Binding molecules against toxin proteins and kits thereof

Abstract

This invention relates to an antitoxin protein binding molecule and a kit thereof. The antitoxin protein binding molecule comprises an antibody or an antigen-binding fragment targeting the antitoxin protein. The binding molecule includes a light chain variable region and a heavy chain variable region. The light chain variable region includes a complementarity-determining region (LCDR), which comprises LCDR1 with the sequence SEQ ID NO:1, LCDR2 with the sequence SEQ ID NO:2, and LCDR3 with the sequence SEQ ID NO:3. The heavy chain variable region includes a complementarity-determining region (HCDR), which comprises HCDR1 with the sequence SEQ ID NO:4, HCDR2 with the sequence SEQ ID NO:5, and HCDR3 with the sequence SEQ ID NO:6.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to antitoxin protein binding molecules and their reagent kits. Background Technology

[0002] Plasmid technology is a novel antibiotic-free microplasmid production technology that utilizes the antagonistic effects of the toxin protein GF_0636 and the antitoxin protein GF_0637 derived from the marine microorganism Roseivirga spongicola_GF047 in Escherichia coli to maintain plasmids and amplify their production. This plasmid production system mainly consists of: an E. coli host cell containing an inducible toxin gene expression toxin protein for self-destruction; and a DNA plasmid containing a replicable structural unit and an antitoxin gene expression cassette [1,2].

[0003] The host cell genome of *E. coli* contains an inducible toxin gene expression cassette. Induced expression of the toxin protein leads to the death of the host bacteria. However, the host bacteria can survive when the plasmid and its expressed antitoxin protein are present in the bacterial cell. This toxin / antitoxin (T / A) protein pairing and its antibacterial mechanism are known to act specifically on prokaryotic microorganisms, but the effects of these proteins on eukaryotes or cells are unknown.

[0004] The production of the cytotoxic plasmid is based on large-scale fermentation and purification. The production process involves the continuous expression of the antitoxin from the cytotoxic plasmid, the selection of toxin-inducing strains, fermentation, and purification. Theoretically, trace amounts of toxin and antitoxin proteins may remain in the final cytotoxic plasmid product. These residual proteins may cause non-specific toxicity or immunogenicity in animals. Therefore, it is necessary to establish quantitative detection methods to monitor product quality.

[0005] According to USP<1047> Gene Therapy Products and T / SHPPA 019-2023 Guidelines for the Quality Management of Plasmid Production for Immunotherapy Products, the residual host protein in plasmid products is generally less than 1 μg / mg. Typically, toxin proteins and antitoxin proteins, as components of the host protein, have even lower concentrations; therefore, the detection method for antitoxin proteins needs to meet a limit of detection lower than the minimum residual amount.

[0006] References:

[0007] 1. CN118374547A, An antibiotic-free microplasmid, its preparation method and application

[0008] 2. Chen Z, Yao J, Zhang P, Wang P, Ni S, Liu T, Zhao Y, Tang K, Sun Y, Qian Q, Wang 2023 Jul 13. PMID: 37451534. Summary of the Invention

[0009] In view of the current state of research, the present invention provides a binding molecule for antitoxin proteins that is highly specific and sensitive.

[0010] The specific technical solution is as follows:

[0011] One objective of this invention is to provide a binding molecule for antitoxin proteins, comprising an antibody or an antigen-binding fragment targeting the antitoxin protein, said binding molecule comprising a light chain variable region and a heavy chain variable region, said light chain variable region comprising a complementarity-determining region (LCDR), said LCDR comprising LCDR1 with sequence SEQ ID NO:1, LCDR2 with sequence SEQ ID NO:2, and LCDR3 with sequence SEQ ID NO:3; or comprising LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:9;

[0012] The heavy chain variable region includes a complementarity determination region (HCDR), which includes HCDR1 with sequence SEQ ID NO:4, HCDR2 with sequence SEQ ID NO:5, and HCDR3 with sequence SEQ ID NO:6; or includes HCDR1 with sequence SEQ ID NO:10, HCDR2 with sequence SEQ ID NO:11, and HCDR3 with sequence SEQ ID NO:12.

[0013] In some specific embodiments, the binding molecule comprises LCDR containing LCDR1 with sequence SEQ ID NO:1, LCDR2 with sequence SEQ ID NO:2, and LCDR3 with sequence SEQ ID NO:3; and HCDR containing HCDR1 with sequence SEQ ID NO:4, HCDR2 with sequence SEQ ID NO:5, and HCDR3 with sequence SEQ ID NO:6.

