An antigen binding protein pair specifically binding to enterotoxin sec and use thereof

Abstract

The present application relates to a kind of antigen binding protein pair specifically binding enterotoxin SEC and its application.The antigen binding protein pair of the present application includes the first antigen binding protein 16E12 combined with enterotoxin SEC and the second antigen binding protein 9B7 combined with enterotoxin SEC combined with the first antigen binding protein 16E12.The antigen binding protein of the present application has unique variable region sequence, the minimum detection limit for enterotoxin SEC is 0.1ng / mL, and there is no cross reaction for enterotoxin SEA, SEB, SED, SEE and common microbial toxin;The antigen binding protein sequence of the present application is clear, can effectively avoid gene mutation, the batch difference of antigen binding protein is small, quality is stable, structure is complete, it is helpful to the development of stable immune analysis detection method.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to an antigen-binding protein pair that specifically binds to enterotoxin SEC and its application. Background Technology

[0002] Staphylococcus aureus (S. aureus) is a common foodborne pathogen, widely distributed in the natural environment. Under suitable conditions, it can produce enterotoxins, causing food poisoning. Reports of food poisoning caused by S. aureus are frequent, accounting for approximately 25% of foodborne microbial food poisoning incidents, and S. aureus has become the third most common foodborne pathogen after Salmonella and Vibrio parahaemolyticus. The enterotoxins secreted by S. aureus are single-chain small protein molecules with a molecular weight of approximately 26-29 kDa. They are heat-stable and damage the human intestine, leading to symptoms such as vomiting and diarrhea. Common enterotoxins include SEA, SEB, SEC, SED, and SEE.

[0003] Currently, the main methods used for detecting Staphylococcus aureus include traditional isolation and identification methods, molecular biological methods, and immunological methods. Traditional isolation and identification methods typically include streak plating and biochemical identification. Molecular biological detection methods typically include PCR, nucleic acid probe technology, and loop-mediated isothermal amplification (LAMP). Immunological detection methods typically include ELISA, immunomagnetic beads, immunochromatographic test strips, and biosensor technology.

[0004] Antibodies are the core reagents for immunoassay. Traditional monoclonal antibody production involves mouse immunization, hybridoma cell fusion, monoclonal screening, cell expansion, and ascites fluid production. Traditional monoclonal antibodies have the following limitations: antibody gene drift or loss can occur during the cryopreservation, thawing, and passage of hybridoma cells, leading to a decline in monoclonal antibody performance; hemolysis can occur during ascites fluid production due to individual mouse differences, affecting antibody quality; furthermore, long-term preservation of hybridoma cells is heavily reliant on liquid nitrogen, requiring constant replenishment and incurring high preservation costs. Recombinant antibodies based on genetic engineering technology can circumvent these limitations. Compared to antibodies prepared using the traditional ascites fluid method, recombinant antibodies offer the following advantages: reproducibility (antibody gene information is permanently preserved and not lost, ensuring accuracy and batch-to-batch consistency); remodeling capability (antibody type or subtype can be altered through gene recombination, enhancing antibody performance); no need for experimental animals, reducing animal harm; and high purity (recombinant antibodies are expressed under serum-free conditions, avoiding serum contamination). Existing recombinant antibodies that recognize enterotoxin SEC can recognize not only SEC but also enterotoxin SEB, exhibiting poor specificity. Furthermore, the detection limit for SEC is only 8 ng / mL, indicating low sensitivity. Therefore, there is an urgent need to develop a recombinant antibody against Staphylococcus aureus enterotoxin SEC with high specificity and 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] To address the aforementioned technical problems, this invention provides an antigen-binding protein pair specifically binding to enterotoxin SEC, named 9B7 and 16E12, respectively. Utilizing the unique variable region sequences of 9B7 and 16E12, 16E12 is used as the capture antibody and 9B7 as the detection antibody. A double-antibody sandwich assay is employed to detect enterotoxin SEC, achieving a limit of detection of 0.1 ng / mL. Furthermore, no cross-reactivity is observed with enterotoxins SEA, SEB, SED, SEE, and common microbial toxins. The antigen-binding protein pair of this invention exhibits high specificity, high sensitivity, accuracy, and batch-to-batch consistency.

[0007] The first objective of this invention is to provide an antigen-binding protein pair that specifically binds to enterotoxin SEC, said 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 to which the first antigen-binding protein 16E12 is bound, wherein:

[0008] The heavy chain variable region of the first antigen-binding protein 16E12 includes heavy chain complementarity-determining regions VH-CDR4, VH-CDR5, and VH-CDR6, with amino acid sequences shown in SEQ ID NO.15-17 or sequences with at least 90% homology to them.

