Antigen binding protein pair specifically binding to enterotoxin SEC and application thereof

Abstract

The invention relates to an antigen binding protein pair specifically binding to enterotoxin SEC and application of the antigen binding protein pair. The antigen-binding protein pair of the present invention comprises a first antigen-binding protein 16E12 that binds to enterotoxin SEC and a second antigen-binding protein 9B7 that binds to enterotoxin SEC to which the first antigen-binding protein 16E12 is bound. The antigen binding protein disclosed by the invention has a unique variable region sequence, the lowest detection limit of the antigen binding protein to enterotoxin SEC is 0.1 ng / mL, and the antigen binding protein has no cross reaction to enterotoxins SEA, SEB, SED and SEE and common microbial toxins; the antigen binding protein disclosed by the invention is clear in sequence, capable of effectively avoiding gene mutation, small in batch difference, stable in quality and complete in structure, and contributes to the development of stable immunoassay detection methods.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, in particular to an antigen binding protein pair specifically binding to enterotoxin SEC and application thereof. Background Art

[0002] Staphylococcus aureus (S.aureus) is a common foodborne pathogen that is widely present in the natural environment. Under appropriate conditions, it can produce enterotoxins and cause food poisoning. Reports of food poisoning caused by S.aureus are endless. Food poisoning caused by S.aureus accounts for about 25% of foodborne microbial food poisoning incidents, and S.aureus has become the third largest foodborne pathogen after Salmonella and Vibrio parahaemolyticus. The enterotoxin secreted by S.aureus is a single-chain small molecule protein with a molecular weight of about 26-29kDa. It is heat-stable and damages the human intestine, causing symptoms such as vomiting and diarrhea. Common enterotoxins mainly include SEA, SEB, SEC, SED, and SEE.

[0003] Currently, the methods used for Staphylococcus aureus detection mainly include traditional separation and identification methods, molecular biology methods and immunological methods. Among them, traditional separation and identification methods usually include plate streaking and biochemical identification. Molecular biology detection methods usually include PCR, nucleic acid probe technology, loop-mediated isothermal amplification technology (LAMP). Immunological detection methods usually include ELISA, immunomagnetic bead method, immunochromatographic test strips, biosensor technology, etc.

[0004] Antibodies are the core reagents of immunoassays. The acquisition of traditional monoclonal antibodies requires the preparation of mouse immunization, hybridoma cell fusion, monoclonal screening, cell expansion and ascites production. Traditional monoclonal antibodies have the following limitations: During the cryopreservation, recovery and passage of hybridoma cells, antibody gene drift or loss may occur, resulting in decreased monoclonal antibody performance; during the ascites production process, due to individual differences in mice, hemolysis may occur, thus affecting the quality of the antibody; in addition, the long-term storage of hybridoma cells is highly dependent on liquid nitrogen, which needs to be replenished at any time, and the storage cost is high. Recombinant antibodies based on genetic engineering technology can circumvent these limitations. Compared with antibodies prepared by the traditional ascites method, the advantages of recombinant antibodies are: repeatability, the information of antibody genes can be permanently maintained without loss, and they have accuracy and batch consistency; remodelability, the type or subtype of antibodies can be changed by genetic recombination, and the performance of antibodies can also be enhanced; no experimental animals are required, reducing harm to animals; high purity, recombinant antibodies are expressed under serum-free conditions to avoid contamination by serum components. The existing recombinant antibodies that recognize enterotoxin SEC can recognize not only SEC but also enterotoxin SEB, but have poor specificity, and the minimum detection limit of SEC is 8 ng / mL, which has low sensitivity. Therefore, it is urgent to develop a recombinant antibody against Staphylococcus aureus enterotoxin SEC with good specificity and high sensitivity. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of recombinant enterotoxin SEC antibodies with good specificity and high sensitivity in the prior art.