[0014] In other specific embodiments, the binding molecule comprises LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:9; and HCDR1 with sequence SEQ ID NO:10, HCDR2 with sequence SEQ ID NO:11, and HCDR3 with sequence SEQ ID NO:12.

[0015] In some embodiments, the light chain variable region is selected from SEQ ID NO:4 or 16, and the heavy chain variable region is selected from SEQ ID NO:10 or 22.

[0016] In some specific implementations, the light chain variable region is SEQ ID NO:4 and the heavy chain variable region is SEQ ID NO:10.

[0017] In some other specific embodiments, the light chain variable region is SEQ ID NO:16 and the heavy chain variable region is SEQ ID NO:22.

[0018] In some embodiments, the binding molecule of the antitoxin protein further includes a mouse IgG Fc fragment, a rabbit IgG Fc fragment, or a human IgG Fc fragment;

[0019] In some embodiments, the binding molecule of the antitoxin protein also includes a signal peptide.

[0020] In some specific embodiments, the light chain signal peptide is selected from SEQ ID NO:17 or 19, and the heavy chain signal peptide is selected from SEQ ID NO:18 or 20.

[0021] In some specific embodiments, the binding molecule of the antitoxin protein comprises a light chain as shown in SEQ ID NO:21 or 23; and a heavy chain as shown in SEQ ID NO:22 or 24.

[0022] A second objective of this invention is to provide an antitoxin protein binding molecule pair, wherein the antitoxin protein binding molecule pair includes a first antitoxin protein binding molecule and a second antitoxin protein binding molecule, wherein the first antitoxin protein binding molecule comprises the following CDRs: LCDR1 shown in SEQ ID NO:1, LCDR2 shown in SEQ ID NO:2, LCDR3 shown in SEQ ID NO:3, and HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, and HCDR3 shown in SEQ ID NO:6;

[0023] The binding molecules of the second antitoxin protein include the following CDRs: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:8, LCDR3 shown in SEQ ID NO:9, and HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, and HCDR3 shown in SEQ ID NO:12.

[0024] In some embodiments, the second antitoxin protein-binding molecule further comprises a marker selected from any one of fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, or enzymes.

[0025] A third objective of this invention is to provide a kit for detecting antitoxin proteins, the kit comprising the binding molecules of the antitoxin proteins described in objective one or the binding molecule pairs described in objective two.

[0026] In some specific embodiments, the kit further includes reagents for detecting the binding of the antitoxin protein to an antitoxin-binding molecule or a pair of antitoxin-binding molecules.

[0027] In some embodiments, the antitoxin protein-binding molecule or antitoxin protein-binding molecule pair is used for sandwich detection.

[0028] In some specific implementations, the first antitoxin protein-binding molecule is coupled to a support, and the second antitoxin protein-binding molecule contains a marker.

[0029] In some embodiments, the kit includes a reaction plate coated with a first antitoxin protein-binding molecule, a biotin-labeled second antitoxin protein-binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

[0030] The fourth objective of this invention is to provide a non-diagnostic method for detecting the presence of antitoxin in a sample, the method comprising: incubating the sample with a binding molecule of the antitoxin protein as described in the first objective, and binding the sample with a reagent for detecting the binding of the antitoxin protein to the binding molecule, thereby determining the presence of antitoxin protein in the sample.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention utilizes recombinant monoclonal antibody production technology to prepare a binding molecule for antitoxin protein. The binding molecule has high sensitivity and high specificity and can be used to detect antitoxin protein present in samples. Attached Figure Description

[0033] Figure 1 The SDS-PAGE image of antitoxin protein 0637 is shown.

[0034] Figure 2 The fitting curve results are shown under the condition of SM02 coating at 5.0 μg / mL.

[0035] Figure 3 The results of the fitting curve for the antitoxin protein standard under dilution scheme ① are shown.

[0036] Figure 4 The results of the fitting curve for the antitoxin protein standard under dilution scheme ② are shown.

[0037] Figure 5 The results of the fitting curve for the antitoxin protein standard under dilution scheme ③ are shown.

[0038] Figure 6 The results of the fitting curve for the antitoxin protein standard under dilution scheme ④ are shown.

