The pASC144-BSA protein was diluted with PBS and coated in a 96-well ELISA plate at 0.2μg/mL and 100μL/well. After overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300μL/well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA PBS solution, 200μL/well, at room temperature for 2 hours, and patted dry after blocking; anti-phosphorylated ASC_Tyr 144 antibody 9S26 diluted to 20ng/mL with PTB was added, and the reaction was carried out in a 37°C incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300μL/well each time. Pat dry after washing. Add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB, react in a 37°C incubator for 1 hour, wash the ELISA plate twice with PBST solution, 300 μL/well each time. Pat dry after washing. Add TMB substrate solution, react at room temperature for 10 minutes at 100 μL/well, and add 100 μL/well of 1M H ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 4. As can be seen from Table 4, the titer of the purified antibody is 1:729000.

Table 4 Antibody titer determination

Example 7: Sequence analysis of monoclonal antibodies

(1) Monoclonal antibody subtype identification

The hybridoma cell line 9S26 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO ₂ ) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube, and 4×10 ⁶ cells were taken out after counting with a hemocytometer. After centrifugation at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN.

The antibody subtype was determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA was used as a PCR reaction template. The primer sequence information used in PCR is shown in Table 5.

Table 5 PCR primer sequence information

Note: In the table, S=C or G, M=A or C, R=A or G, and W=A or T.

PCR reaction solution system: TAKARA Ex Taq (5U/μL, TAKARA, RR001B), 0.25μL; 10×Ex TaqBuffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; add double distilled water to a total volume of 50μL.

PCR reaction temperature program: 94°C for 5 minutes of pre-denaturation, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), and extension at 72°C for 10 minutes.

After the reaction was completed, 10 μL of each PCR product was loaded on a 1% agarose gel for electrophoresis. The electrophoresis diagram is shown in Figure 3, and the order of samples added to each lane is shown in Table 6. As shown in Figure 3, through electrophoresis diagram analysis, lane 1 (Lane 1) and lane 1 (Lane 6) amplified positive bands of 650 bp for IgG1 heavy chain and kappa light chain, respectively. It was determined that the heavy chain of the monoclonal antibody 9S26 obtained in the present invention was IgG1 subtype, and the light chain was kappa subtype. According to the PCR product results, the subtype of the antibody can be inferred (Table 6), and the results are shown in Table 6. As can be seen from Table 6, the monoclonal antibody 9S26 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

Table 6 Sample information corresponding to the lane

Lanes PCR primers Antibody subtype specificity 1 PCR products of primers F-VH and R-VH 1 IgG1 heavy chain 2 PCR products of primers F-VH and R-VH 2A IgG2A heavy chain 3 PCR products of primers F-VH and R-VH 2B IgG2B heavy chain 4 PCR products of primers F-VH and R-VH 3 IgG3 heavy chain 5 DNA molecular weight standard (full gold, 4Kb DNA Ladder, #BM131) N/A 6 PCR products of primers F-VH and R-VK IgKappa light chain 7 PCR products of primers F-VL and R-VL1 IgLambda1 light chain 8 PCR products of primers F-VL and R-VL2 IgLambda2 light chain

(2) Sequencing of the variable region (V region) of the hybridoma cell line 9S26

The fragments obtained after PCR amplification of the V region of the antibody of cell line 9S26 (see above) were cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragments were linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The nucleic acid sequence of the light chain V region of the anti-phosphorylated ASC_Tyr 144 antibody 9S26 provided by the present invention is shown in SEQ ID NO:9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO:10.

Based on the above, the amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the monoclonal antibody 9S26 obtained are shown in Table 7. The CDR sequences of the antibody according to the IMGT database are shown in Table 8. The light chain constant region and heavy chain constant region sequences of the antibody are shown in Table 9. The complete heavy chain and light chain sequences of the antibody are shown in Table 10.

