Affinity-improved ochratoxin A nano antibody

Abstract

The invention discloses an ochratoxin A nano antibody with improved affinity, and belongs to the technical field of biotechnology and immunoassay. The invention provides a nano antibody for detecting ochratoxin A. The nano antibody targets an antigen epitope of the ochratoxin A; the Nb1H and Nb11B are included; the amino acid sequence of the nano antibody Nb1H is as shown in SEQ ID NO. 4; the amino acid sequence of the nano antibody Nb11B is as shown in SEQ ID NO. 5. The nano antibody prepared by the invention has high valence affinity, strong stability and high tolerance to OTA and other small molecule toxins; the preparation method does not need a biological immune step, and the nano antibody can be quickly generated in a short time; not only is an excellent antibody candidate molecule provided for sensitive detection of OTA, but also a method generally applicable to preparation of nano antibodies from low-molecular-weight compounds is established.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and immunoassay, and in particular to an ochratoxin A nanobody with enhanced affinity. Background Technology

[0002] Ochratoxin A (OTA), as a fungal toxin, possesses important toxic characteristics. OTA is produced by various Aspergillus species in temperate and tropical climates and is a common fungal toxin found in everyday foods. Long-term exposure can lead to severe kidney damage. Under warm and humid storage conditions, agricultural products such as grains, coffee, wine, dried fruits, and spices are commonly contaminated with OTA. This toxin exhibits potent nephrotoxicity, carcinogenicity, and immunotoxicity. Therefore, developing rapid and sensitive detection methods is of great value.

[0003] In the field of OTA (Original Toxic Acid) detection, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) are widely used due to their high sensitivity, accuracy, and reliability. However, due to the high cost of equipment and complex operation procedures, these methods are not suitable for on-site testing in underdeveloped areas. In contrast, immunoassay has the advantages of high specificity, simple operation, suitability for large-scale testing, and rapid on-site detection. To achieve the goals of high sensitivity, high accuracy, and rapid detection, two main methods are currently used to improve detection sensitivity. One method is to directly amplify the signal of antigen-antibody interaction using techniques such as nanoparticle amplification, enzyme assembly, and isothermal amplification. The other method is to improve the antibody titer and affinity to further enhance the specificity and strength of antigen capture. Nanobodies (Nb) are single-domain antibody fragments composed only of the heavy chain variable region. Due to their strong stability under different temperatures, organic solvents, pH values, and ionic strengths, and their ease of fusion with additional indicator proteins, they can be used to detect mycotoxins, pesticides, and marine toxins, and are suitable for the detection of complex samples. The CDR3 region of nanobodies exhibits an extended structure, forming a distinctive exposed protruding ring conformation. This unique structure allows nanobodies to penetrate antigen clefts or grooves like "probes," recognizing and binding to epitopes that are difficult for traditional antibodies to reach. In prokaryotic systems, nanobodies can also achieve highly efficient soluble expression, significantly reducing production costs. Based on these properties, nanobodies can be used for rapid on-site detection and food safety monitoring, offering advantages such as sensitivity, stability, and cost-effectiveness.

[0004] However, nanobodies currently suffer from insufficient affinity for small molecule analytes, primarily due to their small antigen-binding sites and missing variable domains. Achieving affinity maturation through sequence modification is a key strategy for improving nanobody performance. Furthermore, the compact molecular structure of nanobodies makes them easier to modify efficiently through molecular engineering compared to traditional monoclonal antibodies. Currently, strategies for in vitro preparation of high-performance nanobodies include DNA shuffling, chain shuffling, error-prone PCR, and chemical and UV mutagenesis. However, due to the unpredictability and mutational bias of mutations, these methods suffer from limited library diversity and time-consuming screening processes, limiting their application in in vitro nanobody preparation. Meanwhile, the accuracy of protein three-dimensional structure is crucial for modeling protein-ligand interactions. Current mainstream techniques for protein structure determination include X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance (NMR), but these also have limitations due to complex sample preparation, high equipment costs, demanding experimental conditions, and difficult data analysis. Based on these shortcomings, constructing nanobodies with high stability, tolerability, and higher titer and affinity has become a pressing technical challenge. Summary of the Invention

[0005] Based on the technical problems to be solved by the present invention, the present invention uses anti-ochratoxin A (OTA) Nb3G as a model antibody, employs AI-enhanced modeling tools to analyze the interaction between OTA and Nb3G, and selects nine key residues to construct a site-directed mutation library. After biological screening and identification, mutants Nb1H and Nb11B with enhanced affinity were obtained, and an ochratoxin A nanobody with enhanced affinity was proposed.