[0039] Figure 7 The results show the fitting curve of the antitoxin protein standard at a lowered starting concentration. Detailed Implementation

[0040] Antibody

[0041] In this article, "antitoxin protein binding molecules" are molecules that recognize and bind to antitoxin proteins, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, microbodies, affinity molecules, receptor target binding regions, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0042] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, particularly antigen-binding fragments, such as Fab, F(ab')2, and Fv. In some embodiments herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0043] A basic tetrameric antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic tetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; while IgA antibodies contain 2-5 basic tetrameric units, which can combine with the J chain to form multivalent assemblies. In the case of IgG, a tetrameric unit is typically about 150,000 Daltons. Each light chain is linked to the heavy chain by a covalent disulfide bond, while two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the homotype of the heavy chains. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for the μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, while the CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant heavy chain domains. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their constant heavy chain domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0044] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0045] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity.

[0046] In some embodiments, the binding molecule of the antitoxin protein includes a light chain variable region and a heavy chain variable region, the light chain variable region comprising a complementarity-determining region (LCDR), the LCDR comprising LCDR1 with sequence SEQ ID NO:1, LCDR2 with sequence SEQ ID NO:2, and LCDR3 with sequence SEQ ID NO:3; or comprising LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:9;

[0047] The heavy chain variable region includes a complementarity determination region (HCDR), which includes HCDR1 with sequence SEQ ID NO:4, HCDR2 with sequence SEQ ID NO:5, and HCDR3 with sequence SEQ ID NO:6; or includes HCDR1 with sequence SEQ ID NO:10, HCDR2 with sequence SEQ ID NO:11, and HCDR3 with sequence SEQ ID NO:12.

[0048] In some specific embodiments, the binding molecule comprises LCDR containing LCDR1 with sequence SEQ ID NO:1, LCDR2 with sequence SEQ ID NO:2, and LCDR3 with sequence SEQ ID NO:3; and HCDR containing HCDR1 with sequence SEQ ID NO:4, HCDR2 with sequence SEQ ID NO:5, and HCDR3 with sequence SEQ ID NO:6.

[0049] In other specific embodiments, the binding molecule comprises LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:9; and HCDR1 with sequence SEQ ID NO:10, HCDR2 with sequence SEQ ID NO:11, and HCDR3 with sequence SEQ ID NO:12.

[0050] In some embodiments, the light chain variable region is selected from SEQ ID NO:4 or 16, and the heavy chain variable region is selected from SEQ ID NO:10 or 22.

[0051] In some specific implementations, the light chain variable region is SEQ ID NO:4 and the heavy chain variable region is SEQ ID NO:10.

[0052] In some other specific embodiments, the light chain variable region is SEQ ID NO:16 and the heavy chain variable region is SEQ ID NO:22.

[0053] In some embodiments, the binding molecule of the antitoxin protein further includes a mouse IgG Fc fragment, a rabbit IgG Fc fragment, or a human IgG Fc fragment.

[0054] In some embodiments, the binding molecule of the antitoxin protein also includes a signal peptide.

[0055] In some specific embodiments, the light chain signal peptide is selected from SEQ ID NO:17 or 19, and the heavy chain signal peptide is selected from SEQ ID NO:18 or 20.

[0056] In some specific embodiments, the binding molecule of the antitoxin protein comprises a light chain as shown in SEQ ID NO:21 or 23; and a heavy chain as shown in SEQ ID NO:22 or 24.

[0057] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of both antibody heavy chains. While the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from amino acid residues at position C226 or P230 of the heavy chain to the carboxyl terminus, where this numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0058] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. Antibody fragments are preferably antigen-binding fragments of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased through chemical modification or incorporation into liposomes. Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, the name reflecting its ease of crystallization. Fab fragments consist of a complete light chain and a variable domain (VH) of the heavy chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Treatment of an antibody with pepsin produces a larger F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, possessing different antigen-binding activities and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment due to the addition of several additional residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the CH1 domain. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain cell types.

[0059] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also includes a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure.

[0060] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0061] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0062] This invention also includes derivatives and analogs of the various antibodies described herein (e.g., nanobodies, heavy chain binding molecules, multivalent nanobodies, multispecific nanobodies, binding molecules). “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the various antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0063] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0064] The various antibody variants described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the various antibodies of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the various antibodies of the present invention.

[0065] In some embodiments, the sequence of the variants described in this invention may have at least 95%, 96%, 97%, 98%, or 99% homology with its source sequence. Sequence homology described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. This invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0066] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques or phage display techniques well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed using sequences encoding the various antibodies of the present invention. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulating polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. In particular, preferred cell lines are selected by identifying which cell lines have high expression levels and produce antibodies with basic antitoxin protein binding properties.