[0006] In order to solve the above technical problems, the present invention provides an antigen binding protein pair that specifically binds to enterotoxin SEC, which are named 9B7 and 16E12 respectively. 9B7 and 16E12 have unique variable region sequences, 16E12 is used as a capture antibody, 9B7 is used as a detection antibody, and a double antibody sandwich detection method is used to detect enterotoxin SEC, and its minimum detection limit is 0.1ng / mL, and there is no cross reaction to enterotoxin SEA, SEB, SED, SEE and common microbial toxins. The antigen binding protein pair of the present invention has good specificity, high sensitivity, accuracy and batch consistency.

[0007] The first object of the present invention is to provide an antigen binding protein pair that specifically binds to enterotoxin SEC, the antigen binding protein pair comprising a first antigen binding protein 16E12 that binds to enterotoxin SEC and a second antigen binding protein 9B7 that binds to enterotoxin SEC bound to the first antigen binding protein 16E12, wherein:

[0008] The heavy chain variable region of the first antigen binding protein 16E12 comprises heavy chain complementary determining regions VH-CDR4, VH-CDR5, and VH-CDR6, and the amino acid sequences are shown in SEQ ID NOs. 15-17 or sequences with a homology of not less than 90% thereto;

[0009] The light chain variable region of the first antigen binding protein 16E12 comprises light chain complementary determining regions VL-CDR4, VL-CDR5, and VL-CDR6, and the amino acid sequences are shown in SEQ ID NOs. 18-20 or sequences with a homology of not less than 90% thereto;

[0010] The heavy chain variable region of the second antigen binding protein 9B7 comprises heavy chain complementary determining regions VH-CDR1, VH-CDR2, and VH-CDR3, and the amino acid sequence is as shown in SEQ ID NO.1-3 or a sequence with a homology of not less than 90% thereto;

[0011] The light chain variable region of the second antigen binding protein 9B7 comprises light chain complementary determining regions VL-CDR1, VL-CDR2, and VL-CDR3, and the amino acid sequence is as shown in SEQ ID NO.4-6 or a sequence with a homology of not less than 90% thereto.

[0012] Furthermore, the antigen binding protein may be an antibody or an antigen binding fragment thereof.

[0013] Furthermore, the heavy chain variable region of the first antigen binding protein 16E12 comprises framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8; the framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8 respectively comprise amino acid sequences as shown in SEQ ID NO.21-24 or sequences with a homology of not less than 90% thereto;

[0014] The heavy chain variable region of the second antigen-binding protein 9B7 comprises framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4; the framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4 respectively comprise amino acid sequences shown in SEQ ID NO.7-10 or sequences with a homology of not less than 90% thereto.

[0015] Furthermore, a complementary determining region is arranged between two adjacent framework regions of the second antigen-binding protein 9B7, so that VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3, and VH-FR4 are arranged in sequence on the heavy chain variable region of the second antigen-binding protein 9B7, and VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3, and VL-FR4 are arranged in sequence on the light chain variable region.

[0016] Furthermore, a complementary determining region is arranged between two adjacent framework regions of the first antigen binding protein 16E12, so that VH-FR5, VH-CDR4, VH-FR6, VH-CDR5, VH-FR7, VH-CDR6, and VH-FR8 are arranged in sequence on the heavy chain variable region of the first antigen binding protein 16E12, and VL-FR5, VL-CDR4, VL-FR6, VL-CDR5, VL-FR7, VL-CDR6, and VL-FR8 are arranged in sequence on the light chain variable region.

[0017] Furthermore, the second antigen binding protein 9B7 also comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.31.

[0018] Furthermore, the first antigen binding protein 16E12 also includes a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.31.

[0019] Furthermore, the species of origin of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans.

[0020] Furthermore, the constant region is selected from one of mouse IgG1, IgG2a, IgG2b, IgG3 or human IgG1, IgG2, IgG3, IgG4, and IgM.

[0021] The second object of the present invention is to provide nucleic acid molecules encoding the above antigen-binding protein pairs.

[0022] The third object of the present invention is to provide an expression vector containing the above nucleic acid molecule.