Table 7 VL and VH sequences of monoclonal antibody 9S26

Table 8 CDR sequences of monoclonal antibody 9S26

LCDR1 QSAQDIGKSLL SEQ ID NO:3 LCDR2 DSSNLWD SEQ ID NO:4 LCDR3 QQWL SEQ ID NO:5 HCDR1 SYG SEQ ID NO:6 HCDR2 KDWSGGSTDYNAAFIS SEQ ID NO:7 HCDR3 RTQISKESLPYFDY SEQ ID NO:8

Table 9 Light chain constant region and heavy chain constant region of monoclonal antibody 9S26

Table 10 Light chain and heavy chain of monoclonal antibody 9S26

Example 8: scFV antibody modification, expression and purification of antibody 9S26

(1) Construction of pCDNA3.1-scFV9S26 plasmid

The nucleic acid sequence of the light chain V region of antibody 9S26 obtained in the early stage is shown in SEQ ID NO.9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.10. Through molecular cloning methods, they are inserted into the BamHI and XbaI restriction sites of pCDNA3.1, and the expression ORF is: signal peptide + VH + (GS) 4 Linker + VL + 6 × His Tag. The constructed expression plasmid is named pCDNA3.1-scFV9S26, and the plasmid map is shown in Figure 4. The inserted sequence between the plasmid BamHI and XbaI restriction sites is shown in Table 11, and the sequence is shown in SEQ ID NO: 27.

Table 11 Insertion sequence information

The light chain variable region (VL) and heavy chain variable region (VH) amino acid sequences of the scFV9S26 antibody are shown in Table 12. The CDR sequences of the antibody according to the Kabat system are shown in Table 13. The amino acid sequences of the antibodies are shown in Table 14.

Table 12 Amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the scFV9S26 antibody

Table 13 CDR sequences of scFV9S26 antibody

LCDR1 QSAQDIGKSLL SEQ ID NO:3 LCDR2 DSSNLWD SEQ ID NO:4 LCDR3 QQWL SEQ ID NO:5 HCDR1 SYG SEQ ID NO:6 HCDR2 KDWSGGSTDYNAAFIS SEQ ID NO:7 HCDR3 RTQISKESLPYFDY SEQ ID NO:8

Table 14 Amino acid sequence of scFV9S26 antibody

(2) Expression and purification of single-chain antibody scFV9S26

The pCDNA3.1-scFV9S26 plasmid constructed above was transfected into CHO-S cells. In transfecting the suspended CHO-S cells, the transfection reagent used was Free Style MAX (Invitrogen, 16447100), and the transfection operation steps were carried out as described in the manufacturer's product manual. The transfected CHO-S cells were transferred to a 125 mL triangular shake flask, and the cell density was 1×10 ⁶ cells/mL in 30 mL FreeStyleCHO medium. The cells were cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) with a shaker speed of 130 rpm. The culture supernatant was collected after 7 days of cell culture, and the activity concentration of the anti-phosphorylated ASC_Tyr 144 single-chain antibody (named scFV9S26) in the supernatant (expressed and secreted by CHO-S cells) was detected by indirect ELISA. In this experiment, pASC144-BSA protein was diluted with PBS as antigen and coated in 96-well ELISA plate at a coating concentration of 1 μg/mL and a volume of 100 μL/well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL/well each time. After washing, pat dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL/well, at room temperature for 2 hours. Pat dry, add the supernatant of cells expressing scFV9S26 diluted with PTB in different ratios, add to the blocked ELISA plate, 100 μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled mouse anti-His antibody (abcam, #ab18184) diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, at room temperature for 10 minutes, then add 100 μL/well of 1M H ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450 nm. See Table 15 for the results. The activity titer of the anti-pASC144-BSA single-chain antibody scFV9S26 in the cell supernatant was detected. The titer of scFV9S26 in the cell supernatant was 72900.