[0067] Nucleic acid

[0068] This invention also provides polynucleotides encoding the various antibodies or fragments thereof described above. This document provides polynucleotides encoding the heavy chain variable region, the light chain variable region, the heavy chain, the light chain, and each CDR. The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0069] As those skilled in the art will understand, due to the degeneracy of the genetic code, a vast number of nucleic acids can be produced, all of which encode the binding molecules of this invention. Therefore, given the identification of specific amino acid sequences, those skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without altering the amino acid sequence encoding the protein. Thus, this invention also relates to polynucleotides that hybridize with the aforementioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. This invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In this invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, hybridizable polynucleotide-encoded peptides have the same biological functions and activities as mature peptides.

[0070] The full-length nucleotide sequences or fragments of the various antibodies of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequences artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0071] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0072] Therefore, the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to in some embodiments as "flanking sequences") typically include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, complete intron sequences containing donor and acceptor splicing sites, sequence encoding a leader sequence for polypeptide secretion, ribosome binding site, polyadenylated sequence, multi-linker regions for inserting nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0073] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0074] Diagnostics, tests and kits

[0075] The binding molecules of this invention, due to their high affinity for antitoxin proteins, can be used for assays, such as binding assays, to detect and / or quantify antitoxin proteins expressed in tissues or cells. Binding molecules, such as monoclonal antibodies, can be used in studies further investigating the role of antitoxin proteins in disease. The method for detecting antitoxin proteins generally involves obtaining cell and / or tissue samples; detecting the level of antitoxin proteins in the samples.

[0076] The antitoxin protein binding molecule of this invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with antitoxin proteins. This invention provides methods for detecting the presence of antitoxin proteins in samples using classical immunohistochemical methods known to those skilled in the art. The detection of antitoxin proteins can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of antitoxin proteins include ELISA, FACS, RIA, etc.

[0077] For diagnostic applications, binders such as monoclonal antibodies are typically labeled with detectable labeling groups. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used in carrying out this invention.

[0078] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to an antitoxin protein. An example of such a determination would involve detecting the amount of free antibody in a solution containing a certain amount of antitoxin protein, in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody not bound to the antitoxin protein) would indicate that the test molecule is able to compete with the antibody for binding to the antitoxin protein. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0079] This invention also provides a detection kit for detecting antitoxin protein levels. The kit includes an antibody that recognizes the antitoxin protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffer solutions, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0080] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0081] I. Experimental Reagents

[0082] 1. Wash buffer: Dilute the 20×Wash Buffer in the ELISA kit to 1×Wash Buffer using deionized water.

[0083] 2. Sample dilution buffer 1×Dilution Buffer: Prepare a blocking solution of 3% BSA using 1×Wash Buffer and BSA (Sigma) to form 1×Dilution Buffer.

[0084] 3. Block Buffer: Prepare a 3% BSA blocking solution using 1×Wash Buffer and BSA (Sigma).

[0085] 4. Preparation of standards: Melt the antitoxin protein standard at room temperature, vortex to mix, and centrifuge. Use 1×Dilution Buffer to serially dilute the antitoxin protein standard.

[0086] 5. Sample preparation: Take the sample to be analyzed and dilute it to the appropriate factor with 1×Dilution Buffer according to actual needs.

[0087] II. Detection Methods

[0088] 1. Plate coating: Take out the coating antibody and thaw it at room temperature. After vortexing and centrifuging, dilute the coating antibody to 5.0 μg / mL using coating buffer (Absin, Abs9289) and add it to 100 μL / well of a 96-well plate. After blocking with sealing membrane, place the plate in a refrigerator at 2~8℃ for overnight coating incubation.

[0089] 2. Sealing: Remove the 96-well plate coated overnight, carefully peel off the sealing film, discard the solution inside the plate, pat dry on absorbent paper, add 300 μL of 1×Wash Buffer to each well, wash the plate three times, 1 min each time, and pat dry as much as possible on the last wash. Add 300 μL of Block Buffer to each well of the 96-well plate, seal the plate with the sealing film, and incubate at 37°C for 2 h.

[0090] 3. Sample loading: After repeating the plate washing step, add 100 μL of standard, test sample and negative control to each well of the 96-well plate, seal with sealing film and incubate at room temperature with shaking for 1 h.

