A nanobody against an epitope of vomitoxin and a method of preparing and using the same

Abstract

This invention provides a vomiting toxin anti-idiotypic nanobody, its preparation method, and its application. The amino acid sequence of the vomiting toxin anti-idiotypic nanobody is selected from any of the following: (a) the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence formed by adding a tag sequence to one or both ends of SEQ ID NO:1. This vomiting toxin anti-idiotypic nanobody can be used as a substitute for vomiting toxin in the immunoassay detection of vomiting toxin. It exhibits high sensitivity and specificity, and can be mass-produced through recombinant expression via genetic engineering, making its preparation simple.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a vomiting toxin anti-idiotype nanobody, its preparation method, and its application. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, belongs to the trichothecene group of type B toxins. It is primarily produced by the *Fusarium graminearum* species complex and is a major pathogen causing wheat scab and corn ear rot. DON is toxic to both animals and humans, exhibiting acute and chronic toxicity, cytotoxicity, dermal toxicity, and immunotoxicity. It also has significant embryotoxic and teratogenic effects and is classified as a Group 3 carcinogen by the IARC. my country has explicitly stipulated that the DON content in wheat, barley, corn, and their products must not exceed 1000 μg / kg.

[0003] The main methods for detecting DON (drug-induced vomiting toxin) include physicochemical analysis and immunoassay. Physicochemical analysis methods mainly include thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), and near-infrared spectroscopy (NIR). These methods all require expensive, large-scale precision instruments, highly skilled personnel, and complex sample pretreatment, which limits their widespread application. Immunoassay methods include enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, and (fluorescent) immunochromatography. Based on the specific binding of antigen and antibody, they offer advantages such as sensitivity, speed, specificity, and simplicity, and have been widely used in the detection of vomitoxin in recent years. However, some immunoassays require the use of vomitoxin as a coating antigen or competing antigen, and some require the use of vomitoxin as a calibrator and standard for calibration and standard curve preparation. The highly toxic vomitoxin poses a potential threat to operators and the environment. Therefore, finding high-quality, non-toxic alternatives to vomitoxin is one way to address this problem and provides core reagents for developing a green method for detecting vomitoxin (DON). Summary of the Invention

[0004] To address the problems existing in the background art, the present invention provides a vomiting toxin anti-idiotypic nanobody, its preparation method and application. This vomiting toxin anti-idiotypic nanobody can replace vomiting toxin as a competitive antigen, coating antigen, calibrator or standard in the immunoassay of vomiting toxin, and has high sensitivity and specificity.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] In a first aspect, the present invention provides a vomiting toxin anti-idiotype nanobody, the amino acid sequence of which is selected from any of the following:

[0007] (a) The amino acid sequence as shown in SEQ ID NO:1;

[0008] (b) An amino acid sequence formed by adding a tag sequence, restriction site sequence or signal sequence to one or both ends of SEQ ID NO:1.

[0009] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned anti-idiotypic nanobody against vomiting toxin.

[0010] Thirdly, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecules.

[0011] Fourthly, the present invention provides a host bacterium that transforms or transfects the above-mentioned expression vector.

[0012] Fifthly, the present invention provides a kit for detecting vomitoxin, comprising the aforementioned vomitoxin anti-idiotype nanobody.

[0013] In a sixth aspect, the present invention provides a method for preparing the above-mentioned anti-idiotypic nanobody of vomitoxin, wherein the anti-idiotypic nanobody of vomitoxin is produced using the above-mentioned host strain, or phage particles displaying the anti-idiotypic nanobody of vomitoxin are obtained by phage amplification.

[0014] In a seventh aspect, the present invention provides the application of the above-mentioned anti-idiotypic nanobody against vomitoxin in the immunoassay detection of vomitoxin.

[0015] According to the above scheme, the immunoassay detection includes enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, colloidal gold immunochromatography, time-resolved fluorescence immunochromatography, electrochemical immunosensing, bioimmunosensing, nanozyme immunoassay, nanoimmunosensing immunodot hybridization, and latex immunoturbidimetry.

[0016] According to the above scheme, the vomitoxin anti-idiotypic nanobody, as an alternative to vomitoxin, is used in immunoassay detection in the form of a competitive antigen, coating antigen, calibrator, quality control product, or standard.

[0017] The beneficial effects of this invention are:

[0018] 1) This invention constructs an alpaca-derived phage-display nanobody library using phage display technology. Through optimized affinity screening conditions, anti-idiotypic nanobodies that can specifically bind to vomitoxin monoclonal antibodies are screened. The obtained vomitoxin anti-idiotypic nanobodies have good specificity and high sensitivity. They can replace vomitoxin as a competitive antigen, coating antigen, calibrator, quality control material, or standard in the field of immunoassay detection of vomitoxin. This method avoids the use of toxic pure toxins, is green, non-toxic, and pollution-free, and is friendly to operators and the environment.