Table 15 Titer detection of anti-pASC144-BSA single-chain antibody scFV9S26 in cell supernatant

Cell supernatant dilution factor scFV9S26 10 2.141 30 1.958 90 1.685 2700 1.352 8100 0.985 24300 0.685 72900 0.325 Background 0.069

The cell supernatant was centrifuged at 3000 × g for 10 minutes, and 1/10 volume of 1M Tris-HCl pH8.0 was added to bring the sample pH to 8.0. The Ni affinity column was balanced with 20 column volumes of 100mM Tris-HCl pH8.0, and the supernatant after adjusting the pH to 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100mM Tris-HCl pH 8.0, and finally the antibody was eluted with 100mM imidazole pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at room temperature for 20 minutes at 3000 × g in a centrifuge (Xiangyi, L550). Batch centrifuged until the solution volume reached 1mL/concentrator tube (2 tubes), 4mL of 10mM PBS pH 7.4 buffer was added, and centrifuged at room temperature for 20 minutes at 3000 × g. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS pH 7.4, add 10mM PBS pH 7.4 to a total volume of 10mL. Finally, 2mL/tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solebo, PC0020), and the concentration of the purified single-chain antibody scFV9S26 was determined to be 2.3mg/mL.

The purified single-chain antibody scFV9S26 was tested for activity titer. In this experiment, pASC144-BSA protein was diluted with PBS as an antigen and coated in a 96-well ELISA plate at a coating concentration of 1μg/mL and a volume of 100μL/well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300μL/well each time. After washing, pat dry, and the ELISA plate was blocked with PBS solution containing 1% BSA, 200μL/well, at room temperature for 2 hours. Pat dry, add different concentrations of single-chain antibody scFV9S26 diluted with PTB, add to the above blocked ELISA plate, 100μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300μL/well each time. Pat dry, add HRP-labeled mouse anti-His antibody (abcam, #ab18184) diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, at room temperature for 10 minutes, and then add 100 μL/well of 1M H ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450nm. The results are shown in Table 16. The titer of the single-chain antibody scFV9S26 in the cell supernatant was 810,000.

Table 16: Titer detection of single-chain antibody scFV9S26 after affinity purification

Antibody dilution factor scFV9S26 10000 2.125 30000 1.925 90000 1.125 270000 0.563 810000 0.214 2430000 0.106 7290000 0.105 Background 0.078

Example 9: Identification of the sensitivity and specificity of antibodies scFV9S26 and 9S26 in detecting phosphorylated ASC_Tyr 144

(1) Identification of the sensitivity of antibodies scFV9S26 and 9S26 in detecting phosphorylated ASC_Tyr 144

The ability of antibodies scFV9S26 and 9S26 to detect phosphorylated ASC_Tyr 144 with sensitivity was evaluated using an indirect ELISA. In this experiment, pASC144-BSA conjugate was diluted in PBS as antigen and coated in a 96-well ELISA plate at a volume of 100 μL/well. After overnight coating at 4°C, the plate was washed twice with PBST solution, 300 μL/well each time. After washing, the plate was patted dry and blocked with PBS solution containing 1% BSA, 200 μL/well, at room temperature for 2 hours. After patting dry, different concentrations of antibody 9S26 and control antibody (rabbit anti-human ASC antibody, abcam, #ab283684) diluted in PTB were added to the above blocked ELISA plate, 100 μL/well, and reacted in a 37°C incubator for 1 hour. After patting dry, the plate was washed 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled mouse anti-His antibody (abcam, #ab18184) diluted 1000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, at room temperature for 10 minutes, and then add 100 μL/well of 1M H ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450 nm. The experimental results are shown in Table 17.

As shown in Table 17, the antibody scFV9S26 has better specific binding to the phosphorylated ASC_Tyr 144 protein than the antibody 9S26 and the control antibody. And the sensitivity of the modified single-chain antibody scFV9S26 is increased by about 10 times compared with the antibody 9S26. The results show that the detection curve of scFV9S26 has R ² =0.9824 and IC50=12.5ng/mL. The standard curve range of this method is 0-3000ng/mL, and the minimum detection limit is 11.2ng/mL, while the detection curve of the control antibody has R ² =0.9127 and IC50=1000ng/mL. The standard curve range of this method is 1000-3000ng/mL, and the minimum detection limit is 500ng/mL. The results are shown in Table 17.