[0091] 4. Preparation and addition of enzyme-labeled antibody: Dilute the enzyme-labeled antibody to 2.5 μg / mL with 1×Dilution Buffer. After repeating the washing steps, add 100 μL of enzyme-labeled antibody to each well of a 96-well plate, seal the plate with sealing film, and incubate at room temperature with shaking for 0.5 h.

[0092] 5. Color Development and Termination: Allow the TMB colorimetric solution (Abcam) to reach room temperature beforehand. After repeating the plate washing steps, add 100 μL of the colorimetric solution to each well of a 96-well plate and incubate at room temperature in the dark for 10-15 min. After color development, add 100 μL of the stop solution (2M H2SO4) to each well, gently shake to mix, and ensure the reaction is completely terminated until the color stabilizes at orange-yellow.

[0093] 6. Reading the plate: After termination, read the OD value using a wavelength of 450nm and export the data.

[0094] 7. Data Calculation

[0095] ① Calculate the OD value: OD value = OD value (sample to be tested / standard solution)

[0096] ② Calculate the CV value: CV = standard deviation of concentration (sample / standard solution) / average concentration (sample / standard solution) * 100%.

[0097] ③ Standard curve fitting and concentration regression: A four-parameter fitting method (Y = (AD) / [1 + (X / C)^B] + D) was used to plot the standard curve with the concentration of the antitoxin protein standard solution on the x-axis and the OD value on the y-axis. (blank is not involved in the standard curve fitting)

[0098] ④ Recovery rate calculation:

[0099]

[0100] Result Judgment Criteria

[0101] ① The R² of the standard curve is greater than or equal to 0.98.

[0102] ② When the sample detection value is lower than the lower limit of quantitation, the antitoxin protein residue result is calculated back from the lower limit of quantitation. ③ The CV of the STD1 replicate concentration value is ≤35%, and the CV of the STD2~STD7 replicate concentration values ​​is ≤20%. If the OD values ​​of the standard solution replicates are all ≤0.1, then no CV requirement is placed.

[0103] Example

[0104] Example 1: Preparation and Identification of Monoclonal Antibodies

[0105] 1. Immunogen preparation: Preparation of His-tagged antitoxin protein (SEQ ID NO: 25).

[0106] 2. Animal Immunization: A standard immunization protocol was used, selecting 5 Balb / c mice aged 6-8 weeks. Antitoxin protein was used as the immunogen, mixed with Freund's complete adjuvant at a 1:1 ratio, and administered as the first immunization at a dose of 50 μg / mouse via multiple subcutaneous injections in the abdomen. A second immunization was administered 2-3 weeks later, with antitoxin protein mixed with Freund's incomplete adjuvant at a 1:1 ratio, and administered as the first immunization at multiple subcutaneous injections in the abdomen. A third immunization was administered 2 weeks later, with antitoxin protein mixed with Freund's incomplete adjuvant at a 1:1 ratio, and administered as the first immunization at a dose of 50 μg / mouse. One week after the three immunizations, serum titers were measured. Mice with acceptable titers were selected for fusion. A booster immunization was performed, and three days after the booster, the spleen was harvested for hybridoma fusion. Animals were confirmed dead by carbon dioxide asphyxiation followed by cervical dislocation, and cell fusion was then performed. The polyclonal antibody titer was detected using an indirect ELISA method.

[0107] 3. Blood collection and titer detection: One week after each immunization, 50-60 μL of blood was collected from the orbital venous plexus of mice, incubated overnight at 4°C, and the supernatant serum was separated by centrifugation for testing;

[0108] Take an appropriate amount of the protein to be detected, dilute it to 5 μg / mL with coating buffer, and then add 100 μL to each well of a 96-well plate using a single-channel pipette. Gently tap the plate to mix the sample, seal it tightly with plastic wrap, and coat it overnight at 4°C. Wash the plate once with 200 μL / well of washing buffer and dry the plate. Block the plate with 300 μL / well of blocking buffer and incubate at room temperature for 1 hour. Wash the plate twice with 400 μL / well of washing buffer and add the serially diluted sample and sample diluent at 100 μL / well. At the same time, add the detection antibody at 100 μL / well to the 96-well plate and incubate at room temperature for 2 hours. Wash the plate five times with 400 μL / well of washing buffer, add 200 μL / well of chromogenic solution, and incubate at room temperature for 12 minutes. Stop the reaction by adding 50 μL / well of stop solution. Detect using an ELISA reader at a wavelength of 450 nm.