[0019] 2) The expression vector containing the gene encoding the anti-idiotypic nanobody containing vomitoxin is transformed or transfected into the host bacteria. After induction of expression, separation, purification and identification, a nanobody in the form of a soluble protein with good performance can be obtained. The anti-idiotypic nanobody containing vomitoxin of the present invention can be prepared in large quantities in the host bacteria, which effectively reduces the production cost of nanobodies. Attached Figure Description

[0020] Figure 1 This is the test result of Example 1 of the present invention used to determine the diversity of phage-displayed nanobody immune libraries;

[0021] Figure 2 This refers to the positive clone identified by phage-ELISA in Example 2 of this invention;

[0022] Figure 3 This describes the binding of VHH DO-7 with monoclonal antibodies against anti-vomiting toxin (DON), ochratoxin A (OTA), T-2 toxin (T-2), serpentine toxin (DAS), fumonisin B1 (FB1), and aflatoxin B1 (AFB1) in Example 4 of the present invention.

[0023] Figure 4 To optimize the VHH DO-7 coating concentration and the working concentration of the vomitoxin monoclonal antibody using the checkerboard method in Example 5 of this invention;

[0024] Figure 5 This is an example of the cross-reactivity of VHH-ELISA based on VHH DO-7 with vomitoxin (DON), ochratoxin A (OTA), T-2 toxin (T-2), serpentine toxin (DAS), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in Example 5 of the present invention.

[0025] Figure 6 This is a schematic diagram of the structure of the colloidal gold immunochromatographic test strip prepared in Example 6;

[0026] The components represented by each number are as follows: 1. Base plate; 2. Absorbent pad; 3. Test pad; 4. Quality control line; 5. Test line; 6. Gold label pad; 7. Sample pad. Detailed Implementation

[0027] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Anti-idiotype antibodies are specific antibodies that target the antigenic determinants (idiotypes) of the variable region of an antibody. Since they are all antigenic, anti-idiotype antibodies have the molecular basis to mimic the binding of natural antigens to their corresponding antibodies. They can block the binding of natural antigens to antibodies and can be used as substitutes for natural antigens in immunoassays.

[0033] Heavy chain antibodies (HCAbs) are antibodies found in the bodies of camels (camels, dromedary camels, and llamas) that consist only of heavy chain dimers. Their antigen-binding site consists of a single domain called the VHH region, hence these antibodies are also called single-domain antibodies (sdAbs). Because these antibodies are variable regions after removing the constant region, their molecular weight is only 12–15 kDa, and their diameter is approximately 10 nanometers, hence they are also called nanobodies (Nbs). Compared to conventional tetrachain antibodies (scFvs), nanobodies have advantages in solubility, stability, resistance to aggregation, foldability, expression yield, and ease of DNA manipulation, library construction, and 3D structure determination.

[0034] To obtain an alternative to vomitoxin for use in immunoassay detection, the inventors constructed an alpaca-derived phage-display nanobody library using phage display technology. Through optimized affinity panning conditions, they screened and obtained a unique nanobody, VHH DO-7, that specifically binds to a monoclonal antibody containing vomitoxin. Its amino acid sequence is as follows:

[0035] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAAS

[0036] GGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNA

[0037] NNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAH

[0038] HSEDPHGQAGQ(SEQ ID NO:1)

[0039] The obtained anti-idiotypic nanobody VHH DO-7 against vomitoxin (DON) exhibits high sensitivity, with an IC50 of [missing information - likely related to DON]. 50 The value was 49.2645 ng / mL, and there was no cross-reactivity with ochratoxin A (OTA), T-2 toxin (T-2), serpentine toxin (DAS), fumonisin B1 (FB1), and aflatoxin B1 (AFB1), indicating good specificity.

[0040] In this art, adding one or more amino acids to the C-terminus and / or N-terminus, such as adding a tag sequence, restriction enzyme site sequence, signal sequence, or secretion signal sequence, generally does not alter the function of the resulting protein. Sequences formed by adding a tag sequence, restriction enzyme site sequence, or signal sequence to one or both ends of the sequence shown in SEQ ID NO:1 are also within the scope of protection of the vomitoxin anti-idiotypic nanobody of this invention.

[0041] In a specific embodiment of the present invention, to facilitate the purification of the vomiting toxin anti-idiotypic nanobody, a histidine tag is added to the C-terminus of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of the vomiting toxin anti-idiotypic nanobody after tagging is shown in SEQ ID NO:2.

[0042] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQHHHHHHGAYPYDVPDYAS(SEQ ID NO:2)

[0043] Those skilled in the art may also choose other labels according to actual needs.

[0044] The nucleic acid molecule encoding the aforementioned anti-idiotypic nanobody VHH DO-7 for vomitoxin is also within the scope of this patent. The nucleic acid molecule of this invention can be in DNA or RNA form. The DNA form includes genomic DNA, cDNA, or artificially synthesized DNA, and the DNA can be a coding strand or a non-coding strand.

[0045] Expression vectors containing the above-mentioned nucleic acid molecules, as well as host bacteria generated by genetic engineering using the vectors of the present invention, are all within the scope of protection of the present invention.

[0046] The vomitoxin anti-idiotypic nanobody VHH DO-7 of the present invention can be mass-produced through phage amplification or recombinant expression via genetic engineering. Phage amplification refers to the large-scale propagation of phages displaying anti-idiotypic nanobody through biological amplification to produce phage particles displaying vomitoxin anti-idiotypic nanobody. Recombinant expression via genetic engineering involves transforming or transducing a recombinant expression vector containing the nucleic acid molecule into a suitable host bacterium, culturing the host bacterium in a suitable culture medium and inducing expression, and then isolating and purifying the nanobody in soluble protein form from the culture medium or bacterial cells.