[0006] One objective of this invention is to provide an ochratoxin A nanobody with enhanced affinity, wherein the nanobody targets the antigenic epitope of ochratoxin A; the nanobody comprises Nb1H and Nb11B; the amino acid sequence of the nanobody Nb1H is shown in SEQ ID NO.4; and the amino acid sequence of the nanobody Nb11B is shown in SEQ ID NO.5.

[0007] A second objective of this invention is to provide an encoding gene for an ochratoxin A nanobody with enhanced affinity, wherein the nucleotide sequence of the nanobody Nb1H is shown in SEQ ID NO.6; and the nucleotide sequence of the nanobody Nb11B is shown in SEQ ID NO.7.

[0008] A third objective of this invention is to provide a recombinant vector containing the encoding gene of an ochratoxin A nanobody with enhanced affinity.

[0009] The fourth objective of this invention is to provide a host cell for a recombinant vector encoding the gene of an ochratoxin A nanobody with enhanced affinity.

[0010] The fifth objective of this invention is to provide a method for detecting ochratoxin A for non-diagnostic / therapeutic purposes. This method utilizes the nanobody described in claim 1 and includes the following steps: S1, conjugating an ochratoxin A hapten with a carrier protein to obtain a complete ochratoxin A antigen, using the complete ochratoxin A antigen as the detection antigen; S2, adding the sample to be tested and the nanobody to a solid-phase carrier coated with the detection antigen, allowing the reaction to proceed fully, discarding the liquid, and washing; S3, adding an enzyme-labeled secondary antibody, allowing the reaction to proceed fully, discarding the liquid, washing, performing a colorimetric reaction, terminating the reaction, and measuring the OD value at 450 nm. The OD value indicates the content of ochratoxin A.

[0011] Furthermore, the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

[0012] The sixth objective of this invention is to provide a method for constructing a synthetic library of ochratoxin A nanobodies with enhanced affinity, comprising: S21, preparation of OTA nanobodies and construction of mutant libraries; S22, biological screening and expression verification; S23, comparative analysis of mutant and non-mutated nanobodies; S24, performance and affinity evaluation, to obtain a synthetic library of nanobodies for detecting ochratoxin A.

[0013] The seventh objective of this invention is to provide an application of an affinity-enhanced ochratoxin A nanobody in the preparation of an ochratoxin A immunological detection kit.

[0014] Furthermore, the immunological detection kit uses ochratoxin A hapten conjugated with a carrier protein to obtain ochratoxin A complete antigen as the detection antigen, and the nanobody is the detection antibody.

[0015] The eighth objective of this invention is to provide an application of an affinity-enhanced ochratoxin A nanobody in the detection of ochratoxin A, wherein the application is in food or non-food products.

[0016] Compared with existing technologies, this invention proposes an ochratoxin A nanobody with enhanced affinity, which has the following beneficial effects: This invention identifies key mutated amino acid sites in the CDR region of the Nb3G gene sequence based on AI-enhanced modeling, introduces key amino acid mutations through SOE-PCR technology, and constructs a phage display nanobody mutation library targeting OTA.

[0017] Furthermore, this invention screened two mutant strains, Nb1H and Nb11B, from a phage display nanobody mutant library targeting OTA for functional optimization and compared them with the parental strain Nb3G. Under matched dilution conditions, the antigen-binding efficiencies of the mutant nanobodies Nb11B (titer 1:5600) and Nb1H (1:2800) were 4-fold and 2-fold higher, respectively, than those of the wild-type 3G (1:1400), confirming that the mutations significantly enhanced their antigen-binding ability. Furthermore, compared with the parental Nb3G, Nb1H and Nb11B exhibited higher detection sensitivity. Furthermore, in terms of tolerance to temperature, pH, salt concentration, and methanol concentration, Nb1H maintained a competitive inhibitory effect comparable to Nb3G, while Nb11B showed superior competitive inhibitory performance compared to Nb3G. Furthermore, surface plasmon resonance analysis showed that the equilibrium dissociation constants (KD = 16.83 nM and 9.19 nM) of the mutant nanobodies Nb1H and Nb11B bound to OTA were 6.27 times and 11.49 times lower, respectively, than those of the wild-type Nb3G (KD = 105.60 nM).

[0018] Furthermore, the nanobodies prepared by this invention exhibit high affinity for small molecule toxins such as OTA, strong stability, and high tolerance; and the preparation method of these nanobodies does not require a biological immunization step, enabling rapid generation of nanobodies in a short time.