[0009] The light chain variable region of the first antigen-binding protein 16E12 includes light chain complementarity-determining regions VL-CDR4, VL-CDR5, and VL-CDR6, with amino acid sequences shown in SEQ ID NO.18-20 or sequences with at least 90% homology to them.

[0010] The heavy chain variable region of the second antigen-binding protein 9B7 includes heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3, with amino acid sequences shown in SEQ ID NO.1-3 or sequences with at least 90% homology to them.

[0011] The light chain variable region of the second antigen-binding protein 9B7 includes light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3, with amino acid sequences shown in SEQ ID NO.4-6 or sequences with at least 90% homology to them.

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

[0013] Furthermore, the heavy chain variable region of the first antigen-binding protein 16E12 includes framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8; the framework regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8 each contain amino acid sequences shown in SEQ ID NO. 21-24 or sequences with at least 90% homology to them;

[0014] The heavy chain variable region of the second antigen-binding protein 9B7 includes frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4; the frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4 each contain amino acid sequences shown in SEQ ID NO. 7-10 or sequences with at least 90% homology to them.

[0015] Furthermore, a complementary determinant region is provided between two adjacent framework regions of the second antigen-binding protein 9B7. Therefore, VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3, and VH-FR4 are sequentially provided 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 sequentially provided on the light chain variable region.

[0016] Furthermore, a complementary determinant region is provided between two adjacent framework regions of the first antigen-binding protein 16E12. Therefore, VH-FR5, VH-CDR4, VH-FR6, VH-CDR5, VH-FR7, VH-CDR6, and VH-FR8 are sequentially provided 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 sequentially provided on the light chain variable region.

[0017] Furthermore, the second antigen-binding protein 9B7 also includes 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 source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, 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, IgM.

[0021] A second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned antigen-binding protein pair.

[0022] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid molecules.

[0023] Furthermore, the expression vector can be a viral vector or a non-viral vector, such as DNA, RNA, viral vectors (e.g., lentivirus, adenovirus, AAV virus, retrovirus or combinations thereof), plasmids, transposons, other gene transfer systems, liposome nanoparticles, etc.

[0024] Furthermore, the host cell can be a prokaryotic or eukaryotic cell, such as plant cells, animal cells, microorganisms, etc. Preferably, the host cell is one of the following: Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293), HeLa cells, young hamster kidney cells, NSO mouse myeloma cells, or other mammalian cells.

[0025] A fifth objective of this invention is to provide monovalent, bivalent, or multivalent antibodies containing the aforementioned antigen-binding protein pairs.

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

[0027] Furthermore, the recombinant protein contains an antigen-binding protein pair and a tag sequence to assist in expression and / or purification.

[0028] Furthermore, the immunoconjugate contains an antigen-binding protein pair and a conjugation portion, such as a detectable marker.

[0029] A seventh object of the present invention is to provide a detection kit for enterotoxin SEC, comprising the antigen-binding protein pair, nucleic acid molecule, expression vector, host cell, monovalent antibody, bivalent antibody, multivalent antibody, recombinant protein or immunoconjugate.

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

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

[0032] The beneficial effects of this invention are:

[0033] This invention has successfully obtained an antigen-binding protein pair that can specifically recognize enterotoxin SEC through continuous screening. The antigen-binding protein pair of this invention has a unique variable region sequence and exhibits good specificity and sensitivity to Staphylococcus aureus enterotoxin SEC antigen. The antigen-binding protein pair of this invention has a well-defined sequence, which can effectively avoid gene mutation. The antigen-binding protein pair has small batch-to-batch variability, stable quality, and intact structure, which is conducive to the development of stable immunoassay detection methods. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

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

[0036] Figure 2 This is an SDS-PAGE protein electrophoresis image of the recombinant antibody in this invention;

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

[0038] Figure 4 This is the specific detection result of the recombinant antibody against SEC in this invention. Detailed Implementation

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

[0040] Terminology Definition

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

[0042] In this invention, the terms "isolated" or "purified" generally refer to molecules (e.g., antibodies, nucleic acids, etc.) that are at least partially isolated from other molecules that are normally bound to them in their natural state. "Isolated or purified polypeptides" or "isolated or purified nucleic acids" are essentially free of other biomolecules such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth media.

[0043] In this invention, the term "antigen-binding protein" is used broadly and refers to a protein comprising a portion that binds to an antigen or target and optionally comprising a framework or frame portion that allows the antigen-binding portion to adopt a configuration that promotes antigen-binding protein binding to an 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 having a transplanted CDR or CDR derivative, alternative protein frameworks, or artificial frameworks. Such frameworks include, but are not limited to: antibody-derived frameworks containing mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein; and fully synthetic frameworks containing, for example, biocompatible polymers.