[0047] In this invention, the host bacterium can be any microorganism suitable for expressing the vomitoxin anti-idiotypic nanobody VHH DO-7, such as bacteria or fungi. In the following examples, *Escherichia coli* Top10F' is preferably used.

[0048] This invention also provides the application of the above-mentioned anti-idiotypic nanobody against vomitoxin in immunological detection and analysis. The immunological detection and analysis methods include enzyme-linked immunosorbent assay (ELISA), immunochromatography (colloidal gold immunochromatography, time-resolved fluorescence immunochromatography), fluorescence polar immunoassay, immunodot hybridization, latex immunoturbidimetry, nanoimmunosensing, electrochemical immunosensing, bioimmunosensing, nanoenzyme immunoassay, and other immunological analysis and detection types based on antigen-antibody specific reactions.

[0049] When applying the anti-idiotypic nanobody of vomitoxin of the present invention, phage particles displaying the anti-idiotypic nanobody of vomitoxin can be obtained by phage amplification and directly used for analysis and detection. Alternatively, the anti-idiotypic nanobody of vomitoxin can be expressed in prokaryotes or eukaryotes and then used as a nanobody in the form of a soluble protein for immunoassay detection.

[0050] Specifically, anti-idiotypic nanobodies against vomitoxin are used as alternatives to vomitoxin in immunoassays in the form of competitive antigens, coating antigens, calibrators, quality control products, or standards.

[0051] The amino acid sequence of the anti-idiotypic nanobody of vomiting toxin of the present invention can be used as a precursor and modified by random mutation or site-directed mutagenesis to obtain mutants with better properties (affinity, stability, etc.).

[0052] The anti-idiotypic nanobody for vomitoxin of the present invention can be linked to various signal materials (such as fluorescent proteins, fluoresceins, enzymes, etc.) by chemical or biological conjugation, and used as a detection antigen in immunoassay.

[0053] The molecular biology experiments in the following examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation. These were primarily conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook et al., published by Science Press. Specific experimental conditions could be determined through simple experiments if necessary. PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments could be made through simple experiments if necessary.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] Vomitoxin monoclonal antibodies can be prepared at home or purchased from the market.

[0056] Example 1: Construction of a phage-displaying nanobody immune library

[0057] 1. Alpaca Immunity

[0058] 200 μg of anti-vomiting toxin monoclonal antibody (dissolved in PBS 7.4) was weighed and emulsified with an equal volume of Freund's incomplete adjuvant. This mixture was then injected subcutaneously at multiple sites into three-year-old male alpacas. Subsequent immunizations were administered every two weeks for a total of eight immunizations. Blood samples were collected after four immunizations. Seven to ten days after the fourth immunization, 10 mL of blood was collected from the jugular vein using an EDTA vacuum blood collection tube. The tube was gently inverted to prevent blood clotting. The blood was then processed using a filter from the LeukoLOCK kit. The filter was then rinsed sequentially with 3 mL of PBS buffer and 3 mL of RNAlater buffer, thus retaining the desired white blood cells. Finally, the filter was sealed and stored at -80°C for RNA extraction.

[0059] 2. Total RNA extraction

[0060] Total RNA was isolated from alpaca blood according to the instruction manual of the Life Technology LeukoLOCK Total RNA Extraction Kit. The specific procedures are as follows:

[0061] (1) Add 70 μL of pH adjustment buffer to 2.5 mL of lysis / binding buffer, and use immediately after preparation;

[0062] (2) Allow the sealed filter to return to room temperature, open the filter cap, and flush away the residual RNAlater buffer in the filter with a 2mL syringe;

[0063] (3) Use a 2.5 mL syringe to draw 2.5 mL of freshly prepared lysis / binding buffer to rinse the filter once, and collect the effluent in a 15 mL RNAse-free centrifuge tube;

[0064] (4) Add 2.5 mL of ddH2O without nuclease, vortex to mix, add 25 μL of proteinase K, and shake the centrifuge tube at room temperature at 250 g for 5 min.

[0065] (5) Take out the RNA-binding magnetic beads stored at 4℃, vortex to mix, and then aspirate 50 μL into the above centrifuge tube. Vortex the centrifuge tube, and then add 2.5 mL of isopropanol to the centrifuge tube. Shake at room temperature for 5 min.

[0066] (6) Centrifuge the above centrifuge tubes at 3200g for 3 minutes, carefully aspirate and discard the supernatant, being careful not to aspirate the precipitated magnetic beads;

[0067] (7) Add 600 μL of washing solution I, repeatedly blow and agitate the precipitated magnetic beads to disperse them evenly, transfer the suspension to a 1.5 mL centrifuge tube, rinse the 15 mL centrifuge tube once with 600 μL of washing solution I, and transfer it to the same 1.5 mL centrifuge tube.

[0068] (8) Centrifuge the 1.5mL centrifuge tube at 16000g for 30s and carefully discard the supernatant;

[0069] (9) Add 2 / 3 of 750 μL washing solution, vortex vigorously for 30 s to disperse the aggregated magnetic beads, centrifuge at 16000 g for 30 s to collect the magnetic beads, and carefully discard the supernatant.