[0019] Furthermore, this invention not only provides superior antibody candidate molecules for the sensitive detection of OTA, but also establishes a universally applicable method for preparing nanobodies from low molecular weight compounds. Attached Figure Description

[0020] Figure 1 The diagram illustrates a screening process for affinity-enhancing nanobodies according to an embodiment of the present invention; wherein, A: OTA structure analysis; B: mutant antibody library design; C: phage display nanobodies library construction; D: bioscreening; E: nanobodies performance evaluation.

[0021] Figure 2 A schematic diagram of an antibody library design according to an embodiment of the present invention is shown.

[0022] Figure 3 The figure shown is an electrophoretic detection result of PCR amplification product according to an embodiment of the present invention; wherein, 1 is the amplification product and M is the marker.

[0023] Figure 4 An image of a positive clone identified by enzyme-linked immunosorbent assay (ELISA) according to an embodiment of the present invention is shown.

[0024] Figure 5 This invention illustrates a standard curve plot of ochratoxin A standard using an indirect competitive ELISA method according to an embodiment of the present invention.

[0025] Figure 6 An image showing a specific detection result according to an embodiment of the present invention is illustrated. Figure 7 The SPR results of ochratoxin A according to an embodiment of the present invention are shown; wherein, (a) is the affinity determination of Nb3G; (b) is the affinity determination of Nb1b; and (c) is the affinity determination of Nb11H.

[0026] Figure 8 A schematic diagram of the amino acid sequence of a nanobody according to an embodiment of the present invention is shown. Detailed Implementation

[0027] Exemplary embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0028] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the reagents and materials in this invention are obtained from the market or other public channels.

[0029] This invention utilizes AI-enhanced modeling tools to analyze the interaction mechanism between nanobodies and OTA, constructs a site-directed mutation library for screening high-performance nanobodies, and employs phage display technology to screen mutants from the nanobodies library that exhibit excellent OTA binding ability and significant inhibitory effects. A nanobody with enhanced affinity for ochratoxin A is proposed. (See [link to related document]). Figure 1 .

[0030] Example 1 This embodiment proposes the construction of an ochratoxin A nanobody synthesis library.

[0031] The first step is structural modeling and comparative analysis.

[0032] The three-dimensional structures of three nanobody-OTA complexes were predicted using the ProteinX software platform (https: / / protenix-server.com). For each model, this embodiment further evaluated the local structure, prediction confidence, and relative positions and orientation errors between residues. When performing structural analysis using PyMOL (v2.5.2), the interaction between the nanobody and OTA was the primary focus. The results revealed structural differences with potential non-covalent binding mechanisms.

[0033] This embodiment first characterizes the interaction between OTA and Nb3G through computational modeling, and the analysis shows (see...) Figure 2 Nine amino acid residues (Thr28, Tyr32, Asp54, Thr57, Trp99, Phe103, Glu106, Trp110, Tyr112) located in the complementarity-determining region (CDR) can form OTA. Stacking, hydrophobicity, and hydrogen bonding interactions result in relevant residues being sequentially distributed in the CDR1, CDR2, and CDR3 regions of Nb3G. Based on these key nucleotide sites, targeted mutations were introduced in this embodiment. After two rounds of polymerase chain reaction (PCR) amplification, a full-length nanobody fragment was obtained.

[0034] The second step is to construct a mutation library.

[0035] Using a two-step SOE PCR technique, the DNA sequence fragments I-IV encoding VHH were assembled into a complete VHH nanobody using Q5 high-fidelity premix and primers. Subsequently, VHH and pCantab 5E plasmids were double-digested with XmaI and KpnI, and then ligated using T4 ligase. The construct was electroporated into *E. coli* TG1 cells at 1.8 kV and 5.9 ms, immediately followed by the addition of 1 mL of preheated SOC medium and incubation at 37°C with shaking for 1 h. The cells were then collected. Serially diluted bacterial cultures were inoculated onto 2YT-Amp plates and incubated overnight at 37°C. The remaining electroporation mixture was supplemented with 10 μL of ampicillin, and the cells were incubated overnight with shaking.

[0036] Step 3: Amplification of the target gene VHH of the nanobody.

[0037] Design from arrive The primer nucleotide sequences are shown in SEQ ID NO.1~3.

[0038] Table 1. List of primer nucleotide sequences The first round of PCR used fragments I, II, and III as PCR templates. The reaction system for the first round of PCR is shown in Table 2.