Table 17 Identification of the sensitivity of antibodies scFV9S26 and 9S26 in measuring pASC144-BSA protein

pASC144-BSA concentration (ng/mL) Control Antibodies 9S26 scFV9S26 3000 0.325 2.103 2.561 1000 0.214 1.852 2.235 300 0.185 1.524 2.124 100 0.154 0.958 1.925 50 0.106 0.452 1.225 25 0.114 0.213 0.535 12.5 0.124 0.113 0.325 0 0.106 0.105 0.089 Background 0.092 0.103 0.054

(2) Identification of the specificity of antibodies scFV9S26 and 9S26 in detecting phosphorylated ASC_Tyr 144

The ability of antibodies scFV9S26 and 9S26 to detect phosphorylated ASC_Tyr 144 specifically was evaluated by indirect Cellbased ELISA. In this experiment, THP-1 cells (positive cells) and the corresponding negative cells Hela cell line were cultured separately. Hela cells do not express ASC protein. After 3 generations of culture using DMEM medium with 10% serum, the cells were transferred to 96-well cell culture plates at a cell density of 100,000 cells/well, cultured overnight, the supernatant was discarded, and 4% paraformaldehyde was added at a volume of 100 μL/well to fix the cells for 30 minutes. PBST solution was washed twice, 300 μL/well each time. After washing, pat dry, and the 96-well plate was blocked with PBS solution containing 1% BSA, 200 μL/well, at room temperature for 2 hours. Pat dry, add different concentrations of antibody scFV9S26 and control antibody (rabbit anti-human ASC antibody, abcam, #ab283684) diluted with PTB, 100 μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled mouse anti-His antibody (used as secondary antibody for scFV9S26 antibody) diluted 5000 times with PTB to the ELISA plate; HRP-labeled goat anti-rabbit antibody (used as secondary antibody for control antibody) diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, and incubate at room temperature for 10 minutes, then add 100 μL/well of 1M H ₂ SO ₄ to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader, and the experiment is shown in Table 18. As shown in Table 18, the antibody scFV9S26 has better specific binding than the antibody 9S26 and the control antibody in detecting the native phosphorylated ASC_Tyr 144 protein. In addition, the sensitivity of the modified single-chain antibody scFV9S26 is increased by about 6 times compared with the antibody 9S26.

Table 18 Identification of the specificity of antibody scFV9S26 in detecting phosphorylated ASC_Tyr 144

Example 10: Application of antibodies scFV9S26 and 9S26 in detecting phosphorylated ASC_Tyr144 in human THP-1 cells by flow cytometry

(1) Fluorescein isothiocyanate (FITC) labeling of antibody scFV9S26 and 9S26 and identification of labeled products

Antibody scFV9S26 and 9S26 (20 nmol, 3 mg) were taken to a volume of 0.5 mL and added to a dialysis bag (10 KDa, width 1 cm), and dialyzed in 2L 10mM PBS solution (pH 7.4) at 4°C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1L 10mM carbonate buffer (pH 9.5) and dialyzed for 2 hours at room temperature, ready for coupling with activated FITC.

At the same time, use an analytical balance to accurately weigh 0.1 mg FITC, add it to the dialyzed antibody solution, add fluorescein while stirring to prevent the powder from adhering to the wall, and place it in a 4°C refrigerator or icehouse and continue stirring for 12 to 18 hours. After the binding is completed, first centrifuge the conjugate at 3000×g for 20 minutes, remove a small amount of precipitate, put it into a dialysis bag, and then dialyze it overnight in a beaker of 10mM PBS solution (pH 7.4). Take the marker that has been dialyzed overnight, pass it through a dextran gel G-25 column, separate the free FITC, collect the labeled fluorescent antibody and store it at 4°C.

(2) Using FTTC-labeled antibodies scFV9S26 and 9S26 to detect phosphorylated ASC_Tyr 144 protein in human THP-1 cells by flow cytometry

In this experiment, Hela and THP-1 cells were fixed in 4% paraformaldehyde at 22°C for 10 minutes. Red blood cells were then lysed for 15 minutes by adding Triton X-100 (final concentration of 0.1%) at 37°C. In this experiment, scFV9S26 was diluted 100 times, incubated at 4°C for 30 minutes, and FITC-labeled mouse anti-His antibody (abcam, ab1206) was added and stained at 4°C for 30 minutes. Using the CytoFLEX instrument, >30,000 total events were collected, using a 50mW argon blue laser (488nm) and a 530/30 bandpass filter. Gating strategy events were collected by the forward and side light scattering properties of living cells, and the antibody scFV9S26 and 9S26 were determined by comparing the FITC values to identify the phosphorylated ASC_Tyr 144 protein in the cell, as shown in Figure 5.