[0070] (10) Open the cap of the centrifuge tube and let it stand in the clean bench for 2 minutes to evaporate 2 / 3 of the residual alcohol in the washing solution. During this time, prepare the TURBODNase master mixture. Take 4 μL of TURBO DNase (20 U / μL) and add it to 296 μL of LeukoLOCKDNase buffer. After mixing well, transfer it to the centrifuge tube. Use a pipette to repeatedly blow and blow the precipitated magnetic beads to disperse them evenly. Shake at 1000g for 10 minutes at room temperature, and gently invert and mix several times during the process.

[0071] (11) Add 300 μL each of lysis / binding buffer (without pH adjustment buffer) and isopropanol to the above centrifuge tube, mix well, centrifuge for 2 seconds, and then incubate at room temperature for 3 minutes.

[0072] (12) Centrifuge at 16000g for 30s, discard the supernatant, add 2 / 3 of 750μL washing solution, vortex vigorously for 30s, centrifuge at 16000g for 30s, discard the supernatant, add 2 / 3 of 750μL washing solution again, vortex vigorously for 30s, centrifuge at 16000g for 1min, discard the supernatant as dry as possible, let stand at room temperature with the container open for 3min to allow the residual washing solution to evaporate. Note that it should not be left for too long to avoid the magnetic beads drying out excessively.

[0073] (13) Add 60 μL of elution buffer, vortex for 30 s, centrifuge at 16000 g for 2 min, transfer the eluted RNA solution to another 1.5 mL centrifuge tube without nuclease, keep 1 μL of RNA solution to measure the concentration with nanodrop, and take about 3 μL of RNA solution for agarose gel electrophoresis analysis. Immediately reverse the remaining RNA solution to cDNA to prevent degradation.

[0074] 3. cDNA Synthesis

[0075] The first strand of cDNA was synthesized according to the reverse transcription kit instructions, following these steps:

[0076] (1) Take two 200μL PCR tubes without nuclease, add 3μL of 10mM dNTPmix, 3μL of 50μM oligo(dT)20 and 24μL of RNA to each tube, and mix gently.

[0077] (2) Heat at 65℃ for 5 minutes to denature the template and open the secondary structure;

[0078] (3) Immediately place on ice to cool for at least 1 minute;

[0079] (4) Prepare a premixed solution for amplifying cDNA:

[0080]

[0081] (5) Add 30 μL of premixed solution to each of the two PCR tubes and mix well by pipetting.

[0082] (6) Heat the reaction mixture: anneal at 50°C for 50 min, then heat at 85°C for 5 min to terminate the reaction;

[0083] (7) Add 3 μL of RNAse H to each of the reaction mixtures and mix well. Heat at 37°C for 20 min to decompose the unreacted RNA.

[0084] (8) The synthesis of the first-strand cDNA is completed. After aliquoting, it is stored at -20℃ for later use.

[0085] 4. Amplification of the VHH gene in the variable region of heavy chain antibody

[0086] Degenerate primer pairs for the VHH gene of the IgG2 and IgG3 variable regions were used to amplify the VHH gene using cDNA as a template via polymerase chain reaction (PCR). Primer pairs F and R2 were used to clone the IgG2 isotype, and primer pairs F and R1 were used to clone the IgG3 isotype. The reaction system is as follows:

[0087]

[0088] The PCR procedure is as follows:

[0089]

[0090] The upstream primers for amplifying IgG2 and IgG3 are VHH-F, and the downstream primers are VHH-R2 and VHH-R1, respectively. The primer sequences are shown in Table 1.

[0091] Table 1. Primer sequence list for VHH antibody gene amplification and sequencing

[0092]

[0093] 5. Construction of phage-displayed nanobody libraries

[0094] The vectors pComb3X and VHH were digested with SfiI, and the digested VHH fragment was ligated with the vector pComb3X using T4 ligase. 3 μL of the ligation product was added to 25 μL of E. coli ER2738 competent cells, gently mixed, and then transferred to a pre-chilled electroporation cuvette (1 mm inner diameter). The cuvette was quickly placed in an electroporator for electroporation. Immediately after electroporation, 1 mL of preheated SOC medium at 37°C was added to the cuvette, and the mixture was gently aspirated and transferred to a shaker tube. The cuvette was then incubated at 37°C with shaking at 250 g for 1 h. The electroporation was repeated 10 times, with 3 μL of the ligation product added each time. The bacterial culture from the 10 transformations was collected, and 1 μL of the culture was diluted 10 times with sterile water and spread on an LB-Amp plate. The culture was then incubated overnight at 37°C to estimate the library capacity. All transformed bacteria were transferred to 200 mL of SB medium, and carbenicillin was added to a concentration of 50 μg / mL and tetracycline to a concentration of 20 μg / mL; the culture was carried out at 37°C and 250 rpm until OD500 was reached. 600 The concentration was 0.6; 1 mL of helper phage (1 × 10⁶) was added. 13 Incubate the phage solution at 37°C for 30 min (pfu / mL), then incubate at 37°C for 2 h (250g), add kanamycin to a concentration of 70 μg / mL, and continue incubation overnight. The next day, centrifuge the bacterial solution at 10000g for 15 min (4°C). Transfer the supernatant to a sterile centrifuge tube, add 1 / 4 volume of PEG / NaCl solution, incubate on ice for 2 h, centrifuge again, discard the supernatant, and resuspend the precipitate in 10 mL of PBS buffer (containing 1× protease inhibitor, 0.02% NaN3, and 0.5% BSA). Filter the phage solution through a 0.22 μm filter to remove residual bacteria. The resulting phage solution is the phage display nanobody library. Aliquot and label the library, and store at -70°C.