[0039] Table 2 List of PCR reaction systems for the first round The reaction program was 98℃ for 30 seconds; 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds, 36 cycles; 72℃ for 5 minutes; 4℃ hold. The first-round PCR amplification product showed a band at 300 bp (see...). Figure 2 ), and then recycle the glue.

[0040] The second round of PCR used the recovered products and fragment IV from the first round of PCR as templates. The reaction system for the second round of PCR is shown in Table 3.

[0041] Table 3 List of Second-Round PCR Reaction Systems The reaction program was 98℃ for 30 seconds; 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds, 36 cycles; 72℃ for 5 minutes; store at 4℃ for later use. The second-round PCR amplification product showed a band at 450 bp (see...). Figure 3 ), and then recycle the glue.

[0042] Step 4: Construction of anti-OTA nanobody gene library.

[0043] (1) Enzyme digestion of VHH target gene and vector The VHH target gene and pCantab 5E vector were digested using XmaI and KpnI-HF enzymes under the following conditions: constant temperature reaction at 37℃ for 12 h.

[0044] The VHH gene and pCantab 5E vector digestion products were directly cleaned and recovered using a DNA recovery kit.

[0045] (2) Ligation of enzyme digestion products The pCantab 5E vector and VHH fragment were mixed (molar ratio 1:3), reacted at 16°C for 12 h, and then cleaned and recovered using a DNA recovery kit.

[0046] (3) Electroshock conversion Add 5 μL of the ligation product to 50 μL of electroporation-competent E. coli TG1 cells, mix gently, and transfer to a 0.2 cm electroporation cuvette for electroporation (1.8 kV). Immediately after electroporation, add 3 mL of preheated SOC medium (37°C) to the cuvette and incubate at 37°C and 250 rpm for 1 hour to revive the cells. Perform serial dilutions of 10 μL of the revived bacterial culture, and spread 50 μL of each dilution onto 90 mm diameter 2YT-Amp culture dishes as counting chambers. Incubate overnight at 37°C. Amplify the remaining undiluted revived bacterial culture overnight at 37°C and 250 rpm.

[0047] Count the number of colonies on the culture dish (e.g.) Figure 4 (As shown), calculate the total number of bacteria in the resuscitation solution, perform multiple electroporation transformations, and accumulate the total number of transformed colonies to reach [a certain value]. The number of CFU or higher represents the library capacity of the nanobody gene library.

[0048] The transgenic E. coli cultured overnight was added to glycerol (v / v) at a final concentration of 18% and stored at -20°C to obtain the ochratoxin A nanobody gene library.

[0049] Step 5: Phage rescue.

[0050] Inoculate cells at more than 10 times the library volume in 200 mL of 2YT (Amp) and incubate at 37°C and 250 rpm until the OD600 reaches approximately 0.4–0.6. Add helper phage M13K07 (20:1 multiplicity of infection), incubate at 37°C for 30 min, then incubate at 250 rpm for 30 min. Add kanamycin (1:1000) and incubate overnight at 30°C and 250 rpm. Centrifuge at 12000 rpm, 4°C for 15 min, collect the supernatant, add 1 / 5 volume of PEG / NaCl (100 g PEG8000 and 73.05 g sodium chloride diluted with water to a final volume of 500 mL), and incubate on ice for 1 h. Centrifuge at 10000 rpm, 4℃, for 15 min, discard the supernatant, resuspend the precipitate in 1 mL PBS, transfer to a 2 mL centrifuge tube, centrifuge at 12000 rpm, 4℃, for 15 min, take 10 μL to determine the volume, add the remainder to a final concentration of 20% glycerol, and store at -80℃.

[0051] Step 6: Affinity screening of nanobodies.