[0023] Furthermore, the expression vector may be a viral vector or a non-viral vector, such as DNA, RNA, a viral vector (such as a lentivirus, adenovirus, AAV virus, retrovirus or a combination thereof), a plasmid, a transposon, other gene transfer systems, liposome nanoparticles, etc.

[0024] Further, the host cell may be a prokaryotic cell or a eukaryotic cell, such as a plant cell, an animal cell, a microorganism, etc. Preferably, the host cell is a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell (HEK293), a HeLa cell, a baby hamster kidney cell, a NS0 mouse myeloma cell, or one of other mammalian cells.

[0025] A fifth object of the present invention is to provide a monovalent antibody, a bivalent antibody or a multivalent antibody comprising the above-mentioned antigen-binding protein pair.

[0026] A sixth object of the present invention is to provide a recombinant protein or immunoconjugate containing the above antigen-binding protein pair.

[0027] Furthermore, the recombinant protein contains an antigen binding protein pair and a tag sequence that assists expression and / or purification.

[0028] Furthermore, the immunoconjugate contains an antigen binding protein pair and a conjugated moiety, such as a detectable label.

[0029] The seventh object of the present invention is to provide a detection kit for enterotoxin SEC, comprising the antigen-binding protein pair, nucleic acid molecules, expression vectors, host cells, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins or immunoconjugates.

[0030] Furthermore, the detection includes flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay (ELISA) detection, etc.

[0031] The eighth object of the present invention is to provide an application of the above-mentioned antigen-binding protein pair, the above-mentioned recombinant protein or immunoconjugate, the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned host cell or the above-mentioned kit in the preparation of enterotoxin SEC detection products.

[0032] Beneficial effects of the present invention:

[0033] The present invention successfully obtains an antigen-binding protein pair capable of specifically recognizing enterotoxin SEC through continuous screening. The antigen-binding protein pair of the present invention has a unique variable region sequence and has good specificity and sensitivity to Staphylococcus aureus enterotoxin SEC antigen; the antigen-binding protein pair of the present invention has a clear sequence and can effectively avoid gene mutation; the antigen-binding protein pair has small batch-to-batch differences, stable quality, and complete structure, which is helpful for developing a stable immunoassay detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0035] Figure 1 is an agarose gel electrophoresis diagram of the antibody gene obtained by PCR amplification in the present invention;

[0036] Figure 2 is the SDS-PAGE protein electrophoresis diagram of the recombinant antibody of the present invention;

[0037] Figure 3 is the sandwich ELISA standard curve of the recombinant antibody of the present invention for SEC (the concentrations from high to low are 150, 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0 ng / mL);

[0038] Figure 4 It is the specific detection result of the recombinant antibody of the present invention to SEC. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Definition of terms

[0041] In the present invention, the term "specific binding" generally refers to the binding of an antibody to an epitope through its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an antigen when it binds to an epitope more readily through its antigen binding domain than it would bind to a random, unrelated epitope.

[0042] In the present invention, the term "isolated" or "purified" generally refers to a molecule (e.g., antibody, nucleic acid, etc.) that is at least partially separated from other molecules with which it is normally associated in its native state. An "isolated or purified polypeptide" or "isolated or purified nucleic acid" is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth medium.

[0043] In the present invention, the term "antigen binding protein" is used in its broad sense and means a protein comprising a portion that binds to an antigen or target and optionally comprising a framework or framework portion that allows the antigen binding portion to adopt a configuration that promotes the binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Antigen binding proteins may include, for example, chimeric antigen receptors with transplanted CDRs or CDR derivatives, alternative protein frameworks or artificial frameworks. Such frameworks include, but are not limited to: antibody-derived frameworks that include mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks that include, for example, biocompatible polymers.

[0044] In the present invention, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies and camelized single domain antibodies (such as heavy chain variable domain antibodies). Antibodies generally have the structure of immunoglobulins, and may include proteins of at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulins are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of the disulfide bonds of the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain according to the difference in the constant region. Each of the five classes of Ig can have κ chain or λ chain.