[0044] In this 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 (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelified single-domain antibodies (e.g., heavy chain variable domain antibodies). Antibodies typically have the structure of immunoglobulins and may comprise proteins, or antigen-binding fragments thereof, containing at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The amino acid composition and sequence of the immunoglobulin heavy chain constant regions differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Based on differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in their heavy chain, Ig can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0045] In this invention, the term "variable" generally refers to the strong variation in certain portions of the sequence of the variable domain of an antibody, which contributes to the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, referred to as complementarity-determining regions (CRDs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domain are referred to as frames (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, mostly in a β-sheet configuration, linked by three CDRs to form loops, and in some cases forming part of a β-sheet structure. The CDRs in each chain are closely clustered together by the FR regions and, together with CDRs from the other chain, form the antigen-binding site of the antibody. Constant regions do not directly participate in antibody-antigen binding. In the art, antibody CDRs can be defined using various 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 this invention is as follows:

[0047]

[0048]

[0049] Example 1: Isolation and identification of the variable region gene of monoclonal antibody against Staphylococcus aureus enterotoxin SEC

[0050] Hybridoma cell lines producing monoclonal antibodies against Staphylococcus aureus enterotoxin SEC were revived and cultured in culture flasks. After expansion, (5-10) × 10⁻⁶ cells were collected. 6 For each cell, add 1 mL of Trizol reagent, mix thoroughly by pipetting, and lyse. Add 200 μL of chloroform to the lysis buffer, shake for 15 s to obtain an emulsion, incubate at 4 °C for 5 min, and centrifuge at 12000 g for 15 min. Take 450 μL of the colorless aqueous phase, add an equal volume of pre-cooled isopropanol, mix by inverting, incubate at 4 °C for 10 min, and centrifuge at 12000 g for 10 min. Discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, centrifuge at 12000 g for 10 min, discard the supernatant, resuspend the precipitate in 100 μL of RNase-free water, and store at -80 °C.

[0051] Using RNA as a template, the RACE 5' / 3' Kit (purchased from Takara) was used for first-strand cDNA synthesis and rapid amplification of cDNA ends. Different gene-specific primers were used for the heavy and light chains of the antibodies. The specific primers for the 9B7 antibody were named H1-5'GSP and L1-5'GSP. 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. 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 and light chains are 9B7 and 16E12 respectively), agarose gel electrophoresis yields bright target bands, which contain VH and VL gene fragments respectively.

[0052] The target gene was purified to 20 μL using a gel extraction kit. The purified product was cloned into a linearized pRACE plasmid using in-fusion and transformed into Stellar competent cells. The cells were then plated on LB solid medium (containing ampicillin). The next day, 6-8 single colonies each of VH and VL were collected, expanded, and sent to a gene sequencing company for sequencing using universal M13-F / R primers. The gene sequences obtained from the sequencing were imported into the Kabat antibody database for comparison and analysis to identify the VH and VL genes, as well as the CDR and backbone regions. The variable regions of the VH and VL gene sequences were 417 bp and 411 bp in length, respectively, preceded by a 57 bp signal peptide sequence. The variable regions of the VL gene sequences were 387 bp and 396 bp in length, respectively, preceded by 60 bp and 66 bp signal peptide sequences.

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

[0054] Based on the heavy and light chain variable region genes of the monoclonal antibody against Staphylococcus aureus enterotoxin SEC obtained through sequencing, specific primers were designed. Using the pRACE heavy and light chain plasmid as a template, the heavy and light chain variable region genes were amplified by PCR and homologously recombined into the pcDNA3.4 backbone plasmid containing the heavy and light chain constant regions, respectively, to obtain recombinant antibody expression plasmids containing the full-length heavy and light chain genes. The heavy and light chain expression plasmids were transformed into Top 10 competent cells, and single colonies were picked and sequenced to verify the sequence accuracy. The single colonies corresponding to the correctly sequenced heavy and light chain expression plasmids were taken, expanded, and the heavy 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 suspended cells from the liquid nitrogen tank; cell count ≥10-1 7 Each sample was rapidly thawed in a 37°C water bath and transferred to 30 mL of preheated culture medium. It was then cultured in 125 mL shake flasks until a final density of approximately 0.3 × 10⁻⁶ was achieved. 6 Cells / mL, cultured on SMM-293TII medium from Sinocare. Incubator conditions were set as follows: 37℃, 125 rpm, 5% CO2, humidity >80%, cultured for 3-4 days. Cells grew to 3 × 10⁶ cells / mL. 6 Cells / mL, viability greater than 95%, continue passage twice to ensure doubling time of about 24 hours, and use as seed cells.