[0052] Ochratoxin A-BSA was diluted to 10 μg / mL with coating buffer and added to the wells of an ELISA plate, 100 μL per well, and incubated overnight at 4°C. The next day, the plate was washed three times with PBST (0.01 M PBS, 0.05% Tween-20), and then 160 μL of 3% skim milk powder-PBS (w / v) solution was added to each well and incubated at 37°C for 1 h. The liquid in the wells was poured out, and the plate was washed three times with PBST (0.01 M PBS, 0.05% Tween-20), patted dry on absorbent paper, and stored at 4°C for later use. BSA was added to the above ochratoxin A nanobody phage library to a final BSA concentration of 1% (w / v). The phage library containing 1% BSA (w / v) was added to three wells containing immobilized antigen, 100 μL per well, and incubated at 37°C for 1 h. Discard any unbound phages from the wells and wash the microwells 10 times with PBST. Elute with 100 μL glycine (0.2 M) for 30 min at 37°C, followed by 5 min of 100 μL Tris-HCl solution. Collect the liquid from the microwells into sterile centrifuge tubes. The phages at this stage are called "bound outputs," and the first round of screening is complete. Take 10 μL of the eluted phages to determine the titer; the remainder is used to infect 20 mL of E. coli TG1 strain grown to the logarithmic growth phase for amplification. The second round of screening follows the same steps as the first. In the third and fourth rounds, the elution reaction uses 100 μL of 50 ng / mL AFB1 standard for competitive elution, followed by 1 h of oscillation at 37°C. Collect the liquid from the microwells into 1.5 mL sterile centrifuge tubes; these phages are called "competitive outputs." The above screening process was carried out in four rounds. In rounds 1-4, the concentration of the coating antigen was reduced from 10 ug / mL to 5 ug / mL, 2.5 ug / mL, and 1.25 ug / mL, respectively.

[0053] Step 7: Identification of positive clones.

[0054] Positive phage clones were identified using an indirect enzyme-linked immunosorbent assay (ELISA). Specifically, 96 single colonies were randomly selected from the output titer assay plates after the fourth round of competitive elution and inoculated into 96-well plates containing 200 μL of 2YT-Amp per well. The plates were incubated overnight at 37°C to serve as the "mother plate." 10 μL of bacterial culture was then transferred from each well of the mother plate to another 96-well deep-well plate containing 100 μL of 2YT-Amp per well, with the well numbers corresponding to the mother plate. The plates were incubated at 37°C and 180 rpm for 1.5 h. M13K07 phage was diluted and added to the wells, and the plates were incubated overnight at 30°C and 180 rpm. The mother plate was stored at 4°C for later use. For antigen immobilization, OTA-BSA was diluted to 10 μg / mL using coating buffer, and BSA standard was diluted to 10 μg / mL. 100 μL of each BSA standard was added to the wells and incubated overnight at 4°C. On the second day, wash the microwells three times with PBST, blot dry the liquid in the wells, add 160 μL of freshly prepared 3% skim milk powder (w / v) with PBS to each well, block at 37°C for 1 h, discard the blocking solution, wash the microwells three times with PBST, blot dry the liquid in the wells, and store at 4°C for later use.

[0055] Centrifuge the deep-well plate at 4000 rpm for 10 min. Take the ELISA plate containing immobilized antigen and aspirate the supernatant from the centrifuged 96-well plate. Add 100 μL to each well of the corresponding numbered ELISA plate. Incubate at 37°C for 60 min. Wash three times with PBST and blot dry. Dilute Anti-M13-HRP secondary antibody 4000-fold with PBS and add 100 μL to each well. Incubate at 37°C for 60 min. Wash three times with PBST (0.01 M PBS, 0.05% Tween-20), blot dry, and add 100 μL of TMB chromogenic solution (pre-mixed with equal volumes of chromogenic solution A and chromogenic solution B) to each well. Incubate at 37°C for 10 min. Add 50 μL of 10% stop solution. The reaction was terminated at (v / v); the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0056] Centrifuge the deep-well plate at 4000 rpm for 10 min. Take the plate containing the immobilized antigen and aspirate the supernatant from the centrifuged 96-well plate. Add 50 μL of the supernatant to each well, along with 50 μL of OTA standard (ng / ml). Incubate at 37°C for 60 min. Wash three times with PBST and blot dry. Dilute Anti-M13-HRP secondary antibody 4000-fold with PBS and add 100 μL to each well. Incubate at 37°C for 60 min. Wash three times with PBST and blot dry. Add 100 μL of TMB chromogenic solution (pre-mixed with equal volumes of chromogenic solution A and B) to each well and incubate at 37°C for 10 min. Add 50 μL of 10% stop solution. The reaction was terminated at (v / v); the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0057] Inhibition rate measured by an enzyme-linked immunosorbent assay (ELISA) reader, such as Figure 4 As shown. The inhibition rate of each positive clone can be calculated using the following formula. Clones with an OD value greater than 3 times that of the negative control wells and exhibiting significant inhibition (inhibition rate > 20%) were selected in the deep-well plate. The corresponding well numbers were recorded, and the bacterial culture from the corresponding wells in the mother plate was transferred to sterile centrifuge tubes, added glycerol, and stored for later use. The phage clones of the nanobodies obtained through indirect competitive ELISA were subjected to gene sequencing. The amino acid sequences of the nanobodies were obtained based on the DNA sequencing results and codon table. Results showed that two ochratoxin A nanobodies were obtained, named Nb1H and Nb11B.