[0045] In the present invention, the term "variable" generally refers to that some parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed in the entire variable region of an antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementary determining regions (CRDs) or hypervariable regions (HVRs). The more highly conserved parts in the variable domains are called frameworks (FRs). The variable domains of natural heavy and light chains each include four FR regions, most of which adopt a β-folded configuration, are connected by three CDRs, form loop connections, and form a part of the β-folded structure in some cases. The CDRs in each chain are closely together through the FR region, and together with the CDRs from another chain, form the antigen binding site of the antibody, and the constant region does not directly participate in the binding of the antibody to the antigen. In the art, the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule, the Chothia definition rule or the IMGT definition rule based on sequence variability.

[0046] The sequence information involved in the present invention is as follows:

[0047]

[0048]

[0049] Example 1: Isolation and identification of variable region genes of monoclonal antibodies against Staphylococcus aureus enterotoxin SEC

[0050] Resuscitate the hybridoma cell line producing anti-Staphylococcus aureus enterotoxin SEC monoclonal antibody in a culture flask, and collect (5-10)×10 6 cells, add 1mL Trizol reagent, blow and mix, mix thoroughly to lyse; add 200μL chloroform to the lysate, shake for 15s to obtain an emulsion, let it stand at 4℃ for 5min, and then centrifuge at 12000g for 15min; take 450μL of the upper colorless aqueous phase, add an equal volume of pre-cooled isopropanol, mix upside down, let it stand at 4℃ for 10min, and then centrifuge at 12000g for 10min; discard the supernatant, add 1mL 75% ethanol to wash the precipitate, and centrifuge at 12000g for 10min; discard the supernatant, resuspend the precipitate with 100μL RNase-free water, and store it at -80℃.

[0051] Using RNA as a template, the first-strand cDNA synthesis and cDNA end rapid amplification were performed using a RACE 5' / 3'Kit kit (purchased from Takara), respectively. The heavy chain and light chain of the antibody corresponded to different gene-specific primers. The specific primers for the 9B7 antibody were named H1-5'GSP and L1-5'GSP, and the sequence of H-5'GSP was GAT TAC GCC AAG CTT CTC AAGTTT TTT GTC CAC CGT GG TGC, and the sequence of L-5'GSP was GAT TAC GCC AAG CTT CTC ATT CCT GTTGAA GCT CTT GAC AAT GGG. The specific primers for the 16E12 antibody were named H2-5'GSP and L2-5'GSP, and the sequence of H2-5'GSP was GAT TAC GCC AAG CTT CTC AAG TTT TTT GTC CAC CGT GGT GC, and the sequence of L2-5'GSP was GAT TAC GCC AAG CTT CTC ATT CCT GTT GAA GCT CTT GAC AAT GGG. like Figure 1 As shown (from left to right, the heavy chain and light chain of 9B7 and 16E12), bright target bands can be obtained by agarose gel electrophoresis, and the target bands contain VH and VL gene fragments, respectively.

[0052] Purify the target gene to 20 μL with a gel recovery kit, clone the purified product into a linearized pRACE plasmid in an In-fusion manner, transform Stellar competent cells, and apply to LB solid medium (containing ampicillin). The next day, 6-8 single colonies were taken from VH and VL, and sent to a gene sequencing company for sequencing after expansion, and the sequencing primer was M13-F / R universal primer. The gene sequence obtained by the above sequencing was imported into the Kabat antibody database for comparison and analysis, and the VH and VL genes, CDR regions, and framework regions were identified, wherein the gene lengths of the variable regions in the VH gene sequence were 417 bp and 411 bp, respectively, with a 57 bp signal peptide sequence in front; the gene lengths of the variable regions in the VL gene sequence were 387 bp and 396 bp, with a 60 bp and 66 bp signal peptide sequence in front.