[0095] 6. Identification of phage-displayed nanobody immunotherapy libraries

[0096] Pick 30 single colonies from the plate and transfer each to 1 mL of SB medium, then incubate at 37°C until the bacterial culture reaches OD500. 600 The bacterial culture was taken out at approximately 0.8 μL. Using gback as primers (sequence shown in SEQ ID NO:6), it was sent to the company for sequencing. The clone sequences were analyzed to determine the diversity of the constructed library. The sequencing results are as follows: Figure 1 As shown, the amino acid sequences of the inserted fragments of the 30 selected clones are all different, indicating that the constructed phage-displaying nanobody library has good diversity and can be used for subsequent screening.

[0097] SEQ ID NO:6: 5'-GCCCCCTTATTAGCGTTTTGCCATC-3'

[0098] Example 2

[0099] 1. Selection of anti-idiotypic nanobodies against vomiting toxins

[0100] Using vomitoxin monoclonal antibodies as the target, affinity enrichment panning was performed by successively reducing the concentration of the coating agent, competitively eluting the concentration of vomitoxin standards, and alternating the use of blocking reagents to obtain antibodies against vomitoxin monoclonal antibodies, i.e., anti-antibodies, also known as anti-idiotype antibodies. The panning steps are as follows:

[0101] (1) Coating: Coat with vomitoxin monoclonal antibody, 50 μg / mL, 100 μL / well, 6 wells, diluted with coating buffer; remove adsorption wells, coat with 3% BSA / PBS, 300 μL / well, 6 wells; overnight coating at 4℃ is better than incubation at 37℃ for 2h.

[0102] (2) Blocking: 3% PBS™, 300 μL / well, incubate at 37°C for 2 h, then hand wash the plate 3 times;

[0103] (3) Add the constructed phage display nanobody library to the antigen-coated wells: 100 μL / well, react at 37°C for 1 h, then react at room temperature on a shaker for 1 h, and then hand wash these 6 wells with a pipette tip with a filter cartridge, and wash the plate 10 times.

[0104] (4) Elution: Prepare 100 ng / mL vomitoxin standard and react on a shaker at room temperature for 30 min;

[0105] (5) Desorption: Transfer the eluent to the desorption well, 100 μL / well, and react at room temperature for 1 h;

[0106] (6) Collect 600 μL of eluent, which is called "1st output". The first round of panning is completed. The 1st output needs to be amplified before it can be used for the second round of panning.

[0107] (7) Determine the titer of “1st output”: Take 10 μL of “1st output” and serially dilute it to 10 μL. 3 10 4 10 5 Take 10 μL of each of the three dilutions to infect 90 μL of ER2738 (OD = 0.8), let stand at 37°C for 30 min, spread on LB-Amp (ampicillin) plates, and incubate overnight at 37°C. Count the number of single colonies on the plates the next day to estimate the titer.

[0108] In the subsequent panning, the concentration of the coated antibody was gradually reduced, and the concentration of vomitoxin in the eluent was gradually reduced. Five rounds of panning were carried out. The panning process is shown in Table 2, and the panning enrichment results are shown in Table 3.

[0109] Table 2 Selection of Phage Nanobody Library

[0110]

[0111] Table 3. Phage enrichment results for each round of selection.

[0112]

[0113] 2. Screening of positive phage clones

[0114] (1) Randomly select 30 single clones from the output titer plates of the last round of selection, preserve them on LB-Amp plates, and inoculate them with 3 mL of SB medium at the same time;

[0115] (2) Incubate at 37℃ in a shaker for 4-5 hours until the OD value is 0.8. Add 30 μL of M13KO7 helper phage and incubate at 37℃ for 30 minutes.

[0116] (3) Incubate at 37℃ in a shaker for 1 h, add Kana to a final concentration of 70 μg / mL, and incubate at 37℃.

[0117] (4) The next day, take 500 μL of bacterial culture and centrifuge at 5000g for 10 min. Use the supernatant directly for Phage-ELISA.

[0118] (5) Block the ELISA plate with 3% PBS™, 300 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times.

[0119] (6) Add the phage supernatant of each clone to the corresponding 3 wells, 50 μL per well. Add 50 μL of standard to columns 1, 4, 7 and 10, and add 50 μL of 10% methanol / PBS buffer to columns 2, 3, 5, 6, 8, 9, 11 and 12. After adding, shake the microplate reader to mix.

[0120] (7) Let stand at 37℃ for 1 hour, then hand wash the board 10 times;

[0121] (8) Secondary antibody, Anti-M13 horseradish peroxidase, diluted 1:5000, incubated at 37℃ for 1h;

[0122] (9) Develop color, incubate at 37℃ for 15 min, and detect the absorbance at 450 nm.