[0058] Example 2 This invention proposes a method for preparing nanobodies Nb1H and Nb11B.

[0059] Mainly includes: Nanobodies Nb1H and Nb11B were prepared in the form of protein expression. The specific method was as follows: Phage clones of the obtained nanobodies Nb1H and Nb11B were used to extract plasmids, which were then chemically transformed into *E. coli* Trans B (DE3). A single colony was picked from the transformation plate and inoculated into 5 mL of 2YT (Amp) medium, and cultured at 37°C and 220 rpm for 6 h. The culture was then inoculated into 200 mL of 2YT (Amp) medium and cultured at 37°C and 250 rpm until the OD600 reached approximately 0.4–0.6. IPTG was added to a working concentration of 0.1 mM, and the culture was incubated overnight at 37°C and 250 rpm. The next day, the cells were centrifuged at 4°C and 8000 g for 10 min, and the cell pellet was collected. The pellet was then sonicated and centrifuged again at 8000 g for 10 min. The supernatant was collected and purified by nickel gravity column chromatography to obtain nanobodies Nb10E.

[0060] Nanobodies Nb1H and Nb11B were obtained, wherein the amino acid sequence of nanobodies Nb1H is shown in SEQ ID NO.4: QVPLVQSGGGSVETGGSLRLSCSASGYSYSGYCVGWFRQAPGKEREGVAGISSDGRYLDPVKGRFTISVDNAKKTLTLQMNNLKPEDTAMYYCAAKWTPCRTGELATWLSGNWGQGTLVTVSS.

[0061] The amino acid sequence of the nanobody Nb11B is shown in SEQ ID NO.5: QVPLVQSGGGSVETGGSLRLSCSASGYSYSGYCVGWFRQAPGKEREGVAGISSDGRTRYLDPVKGRFTISVDNAKKTLTLQMNNLKPEDTAMYYCAAKWTPCNTGELATWLNGNWGQGTLVTVSS.

[0062] Furthermore, in the encoding gene of the nanobody for detecting ochratoxin A, the nucleotide sequence of nanobody Nb1H is shown in SEQ ID NO. 6: CAGGTTCCGCTGGTTCAGTCTGGTGGTGGTTCTGTTGAAACCGGTGGTTCTCTGCGTCTGTCTTGCTCTGCTTCTGGTTACTCCTACTCTGGTTACTGCGTTGGTTGGTTCCGTCAGGCTCCGGGTAAAGAACGTGAAGGTGTTGCTGGTATCTCTTCTGACGGTCGTTACCGTTACCTGGACCCGGT TAAAGGTCGTTTCACCATCTCTGTGACAACGCTAAAAAAACCCTGACCCTGCAGATGAACAACCTGAAACCGGAAGACACCGCTATGTACTACTGCGCTGCTAAATGGACCCCGTGCCGCACCGGTGAGCTGGCTACCTGGCTGTCCGGTAACTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCT.

[0063] The amino acid sequence of the nanobody Nb11B is shown in SEQ ID NO.7: CAGGTTCCGCTGGTTCAGTCTGGTGGTGGTTCTGTTGAAACCGGTGGTTCTCTGCGTCTGTCTTGCTCTGCTTCTGGTTACTCCTACTCTGGTTACTGCGTTGGTTGGTTCCGTCAGGCTCCGGGTAAAGAACGTGAAGGTGTTGCTGGTATCTCTTCTGACGGTCGTACCCGTTACCTGGACCCGGT TAAAGGTCGTTTCACCATCTCTGTTGACAACGCTAAAAAAACCCTGACCCTGCAGATGAACAACCTGAAACCGGAAGACACCGCTATGTACTACTGCGCTGCTAAATGGACCCCGTGCAACACCGGTGAGCTGGCTACCTGGCTGAACGGTAACTGGGGTCAGGGGACCCTGGTTACCGTTTCTTCT.

[0064] Example 3 This invention proposes a method for determining the activity and sensitivity of nanobodies Nb1H and Nb11B.