[0053] Example 2: Construction of recombinant antibody expression plasmid

[0054] According to the heavy chain and light chain variable region genes of the anti-Staphylococcus aureus enterotoxin SEC monoclonal antibody obtained by sequencing, specific primers were designed, and the heavy chain and light chain variable region genes were amplified by PCR using the pRACE heavy and light chain plasmids as templates. They were homologously recombined into the pcDNA3.4 backbone plasmids containing the heavy chain constant region and the light chain constant region, respectively, to obtain recombinant antibody expression plasmids containing full-length heavy chain and light chain genes. The heavy chain and light chain expression plasmids were transformed into Top 10 competent cells, and single colonies were picked and sequenced to verify the accuracy of the sequence. The single colonies corresponding to the heavy chain and light chain expression plasmids with correct sequencing were taken, expanded, and the heavy chain and light chain expression plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0055] Example 3: Expression and purification of recombinant antibodies

[0056] Take a tube of HEK293F suspension cells from the liquid nitrogen tank. The number of cells should be ≥ 10. 7 After rapid thawing in a 37°C water bath, transfer to 30 mL of pre-warmed culture medium and culture in a 125 mL shake flask to a final density of approximately 0.3 × 10 6 cells / mL, the culture medium is Sino Biological's SMM-293TII culture medium. The incubator conditions are set at: 37°C, 125rpm, 5% CO2, humidity>80%, and culture for 3-4 days. The cells can grow to 3×10 6 cells / mL, with a viability greater than 95%, continue to subculture for 2 more times, ensuring a doubling time of about 24h, and use them as seed cells.

[0057] One day before transfection, 1.5 × 10 6 HEK293F cells were inoculated into a 500 mL shake flask at a density of 120 mL. The next day, the cells grew to 3×10 6 cells / mL, cell viability>95%, start cell transfection. The total amount of heavy chain and light chain expression plasmids is 4μg per ml of culture volume, the ratio of heavy chain to light chain is 1:1.5, the transfection reagent is PEI, and the amount of PEI is 2.5 times the amount of plasmid. Mix the plasmid and PEI in fresh culture medium, let stand at room temperature for 15-20 minutes, slowly add to the shake flask, and then add appropriate amount of fresh culture medium to dilute the final cell density to 2×10 6 cells / mL, and return to the shaker incubator for culture. Add 3.5% of the culture volume of feed solution 20-24 hours after transfection, and continue to add 3.5% of the culture volume of feed solution on the 3rd and 5th days after transfection. Harvest the cell culture supernatant on the 7th day after transfection or when the cell viability is <60%.

[0058] The cell culture supernatant was collected by high-speed centrifugation to remove cells and cell debris. The culture supernatant was filtered through a 0.45 μm filter membrane and the antibody was purified using a Protein G affinity chromatography column. The antibody was dialyzed into 0.01 M PBS buffer and stored at -20°C. A small amount of antibody was taken for reducing SDS-PAGE electrophoresis verification. Figure 2 As shown, there are 2 protein bands, one of which is a heavy chain with a molecular weight of about 50 kDa, and the other is a light chain with a molecular weight of about 25 kDa.

[0059] Example 4: Performance testing of recombinant antibodies

[0060] The above recombinant antibodies were used for SEC sandwich ELISA detection, and the specific steps were as follows: the capture antibody 16E12 was diluted to 4 μg / mL with 0.05M carbonate buffer (pH 9.6), added to a 96-well ELISA plate at 100 μL / well, and incubated at 37°C for 2 hours; washed with PBST washing solution 3 times, 3 minutes each time; added 300 μL / well blocking solution, and incubated at 37°C for 2 hours; added gradient diluted SEC standards (100 μL / well) and detection antibody 9B7 (4 μg / mL, 100 μL / well), and incubated at 37°C for 1 hour; after washing, HRP goat anti-mouse IgG (100 μL / well) was added, and incubated at 37°C for 1 hour; after washing, TMB substrate solution (100 μL / well) was added, and reacted at 37°C for 15 minutes; the stop solution (50 μL / well) was added to stop the reaction, and the absorbance at 450 nm was measured with an ELISA reader.