[0109] 4. Cell fusion and selection: All spleen cells from immunized mice were collected and mixed with mouse myeloma SP2 / 0 cells at a 1:1 ratio. The cells were then fused using an electrofusion method to obtain hybridoma cells. The activity of the supernatant antibody was detected by ELISA, and positive clones were screened.

[0110] 5. Beijing Sinocare Medical Technology Co., Ltd. was commissioned to sequence the positive clones and construct the recombinant plasmids. Large-scale production and purification of recombinant antibodies were then carried out, yielding two purified recombinant monoclonal antibodies. The CDR sequences and corresponding numbers of the antibodies are shown in Table 1.

[0111] Table 1. CDR sequences of monoclonal antibodies binding to antitoxin proteins

[0112]

[0113] Example 2 Detection of antitoxin protein

[0114] An ELISA method for detecting antitoxin protein was established based on the aforementioned monoclonal antibody. The SDS-PAGE identification results of the antitoxin protein are shown below. Figure 1 .

[0115] Experiment 1:

[0116] The antitoxin protein standard was diluted starting at 1000 ng / mL, with 8 concentration points obtained through 3-fold dilutions. HRP-labeled biotin-labeled SM10 (Sapex Biotechnology) was selected as the detection antibody (SM10-HRP). The working solution was prepared by diluting the antibody to 2.5 μg / mL using 1×Dilution buffer and incubating at room temperature for 30 min. TMB color development was terminated after 10 min. Results showed... Figure 2 Using SM02 as the coating antibody, at a concentration of 5.0 μg / mL, the fitting curve showed a good upper plateau with few lower plateau points, indicating a good fitting effect and a high correlation coefficient R. 2 : 0.998.

[0117] Experiment 2:

[0118] The antitoxin-coated antibody SM02 was coated at a concentration of 5.0 μg / mL. The SM10-HRP detection antibody was diluted to 2.5 μg / mL and incubated for 30 min, followed by TMB color development for 10 min before termination of the color development. The antitoxin protein was diluted to 800 ng / mL using 1×Dilution Buffer and labeled as Solution 1. Four dilution schemes were established: ① starting at 400 ng / mL, with 2-fold dilutions resulting in 11 concentration points; ② starting at 400 ng / mL, with 2.5-fold dilutions resulting in 11 concentration points; ③ starting at 400 ng / mL, with 3-fold dilutions resulting in 11 concentration points; ④ starting at 400 ng / mL, with 3.5-fold dilutions resulting in 11 concentration points. The distribution of the upper and lower plateaus of the fitted curve under different dilution schemes of the antitoxin protein standard was verified. Results are shown below. Figure 3 ,in Figure 5 (Scheme ③) has small differences between low concentration points on the standard curve, making it easier for the fitted curve to cover all points (although the mathematical R² is higher), but the actual discrimination is insufficient and the detection sensitivity is low. Figure 3 (Scheme ①) The distribution of the standard curve points is relatively uniform. Based on the comparison of the distribution of the fitted curve points under each dilution scheme, Scheme ① (see Table 2 for specific information on the standard curve) was selected for subsequent experiments.

[0119] Table 2

[0120] Standard track name Concentration (ng / mL) Original liquid volume Add the volume (μL) of diluent. STD1 400.00 500 μL solution 1 500 STD2 200.00 500 μL STD1 500 STD3 100.00 500 μL STD2 500 STD4 50.00 500 μL STD3 500 STD5 25.00 500 μL STD4 500 STD6 12.50 500 μL STD5 500 STD7 6.25 500 μL STD6 500 STD8 3.13 500 μL STD7 500 STD9 1.56 500 μL STD8 500 STD10 0.78 500 μL STD9 500 STD11 0.39 500 μL STD10 500

[0121] Experiment 3:

[0122] The antitoxin-coated antibody SM02 was coated at a concentration of 5.0 μg / mL. The SM10-HRP detection antibody was diluted to 2.5 μg / mL and incubated for 30 min. TMB color development was then terminated after 10 min. The antitoxin protein was diluted to 50 ng / mL using 1×Dilution Buffer and recorded as Solution 1. A total of 13 concentration points were obtained through 2-fold dilutions. OD450 data are shown in Table 3. It can be seen that a clear upper plateau was reached at 12.5 ng / mL, and a lower plateau was reached at 0.05 ng / mL. The inflection points for the upper and lower plateaus were considered to be 6.25 ng / mL and 0.097 ng / mL, respectively. The standard curve range was set to 6.25 ng / mL to 0.097 ng / mL. The fitted curve for this range is shown in Table 3. Figure 7 R 2 =0.997.