[0065] Mainly includes: (1) Coat the microplate with 100 μL / well of OTA-BSA (125 ng / mL) and incubate overnight at 4°C or for 2 hours at 37°C. After washing with PBST, block with 100 μL / well of 3% skim milk at 37°C for 1 hour. Add 50 μL of PBS to the test wells to dilute the antibody and 50 μL of OTA standard; add antibody and 50 μL of 10% methanol-PBS to the control wells. After incubation at 37°C for 1 hour and washing, add 50 μL of HRP-conjugated anti-His secondary antibody diluted 1:3000 and incubate at 37°C for 1 hour. After final washing, add 100 μL / well of TMB substrate and react for 15 minutes. Terminate the reaction with 50 μL / well of stop solution. Measure the absorbance using a microplate reader. Under the above experimental conditions, the working concentrations of Nb1H and Nb11B were 4000-fold diluted, and the working concentration of the antigen was 125 ng / mL.

[0066] (2) Indirect competitive ELISA to construct a standard curve. Ochratoxin A standard was diluted with 10% methanol PBS to obtain a series of ochratoxin A solutions of different concentrations. 50 μL was added to each well of an ELISA plate, with three replicates per concentration. A three-well blank control group (50 μL PBS) was also prepared. The nanobody Nb10E was diluted 500 times to its working concentration with PBS, and 50 μL of the diluted antibody was added to each well. The plate was incubated at 37°C for 30 min, washed three times with PBST, and dried on absorbent paper. 100 μL of Anti-VHH-HRP secondary antibody diluted 3000 times with PBST was added to each well. The plate was incubated at 37°C for 60 min, washed three times with PBST, and dried on absorbent paper. 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated at 37°C in the dark for 10 min. 50 μL of 10% stop solution was added to each well. (v / v) OD values ​​were read at 450 nm using a microplate reader. The average OD450 value of the drug blank group was denoted as B0, and the average OD450 value at different drug concentrations was denoted as Bx. The Bx / B0 ratio and the standard deviation of each parallel data set were calculated using Excel. A scatter plot was created in Origin software with drug concentration on the x-axis and the Bx / B0 ratio on the y-axis, and an indirect competition standard curve was established by fitting the curve using the Boltzmann function.

[0067] The indirect competitive ELISA standard curve established based on antibodies Nb1H and Nb11B is as follows: Figure 5 As shown, the standard curve exhibits an S-shape, indicating good linear correlation. The detection ranges are 0.10-1.34 ng / mL and 0.04-1.24 ng / mL, respectively. The concentrations were 0.31 ng / mL and 0.25 ng / mL, respectively, which meet the maximum detection limit requirements. Furthermore, cross-reactivity (CR) assessments of ochratoxin A analogs (OTB and OTC) and cereal mycotoxins (FB1, AFB1, DON, ZEN) were performed using immunochemiluminescent immunoassay (ic-ELISA). Figure 6 (As shown). Using 1 ng / mL ochratoxin A and 10 ng / mL of other mycotoxins, the nanobodies exhibited high sensitivity to OTB and OTC, with almost no cross-reactivity to other mycotoxins. Standard curves for OTB and OTC were established to validate their specificity for ochratoxin derivatives. These nanobodies offer broad-spectrum detection advantages, demonstrate high specificity to other ochratoxins, and highlight their potential for detecting mixed ochratoxin contamination. Structural analysis suggests that deeper binding cavities may facilitate more efficient encapsulation of OTA, thereby improving detection sensitivity.

[0068] Example 4 This invention proposes a method for determining the affinity of nanobodies Nb1H and Nb11B.

[0069] OTA-BSA protein was immobilized on a Biacore CM5 (Cytiva, CN, BR100530) sensor chip via primary amine groups. The compound was flowed at 30 μL / min for 120 s to promote binding, followed by dissociation from the immobilized protein for 300 s in PBS running buffer (PBS, 0.2% surfactant P20, pH 7.4). Binding assays of the anti-OTA nanobody were performed at concentrations ranging from 6.25 nM to 400 nM. Data were analyzed using evaluation software to calculate the equilibrium dissociation constant (KD).

[0070] After kinetic fitting, the binding rate constant (Ka) and dissociation rate constant (Kd) of the three nanobodies are shown in Table 3. SPR sensor plots show that the KD values ​​of Nb3G, Nb1H, and Nb11B nanobodies are 105.60, 16.83, and 9.19 nM, respectively. Compared with the parent nanobody, the binding strength of Nb1H and Nb11B to OTA-BSA increased by 6.27-fold and 11.49-fold, respectively. Combining KD values ​​and half-inhibitory concentration results, the affinity of Nb1H and Nb11B is superior to that of Nb3G, with Nb11B showing the most significant enhancement effect. Mutations in the CDR region enhance affinity by increasing binding capacity and improving shape complementarity, such as... Figure 7 As shown.