As shown in Figure 5, the peak range (10 ² -10 ³ ) of 488nm fluorescence value of antibody scFV9S26 and 9S26 for negative cells (Hela) is comparable, and the peak value of 488nm fluorescence value of positive cells (THP-1) is higher for antibody scFV9S26 than for 9S26, which proves that antibody scFV9S26 can specifically recognize phosphorylated ASC_Tyr 144 protein in cells and is superior to antibody 9S26.

The present invention provides monoclonal antibodies 9S26 and/or scFV9S26 against phosphorylated ASC_Tyr 144, which have the characteristics of high affinity and specificity, and also provides a method for detecting phosphorylated ASC_Tyr 144 in human THP-1 cells by flow cytometry based on the above scFV9S26 antibody. The present invention solves the problem of relative shortage of antibody raw materials for phosphorylated ASC_Tyr 144 detection projects in the current market.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

The above description 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 as above, 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 by using the methods and technical contents disclosed above 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 fall within the scope of the technical solution of the present invention.

Claims (12)

1. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO:3-5, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO:6-8, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto. 2. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to claim 1, wherein the amino acid sequence of the VL is as shown in SEQ ID NO: 1 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto. 3. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to claim 2, wherein the amino acid sequence of the VL comprises LCDR1, LCDR2 and LCDR3 sequences having a light chain variable region as shown in SEQ ID NO: 1 or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof; the amino acid sequence of the VH comprises HCDR1, HCDR2 and HCDR3 sequences having a heavy chain variable region as shown in SEQ ID NO: 2 or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof. 4. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to any one of claims 1-3, further comprising a light chain constant region CL and/or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 14. 5. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from a single-chain antibody; The antibody is a mouse monoclonal antibody or a single-chain antibody. 6. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to claim 5, wherein the antibody is a monoclonal antibody with clone number 9S26, which further comprises a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16. 7. An antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to claim 5, wherein the antibody is a single-chain antibody, clone number is scFV9S26, and it comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:17. 8. An application, comprising application as a nucleic acid, a vector, a host cell or a hybridoma cell; the nucleic acid comprises an antibody or an antigen-binding fragment thereof encoding the antibody specifically binding to phosphorylated ASC_TYR 144 according to any one of claims 1 to 7; The vector comprises the nucleic acid; The host cell comprises the nucleic acid or the vector, wherein the host cell is a eukaryotic cell; The host cell is a mammalian cell, including but not limited to 293F cells and CHO cells; and The hybridoma cell can produce the antibody or antigen-binding fragment that specifically binds to phosphorylated ASC_Tyr 144. 9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: culturing the host cell or hybridoma cell according to claim 8 under conditions that allow the antibody or antigen-binding fragment thereof to be expressed. 10. A detection reagent comprising an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr144 according to any one of claims 1 to 7; The antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes phosphorylated ASC_Tyr 144, wherein the substances used for fluorescent labeling include but are not limited to PE, PerCP, FITC, and APC. 11. Use of the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to any one of claims 1 to 7, or the detection reagent according to claim 10, in the preparation of a reagent or product for detecting the content of phosphorylated ASC_Tyr144 in a sample, a reagent or product for diagnosing a disease associated with phosphorylated ASC_Tyr 144, or a drug for preventing and/or treating a disease associated with phosphorylated ASC_Tyr 144; The phosphorylated ASC_Tyr 144-related disease is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson's disease. 12. Use of the antibody or antigen-binding fragment thereof that specifically binds to phosphorylated ASC_Tyr 144 according to any one of claims 1 to 7, the nucleic acid, vector, host cell or hybridoma cell according to claim 8 in the preparation of any of the following products: (1) Detection of phosphorylated ASC_Tyr 144 products; (2) Products that stimulate or enhance the immune response; (3) Products for preventing and/or treating diseases related to phosphorylated ASC_TYR 144.