[0123] Table 3

[0124]

[0125]

[0126] Experiment 4:

[0127] The antitoxin-coated antibody SM02 was coated at a concentration of 5.0 μg / mL. The SM10-HRP detection antibody was diluted to 2.5 μg / mL and incubated for 30 min. TMB color development was then terminated after 10 min. Antitoxin protein was diluted to 12.5 ng / mL using 1×Dilution Buffer and recorded as Solution 1. Seven concentration points were obtained through 2-fold dilutions, with a standard curve range of 6.25 ng / mL to 0.097 ng / mL. Antitoxin protein was added to commonly used nucleic acid product buffers (TE buffer, sterile water for injection) and plasmid samples (preparation steps refer to patent CN118374547A) for detection. The results are shown in Table 4; the recovery rate was within 75% to 125%, demonstrating that this method can accurately determine the true content of antitoxin protein in the sample with minimal influence from the sample matrix. The coefficient of variation (CV, SD / mean × 100%) represents the inter-well variation and reflects the repeatability of the detection; a large CV (>35%) indicates unreliable results.

[0128] Table 4

[0129]

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0131] sequence of this article

[0132] SEQ ID NO: 1 SM02 Light Chain CDR1

[0133] WASSSVSH

[0134] SEQ ID NO: 2 SM02 Light Chain CDR2

[0135] DTT

[0136] SEQ ID NO: 3 SM02 Light Chain CDR3

[0137] QQWSSSPLT

[0138] SEQ ID NO: 4 SM02 Heavy chain CDR1

[0139] GFTFSSYG

[0140] SEQ ID NO: 5 SM02 Heavy chain CDR2

[0141] ISRGASYT

[0142] SEQ ID NO: 6 SM02 Heavy chain CDR3

[0143] ARQQYGNNYAWFAY

[0144] SEQ ID NO: 7 SM10 Light Chain CDR1

[0145] QSLVHSNGNTY

[0146] SEQ ID NO: 8 SM10 Light Chain CDR2

[0147] KVS

[0148] SEQ ID NO: 9 SM10 Light Chain CDR3

[0149] SQSTHVPHT

[0150] SEQ ID NO: 10 SM10 Heavy chain CDR1

[0151] GYIFTSYW

[0152] SEQ ID NO: 11 SM10 Heavy chain CDR2

[0153] IFPGTGIT

[0154] SEQ ID NO: 12 SM10 Heavy chain CDR3

[0155] ARSYSFYFDV

[0156] SEQ ID NO: 13 SM02 Light chain variable region

[0157] QIVLSQSPAILSASPGEKVTMTCWASSSVSHMHWYQQKPGSSPKPWIYDTTSLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSSPLTFGAGTKLELK

[0158] SEQ ID NO: 14 SM02 Heavy chain variable region

[0159] EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVASISRGASYTYYPDSVKGRFTISRDNAKNTLYLQMTSLKSEDTAMYYCARQQYGNNYAWFAYWGQGTLVTVSA

[0160] SEQ ID NO: 15 SM10 Light Chain Variable Region

[0161] DVVMTQNPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPHTFGGGTKLEIK

[0162] SEQ ID NO: 16 SM10 Heavy chain variable region

[0163] QVQLKQPGAELVRPGASVKLSCKTSGYIFTSYWIHWVKQRSGQGLEWIARIFPGTGITYYNEKFKDKATLTADKSSSTAYMQLSSLKSEDSAVYLCARSYSFYFDVWGAGTTVTVSS

[0164] SEQ ID NO: 17 SM02 Light Chain Signal Peptide

[0165] MDFQVQIFSFLLISASVIMSRG

[0166] SEQ ID NO: 18 SM02 Heavy chain signal peptide

[0167] MNFGLSLIFLALILKGVQC

[0168] SEQ ID NO: 19 SM10 light chain signal peptide

[0169] MKLPVRLLVLMFWIPVSSS

[0170] SEQ ID NO: 20 SM10 heavy chain signal peptide

[0171] MGWSWVFLFLLSGTAGVLC

[0172] SEQ ID NO: 21 SM02 Light Chain

[0173] MDFQVQIFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCWASSSVSHMHWYQQKPGSSPKPWIYDTTSLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSSPLT FGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0174] SEQ ID NO: 22 SM02 Heavy chain