[0071] Table 4. List of binding parameters for preparing nanobodies Figure 8 A schematic diagram of the amino acid sequence of nanobodies is provided.

[0072] In summary, this invention identifies key mutated amino acid sites in the CDR region of the Nb3G gene sequence based on AI-enhanced modeling, introduces key amino acid mutations using SOE-PCR technology, and constructs a phage-display nanobody mutant library targeting OTA. Furthermore, this invention screens two mutant strains, Nb1H and Nb11B, from the OTA-targeting phage-display nanobody mutant library for functional optimization. The antigen-binding efficiencies of the mutant nanobodies Nb11B and Nb1H are 4-fold and 2-fold higher than that of wild-type Nb3G, respectively, confirming that the mutations significantly enhance their antigen-binding ability. Furthermore, compared with the parental Nb3G, Nb1H and Nb11B exhibit higher detection sensitivity. Furthermore, in terms of tolerance to temperature, pH, salt concentration, and methanol concentration, Nb1H maintains a competitive inhibitory effect comparable to Nb3G, while Nb11B shows superior competitive inhibitory performance compared to Nb3G. Furthermore, surface plasmon resonance analysis showed that the equilibrium dissociation constants of the mutant nanobodies Nb1H and Nb11B binding to OTA were 6.27 times and 11.49 times lower, respectively, than those of the wild-type Nb3G. Furthermore, the nanobodies prepared by this invention exhibit high titer affinity, strong stability, and high tolerance to small molecule toxins such as OTA; moreover, the preparation method of this nanobodies does not require a biological immunoassay step and can rapidly generate nanobodies in a short time. Furthermore, this invention not only provides superior antibody candidate molecules for the sensitive detection of OTA, but also establishes a universally applicable method for preparing nanobodies from low molecular weight compounds.

[0073] It should be noted that the term "comprising," or any other variation thereof, is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0074] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An ochratoxin A nanobody with enhanced affinity, characterized in that, The nanobody targets the antigenic epitope of ochratoxin A; The nanobody contains Nb1H and Nb11B; The amino acid sequence of the nanobody Nb1H is shown in SEQ ID NO.4; The amino acid sequence of the nanobody Nb11B is shown in SEQ ID NO.

5.

2. The encoding gene of the affinity-enhanced ochratoxin A nanobody as described in claim 1, characterized in that, The nucleotide sequence of the nanobody Nb1H is shown in SEQ ID NO.6; The nucleotide sequence of the nanobody Nb11B is shown in SEQ ID NO.

7.

3. A recombinant vector comprising the encoding gene of the ochratoxin A nanobody with enhanced affinity as described in claim 2.

4. A host cell comprising a recombinant vector encoding the gene of the affinity-enhanced ochratoxin A nanobody as described in claim 3.

5. A method for detecting ochratoxin A for non-diagnostic / therapeutic purposes, characterized in that, The method utilizes the nanobody according to claim 1, and includes the following steps: S1. Ochratoxin A complete antigen is obtained by coupling the ochratoxin A hapten with a carrier protein, and the ochratoxin A complete antigen is used as the detection antigen. S2. Add the sample to be tested and the nanobody to a solid-phase carrier coated with the detection antigen, and after the reaction is complete, discard the liquid and wash. S3. After adding enzyme-labeled secondary antibody and allowing it to react fully, discard the liquid and wash the sample. Then perform the colorimetric reaction, terminate the reaction, and measure the OD value at 450 nm. Obtain the content of ochratoxin A based on the OD value.

6. The method for detecting ochratoxin A for non-diagnostic / therapeutic purposes according to claim 5, characterized in that, The enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

7. A method for constructing a synthetic library of ochratoxin A nanobodies with enhanced affinity as described in claim 1, characterized in that, include: Preparation of S21 and OTA nanobodies and construction of mutant libraries; S22, biological screening and expression validation; Analysis and comparison of S23, mutant and non-mutated nanobodies; S24. Analyze the performance and affinity of the nanobodies to obtain a synthetic library of nanobodies for the detection of ochratoxin A.

8. The use of the affinity-enhanced ochratoxin A nanobody as described in claim 1 in the preparation of an ochratoxin A immunological detection kit.

9. The application according to claim 8, characterized in that, The immunological detection kit uses ochratoxin A hapten conjugated with a carrier protein to obtain ochratoxin A complete antigen as the detection antigen, and the nanobody is the detection antibody.

10. The application of the affinity-enhanced ochratoxin A nanobody as described in claim 1 in the detection of ochratoxin A, characterized in that, The application may be food or non-food.

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