[0123] Phage-ELISA was performed on 30 clones, and significant differences were found in their binding reactions with monoclonal antibodies against vomitoxin. In the Phage-ELISA assay, the OD (dose dissociation) of the antibody reaction was [not specified]. 450 The value is relatively high, and the OD value is also higher after adding DON. 450 Phage clones showing a significant decrease in pH were considered positive, as shown in the following results. Figure 2As shown, a total of 12 positive clones were obtained through five rounds of panning (clones 2, 7, 8, 17, 18, 19, 21, 22, 26, 28, 29, and 30, respectively). Based on the above ELISA results, the positive clones were picked from the preservation plates, activated, and then sent to Shanghai Sangon Biotech Co., Ltd. for sequence analysis. The sequencing primers were gback. Gene sequencing was performed on the 12 positive clones, and the phage-displayed vomitoxin nanobody VHH DO-7 with the highest OD value was selected. It has a purification tag, and its amino acid sequence is shown in SEQ ID NO:2.

[0124] Example 3: Expression and purification of anti-idiotypic nanobodies against vomiting toxin

[0125] 1. Preparation of Top10F' competent cells

[0126] (1) Select an appropriate amount of Top10F' and streak it on an LB-tetracycline plate, and incubate at 37°C overnight;

[0127] (2) Pick a Top10F' single colony into 5 mL of LB medium and incubate at 37°C and 250 rpm until OD. 600 It ranges from 0.6 to 0.8;

[0128] (3) Place the bacterial solution on ice and let it stand for 30 minutes;

[0129] (4) After centrifuging at 5000g for 5 min at 4℃, quickly place it back on ice, discard the supernatant, add 1 mL of pre-cooled 0.1M CaCl2 solution to resuspend the bacterial cells, mix well by pipetting, and then incubate on ice for 7 min.

[0130] (5) Repeat the above operation once;

[0131] (6) After centrifuging at 5000g for 5 minutes at 4℃, the cells were quickly placed back on ice and the liquid was completely removed. The precipitate was resuspended in 100μL of pre-cooled 0.1M CaCl2 solution, which is the Top10F' competent cells.

[0132] 2. Extraction and transformation of positive phage plasmids

[0133] (1) Select an appropriate amount of glycerol bacteria from VHH DO-7 / ER2738 and streak it on LB-ampicillin plates. Incubate overnight at 37°C.

[0134] (2) Pick a single colony of VHH DO-7 / ER2738 and add it to 2 mL of SB medium. Incubate at 37°C until OD500. 600 The value was 0.8, and the plasmid of VHH DO-7 was extracted;

[0135] (3) On ice, add 1.5 μL of VHH DO-7 plasmid to 100 μL of Top10F' competent cells prepared above, mix gently, and place on ice for 30 min;

[0136] (4) Heat shock at 42℃ for 90 seconds, then quickly return to ice and leave for 5 minutes;

[0137] (5) In the clean bench, add 700 μL of LB medium to each centrifuge tube and incubate at 37°C and 200 rpm for 45 min.

[0138] (6) Centrifuge at 12000g for 4 min at 4℃, discard the supernatant, add 100μL LLB medium to the centrifuge tube to resuspend the precipitate, mix well and spread on LB-ampicillin plates, and incubate overnight at 37℃.

[0139] 3. Induction, expression, and purification of nanobodies

[0140] On a VHH DO-7 / Top10F' plate, pick a single colony and transfer it to 3 mL of SB medium. Incubate overnight at 37°C with a shaker at 250 rpm. Then, transfer 200 μL of the overnight colony to 200 mL of SB medium and continue culturing until OD. 600 The concentration was 0.6. 200 μL of 1 M IPTG was added, and the mixture was induced overnight at 37°C. The cells were collected by centrifugation at 8000 g for 15 min at 4°C. Based on the precipitate mass, lysis buffer B-PER was added at 20 mL / g. After thorough dispersion, the precipitate was gently shaken at room temperature for 10 min, followed by centrifugation at 12000 g for 20 min. The supernatant was the crude extract of the nanobody. The crude extract was dialyzed against 0.01 M PBS 7.4, and then filtered through a 0.22 μm aqueous filter membrane for nickel column purification.

[0141] The crude nanobody extract was purified using the Ni-NTA His·Bind Resin kit, following these steps:

[0142] (1) Equilibrate the column sequentially with 10 column volumes of sterile ddH2O and 0.01 mol / L PBS 7.4;

[0143] (2) Add the filtered and sterilized crude extract to the chromatography column, mix it with the packing material, and shake it at room temperature for 1 hour to allow the nanoantibody to fully bind with the packing material.

[0144] (3) Load the mixture into the column and equilibrate the column with 10 column volumes of 0.01 mol / L PBS 7.4;

[0145] (4) Prepare 10 mL of 20 mM, 40 mM and 300 mM imidazole-PBS 7.4 with 1 M imidazole, filter through a 0.22 μm aqueous phase filter membrane, use as elution buffer for gradient elution, and collect the eluent in a 2 mL centrifuge tube.

[0146] (5) Pass 10 mL of 20 mM imidazole-PBS 7.4 through the column, without collecting the eluent;

[0147] (6) Pass 10 mL of 40 mM imidazole-PBS 7.4 through the column and collect the first 3 mL of eluent;

[0148] (7) Pass 10 mL of 300 mM imidazole-PBS 7.4 through a column and collect all the eluent;

[0149] (8) Wash the column with 10 column volumes of 1M imidazole-PBS 7.4 without collecting the eluent, so that all non-specifically bound proteins are eluted.