[0061] The standard curve of recombinant antibody for SEC is as follows Figure 3 As shown, the recombinant antibody pair can detect SEC at 0.21 ng / mL, and the theoretical value of its minimum detection limit is 0.1 ng / mL (LOD is OD blank ×2.1 absorbance value corresponding to the antigen concentration), indicating that the recombinant antibody has good sensitivity to SEC.

[0062] Cross-reaction results such as Figure 4 As shown, the recombinant antibody of the present invention is specific only to enterotoxin SEC, and has no cross-reaction to enterotoxins SEA, SEB, SED, SEE and common microbial toxins such as Bacillus cereus hemolysin Hbl B, non-hemolysin Nhe A, Nhe C and Listeria monocytogenes hemolysin LLO. The recombinant antibody of the present invention has good specificity.

[0063] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An antigen binding protein pair that specifically binds to enterotoxin SEC, characterized in that: The antigen binding protein pair comprises a first antigen binding protein 16E12 that binds to enterotoxin SEC and a second antigen binding protein 9B7 that binds to enterotoxin SEC bound to the first antigen binding protein 16E12, wherein: The heavy chain variable region of the first antigen binding protein 16E12 comprises heavy chain complementary determining regions VH-CDR4, VH-CDR5, and VH-CDR6, and the amino acid sequences are shown in SEQ ID NOs. 15-17 or sequences with a homology of not less than 90% thereto; The light chain variable region of the first antigen binding protein 16E12 comprises light chain complementary determining regions VL-CDR4, VL-CDR5, and VL-CDR6, and the amino acid sequences are shown in SEQ ID NOs. 18-20 or sequences with a homology of not less than 90% thereto; The heavy chain variable region of the second antigen binding protein 9B7 comprises heavy chain complementary determining regions VH-CDR1, VH-CDR2, and VH-CDR3, and the amino acid sequence is as shown in SEQ ID NO.1-3 or a sequence with a homology of not less than 90% thereto; The light chain variable region of the second antigen binding protein 9B7 comprises light chain complementary determining regions VL-CDR1, VL-CDR2, and VL-CDR3, and the amino acid sequence is as shown in SEQ ID NO.4-6 or a sequence with a homology of not less than 90% thereto.

2. The antigen-binding protein according to claim 1, characterized in that The heavy chain variable region of the first antigen binding protein 16E12 comprises framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8; the framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8 respectively comprise amino acid sequences as shown in SEQ ID NO.21-24 or sequences with a homology of not less than 90% thereto; The heavy chain variable region of the second antigen-binding protein 9B7 comprises framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4; the framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4 respectively comprise amino acid sequences shown in SEQ ID NO.7-10 or sequences with a homology of not less than 90% thereto.

3. The antigen-binding protein according to claim 1, characterized in that The light chain variable region of the first antigen binding protein 16E12 comprises framework regions VL-FR5, VL-FR6, VL-FR7, and VL-FR8; the framework regions VL-FR5, VL-FR6, VL-FR7, and VL-FR8 respectively comprise amino acid sequences as shown in SEQ ID NO.25-28 or sequences with a homology of not less than 90% thereto; The light chain variable region of the second antigen-binding protein 9B7 comprises framework regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4; the framework regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4 respectively comprise amino acid sequences shown in SEQ ID NO.11-14 or sequences with a homology of not less than 90% thereto.

4. The antigen-binding protein according to claim 1, characterized in that The second antigen binding protein 9B7 further comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.

31.

5. The antigen-binding protein according to claim 1, characterized in that The first antigen binding protein 16E12 further includes a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.

31.

6. A nucleic acid molecule encoding the antigen binding protein pair according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid molecule according to claim 6.

8. A host cell containing the antigen binding protein pair according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or the expression vector according to claim 7.

9. A kit, characterized in that: The kit comprises the antigen binding protein pair according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 or the host cell according to claim 8.

10. Use of the antigen binding protein pair according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 or the host cell according to claim 8 or the kit according to claim 9 in the preparation of an enterotoxin SEC detection product.

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