[0057] The day before transfection, administer 1.5 × 10 6 HEK293F cells were seeded at a density of 120 mL in a 500 mL shake flask. The cells grew to 3 × 10⁶ cells / mL the following day. 6 Cell transfection was initiated when the cell density reached 95% (cells / mL) and cell viability was >95%. The total amount of heavy and light chain expression plasmids used was 4 μg per mL of culture volume, with a heavy-to-light chain ratio of 1:1.5. PEI was used as the transfection reagent, with the amount of PEI being 2.5 times the amount of plasmid. The plasmid and PEI were thoroughly mixed in fresh culture medium and incubated at room temperature for 15-20 minutes. The mixture was then slowly added to a shake flask, followed by dilution with an appropriate amount of fresh culture medium to a final cell density of 2 × 10⁶ cells / mL. 6 Cells / mL, return to shake incubator for further culture. Add 3.5% culture volume of feed solution 20-24 hours after transfection, and continue adding 3.5% culture volume of feed solution on days 3 and 5 post-transfection. Harvest the cell culture supernatant on day 7 post-transfection or when cell viability is <60%.

[0058] Cell culture supernatant was collected by high-speed centrifugation to remove cells and cell debris. The supernatant was filtered through a 0.45 μm filter and then 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 tested by reducing SDS-PAGE electrophoresis for verification. Figure 2 As shown, there are two 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 antibody pair was applied to the sandwich ELISA detection of SEC, and the specific steps are as follows: The capture antibody 16E12 was diluted to 4 μg / mL with 0.05M carbonate buffer (pH 9.6), and 100 μL / well was added to a 96-well ELISA plate and incubated at 37°C for 2 h; washed 3 times with PBST washing buffer for 3 min each time; 300 μL / well blocking buffer was added and incubated at 37°C for 2 h; serially diluted SEC standard (100 μL / well) and detection antibody 9B7 (4 μg / mL, 100 μL / well) were added and incubated at 37°C for 1 h; after washing, HRP goat anti-mouse IgG (100 μL / well) was added and incubated at 37°C for 1 h; after washing, TMB substrate solution (100 μL / well) was added and reacted at 37°C for 15 min; stop solution (50 μL / well) was added to stop the reaction, and the absorbance at 450 nm was measured using an ELISA reader.

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

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

[0063] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antibody pair that specifically binds to enterotoxin SEC, characterized in that, The antibody pair includes a first antibody 16E12 that binds to enterotoxin SEC and a second antibody 9B7 that binds to enterotoxin SEC to which the first antibody 16E12 is bound, wherein: The heavy chain variable region of the first antibody 16E12 includes heavy chain complementarity-determining regions VH-CDR4, VH-CDR5, and VH-CDR6, with amino acid sequences as shown in SEQ ID NO.15-17, respectively. The light chain variable region of the first antibody 16E12 includes light chain complementarity-determining regions VL-CDR4, VL-CDR5, and VL-CDR6, with amino acid sequences as shown in SEQ ID NO.18-20, respectively. The heavy chain variable region of the second antibody 9B7 includes heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3, with amino acid sequences as shown in SEQ ID NO.1-3, respectively. The light chain variable region of the second antibody 9B7 includes light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3, with amino acid sequences as shown in SEQ ID NO.4-6, respectively.

2. The antibody pair according to claim 1, characterized in that, The heavy chain variable region of the first antibody 16E12 includes frame regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8; the amino acid sequences of the frame regions VH-FR5, VH-FR6, VH-FR7, and VH-FR8 are shown in SEQ ID NO.21-24, respectively. The heavy chain variable region of the second antibody 9B7 includes frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4; the amino acid sequences of the frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4 are shown in SEQ ID NO.7-10, respectively.

3. The antibody pair according to claim 1, characterized in that, The light chain variable region of the first antibody 16E12 includes frame regions VL-FR5, VL-FR6, VL-FR7, and VL-FR8; the amino acid sequences of frame regions VL-FR5, VL-FR6, VL-FR7, and VL-FR8 are shown in SEQ ID NO.25-28, respectively. The light chain variable region of the second antibody 9B7 includes frame regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4; the amino acid sequences of frame regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4 are shown in SEQ ID NO.11-14, respectively.

4. The antibody pair according to claim 1, characterized in that, The second antibody 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 antibody pair according to claim 1, characterized in that, The first antibody 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 antibody pair according to any one of claims 1-5.

7. An expression vector containing the nucleic acid molecule of claim 6.

8. A host cell containing the antibody pair of any one of claims 1-5, the nucleic acid molecule of claim 6, or the expression vector of claim 7, characterized in that, The host cell does not include plant cells.

9. A reagent kit, characterized in that, The kit contains the antibody pair as described in any one of claims 1-5 or the nucleic acid molecule as described in claim 6.

10. The use of the antibody pair of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the host cell of claim 8 in the preparation of an enterotoxin SEC detection kit.

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