[0150] (9) Wash the column sequentially with 10 column volumes of PBS, 10 column volumes of ddH2O, and 10 column volumes of 20% ethanol, and finally seal the column with an equal volume of 20% ethanol water.

[0151] The effluent from each tube was analyzed by SDS-PAGE electrophoresis. The effluent with obvious nanobody bands were mixed and dialyzed overnight in PBS buffer at 4°C. The solution was then concentrated using an ultrafiltration tube to obtain the VHH DO-7 nanobody solution, which was aliquoted and stored at -20°C.

[0152] Example 4: Specificity analysis of anti-idiotypic nanobody VHH DO-7

[0153] (1) Prepare 0.2 μg / mL concentrations of DON-BSA (vomiting toxin antigen), OTA-BSA (ochratoxin A antigen), T-2-BSA (T-2 toxin antigen), DAS-BSA (serpentine toxin antigen), FB1-BSA (fumonisin B1 antigen), and AFB1-BSA (aflatoxin antigen) using coating buffer, and coat the microplates overnight at 4°C. The next day, block the microplates with 3% skim milk powder / conventional phosphate buffer / conventional phosphate-Tween buffer solution, 300 μL / well, and incubate at 37°C for 1 h. Serially dilute the six monoclonal antibodies against DON, OTA, T-2, DAS, FB1, and AFB1 starting at 10 μg / mL with conventional phosphate buffer and incubate at 37°C for 1 h. Add horseradish peroxidase-labeled goat anti-mouse monoclonal antibody and incubate at 37°C for 1 h. Add chromogenic solution and develop at 37°C for 15 min. Add stop solution and measure OD. 450 .

[0154] After coating and blocking as described above, the anti-idiotypic nanobody VHH DO-7 for vomitoxin was serially diluted three-fold with standard phosphate buffer. Six monoclonal antibodies against DON, OTA, T-2, DAS, FB1, and AFB1 were prepared at the optimized concentrations. 50 μL of VHH DO-7 dilution buffer and 50 μL of the corresponding monoclonal antibody were added to each well, and the mixture was shaken and incubated at 37°C for 1 h. Horseradish peroxidase-labeled goat anti-mouse antibody was then added at a 1:5000 dilution and reacted at 37°C for 1 h. Color development was performed using the same method, and OD was measured. 450 value.

[0155] Test results are as follows Figure 3 As shown, Figure 3 The results indicate that VHH DO-7 can inhibit the binding of vomitoxin monoclonal antibody to antigen DON-BSA. The inhibitory effect becomes more and more obvious as the concentration of VHH DO-7 increases. However, it has no inhibitory effect on the binding of anti-OTA, T-2, DAS, FB1 and AFB1 monoclonal antibodies to their respective antigens, indicating that VHH DO-7 has good selectivity and specifically binds only to the variable region of vomitoxin monoclonal antibody.

[0156] Example 5: Establishment of an ELISA method using the anti-idiotypic nanobody VHH DO-7 of vomitoxin as a substitute antigen for vomitoxin (VHH-ELISA).

[0157] The procedure for competitively inhibiting the ELISA reaction using the vomitoxin anti-idiotypic nanobody VHH DO-7 as a substitute antigen for vomitoxin is as follows:

[0158] 1. VHH-ELISA steps

[0159] (1) VHH DO-7 anti-idiotype nanobody for vomitoxin was diluted and used to replace the vomitoxin antigen to coat the microplate. The coating was carried out overnight at 4°C. The next day, the plate was blocked with blocking buffer and incubated at 37°C for 1 hour.

[0160] (2) Add methanol / PBS 7.4 buffer to the enzyme label wells, dilute the vomitoxin monoclonal antibody and the competitor with PBS 7.4 buffer and add them to the enzyme label wells, and incubate at 37°C for 1 h;

[0161] (3) Add horseradish peroxidase-labeled goat anti-mouse antibody and incubate at 37°C for 1 hour;

[0162] (4) Add the colorimetric reagent, react at 37°C for 15 min, and then measure the OD. 450 .

[0163] 2. Optimization of VHH-ELISA conditions

[0164] Optimal VHH coating concentration and working concentration of vomitoxin monoclonal antibody: VHH DO-7 was sequentially diluted to concentrations of 20, 10, 5, and 1 μg / mL, coated onto ELISA plates, and the VHH DO-7 nanobody coating concentration and the working concentration of vomitoxin monoclonal antibody were optimized using the checkerboard method. OD values ​​were then collected. 450 The optimal working concentrations for both nanobody and vomitoxin monoclonal antibody were around 1.0. The vomitoxin monoclonal antibody was diluted three-fold to concentrations of 100.00, 33.33, 11.11, 3.70, 1.23, 0.41, and 0.14 μg / mL. Indirect ELISA analysis was performed at these concentrations, and a standard curve was established. Figure 4 Based on the different colorimetric values ​​of each combination, the final working concentration of VHH DO-7 was selected as 5 μg / mL, corresponding to a concentration of 8.39 μg / mL for the vomitoxin monoclonal antibody.