[0175] MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVASISRGASYTYYPDSVKGRFTISRDNAKNTLYLQMTSLKSEDTAMYYCARQQYGNNYAWFAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK-

[0176] SEQ ID NO: 23 Light chain of SM10

[0177] MKLPVRLLVLMFWIPVSSSDVVMTQNPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPHTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0178] SEQ ID NO: 24 Heavy chain of SM10

[0179] MGWSWVFLFLLSGTAGVLCQVQLKQPGAELVRPGASVKLSCKTSGYIFTSYWIHWVKQRSGQGLEWIARIFPGTGITYYNEKFKDKATLTADKSSSTAYMQLSSLKSEDSAVYLCARSYSFYFDVWGAGTTVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0180] SEQ ID NO: 25 Antitoxin Protein Standard 0637

[0181] MSTDERKLEIIRKVSSDIDEPTLEAIEYLLSTPSDIPEPILRVIEQAMAEHHHHHH

Claims

1. An anti-toxin protein binding molecule, the binding molecule being an antibody or an antigen-binding fragment thereof targeting an anti-toxin protein as set forth in MSTDERKLEIIRKVSSDIDEPTLEAIEYLLSTPSDIPEPILRVIEQAMAE, the binding molecule comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions LCDRs, the LCDRs comprising an LCDR1 of SEQ ID NO: 1, an LCDR2 of SEQ ID NO: 2, and an LCDR3 of SEQ ID NO: 3; the heavy chain variable region comprising complementarity determining regions HCDRs, the HCDRs comprising an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO:

6. the light chain variable region is SEQ ID NO: 13 and the heavy chain variable region is SEQ ID NO:

14.

2. The binding molecule against a toxin protein of claim 1, wherein, the binding molecule further comprises a murine IgG Fc segment, a rabbit IgG Fc segment, or a human IgG Fc segment.

3. The binding molecule against a toxin protein of claim 1, wherein, the binding molecule further comprises a signal peptide.

4. The binding molecule against a toxin protein of claim 1, wherein, the light chain signal peptide is selected from SEQ ID NO: 17 and the heavy chain signal peptide is selected from SEQ ID NO:

18.

5. The binding molecule against a toxin protein of claim 4, wherein, the anti-toxin protein binding molecule pair comprises a first anti-toxin protein binding molecule and a second anti-toxin protein binding molecule, the binding molecules being antibodies or antigen-binding fragments thereof targeting an anti-toxin protein as set forth in MSTDERKLEIIRKVSSDIDEPTLEAIEYLLSTPSDIPEPILRVIEQAMAE, wherein, 6. A pair of binding molecules against a toxin protein, characterized in that, the first anti-toxin protein binding molecule comprises the following CDRs: an LCDR1 of SEQ ID NO: 1, an LCDR2 of SEQ ID NO: 2, an LCDR3 of SEQ ID NO: 3, and an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO: 6; the second anti-toxin protein binding molecule comprises the following CDRs: an LCDR1 of SEQ ID NO: 7, an LCDR2 of SEQ ID NO: 8, an LCDR3 of SEQ ID NO: 9, and an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO:

12. the second anti-toxin protein binding molecule further comprises a label selected from any one of a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron-dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, or an enzyme.

7. The pair of binding molecules against a toxin protein of claim 6, wherein, the anti-toxin protein binding molecule of any one of claims 1-5 or the binding molecule pair of claim 6 or 7.

8. A kit for detecting an anti-toxin protein, characterized by, ​ 9. The kit of claim 8, further comprising reagents for detecting binding of anti-toxin protein to an anti-toxin protein binding molecule or pair of anti-toxin protein binding molecules.

10. The kit of claim 8, wherein The anti-toxin protein binding molecule or pair of anti-toxin protein binding molecules are for use in a sandwich assay.

11. The kit of claim 10, wherein the first anti-toxin protein binding molecule is coupled to a support and the second anti-toxin protein binding molecule comprises a label.

12. The kit of claim 8, wherein The kit comprises a reaction plate coated with the first anti-toxin protein binding molecule, a biotin-labeled second anti-toxin protein binding molecule, an avidin-labeled catalyzing enzyme, and a substrate.

13. A non-diagnostic method of detecting the presence of an anti-toxin in a sample, said method comprising: The binding molecule of any one of claims 1-5 is incubated with a sample, and reagents are detected that bind to the anti-toxin protein and the binding molecule, thereby determining the presence of anti-toxin protein in the sample.

Images