[0165] 3. Cross-reactivity of the anti-idiotypic nanobody VHH DO-7 (vomiting toxin) as a coating antigen in VHH-ELISA

[0166] Using VHH DO-7 as a substitute antigen, and based on optimized conditions, six mycotoxins—DON, OTA, T-2, DAS, FB1, and AFB1—were selected as competitors. The cross-reactivity rate of VHH-ELISA was determined, and the results are as follows: Figure 5 As shown. By Figure 5 It can be seen that this ELISA has high sensitivity to DON, and the IC of VHH DO-7 is [missing information]. 50 The value reached 49.2645 ng / mL, and there was no cross-reactivity with OTA, T-2, DAS, FB1 and AFB1. Therefore, VHH-ELISA has good specificity and can specifically detect vomitoxin.

[0167] Example 6: Establishment of a colloidal gold immunochromatographic assay (VHH-GICA) using the anti-idiotypic nanobody VHH DO-7 as a substitute antigen for vomitoxin.

[0168] The establishment of a colloidal gold immunochromatographic assay (VHH-GICA) using the anti-idiotypic nanobody VHH DO-7 as a substitute antigen for vomitoxin includes the following steps:

[0169] 1. Establishment of a colloidal gold immunochromatographic test strip using VHH DO-7 as a substitute antigen for vomitoxin (VHH-GICA)

[0170] The structure of the test strip is as follows Figure 6As shown, the apparatus includes a base plate 1 and a sample pad 7, a gold-labeled pad 6, a detection pad 3, and an absorbent pad 2 sequentially overlapped on the base plate 1. The detection pad 3 has a detection line 5 and a control line 4. The detection line 5 is located on the detection pad 3 near the gold-labeled pad 6, and the control line 4 is located on the detection pad 3 near the absorbent pad 2. The detection line 5 is coated with VHH DO-7 prepared in Example 3, the control line is coated with rabbit anti-mouse polyclonal antibody, and the gold-labeled pad 6 is bound with a gold nanoparticle-labeled anti-vomiting toxin monoclonal antibody. The specific preparation method is as follows:

[0171] (1) Preparation of absorbent pad 2

[0172] Cut the absorbent paper to get absorbent pad 2;

[0173] (2) Preparation of test pad 3

[0174] Coating of detection line 5:

[0175] VHH DO-7 was prepared into coating solutions of 0.25–0.5 mg / mL using coating buffer, and then coated onto nitrocellulose membranes to obtain detection lines 5. The membranes were then dried at 37–40°C for 30–60 minutes. The required coating amount of VHH DO-7 per centimeter of detection line 5 was 100–300 ng. The distance between detection line 5 and the upper edge of the nitrocellulose membrane was 15–20 mm. Each 10 mL of the coating buffer contained: 0.08 g sodium chloride, 0.002 g potassium chloride, 0.002 g potassium dihydrogen phosphate, 0.1–0.2 g ovalbumin, and 0.029 g disodium hydrogen phosphate dodecahydrate.

[0176] Coating of control line 4:

[0177] Rabbit anti-mouse polyclonal antibody was prepared into a coating solution of 0.2-0.4 mg / mL using coating buffer. It was then coated horizontally onto a nitrocellulose membrane to obtain control line 4. The amount of rabbit anti-mouse polyclonal antibody required per centimeter of control line 4 was 50-200 ng. The membrane was then dried at 37-40°C for 1-2 hours. The coating buffer was the same as above.

[0178] (3) Preparation of sample pad 7

[0179] The glass fiber membrane was immersed in the blocking solution, removed, and dried at 37-40°C for 6-10 hours to obtain sample pad 7. It was then stored in a desiccator at room temperature. The blocking solution used contained the following per 100 mL: 2-5 g sucrose, 0.8 g sodium chloride, 0.02 g potassium chloride, 0.02-0.05 g sodium azide, 0.02 g potassium dihydrogen phosphate, 1-2 g bovine serum albumin, and 0.29 g disodium hydrogen phosphate dodecahydrate.

[0180] (4) Preparation of gold-labeled pad 6

[0181] The glass fiber membrane was immersed in the blocking solution and then dried at 37-40°C for 6-10 hours. The nano-gold labeled anti-DON monoclonal antibody solution was then sprayed laterally onto the dried glass fiber membrane using a dot spraying method. The amount of nano-gold labeled anti-vomiting toxin monoclonal antibody required per centimeter of spraying length was 100-200 ng. The membrane was then freeze-dried under vacuum for 2-4 hours and stored at room temperature in a desiccator.

[0182] (5) Assembly of test strips

[0183] The absorbent pad 2, detection pad 3, gold label pad 6, and sample pad 7 are pasted onto the base plate 1 from top to bottom, overlapping each other by 1-2 mm, thus obtaining the immunochromatographic test strip for detecting DON.

[0184] 2. Application of VHH DO-7 as a substitute antigen for vomitoxin in the detection of vomitoxin (DON) content using colloidal gold immunochromatographic (VHH-GICA) test strips.

[0185] The test strip prepared above was found to have a detection limit of 5 ng / mL for vomitoxin, which is far lower than the national standard of 500 ng / mL.

[0186] The specific detection method is as follows: After diluting the sample extract with the test sample sustained-release solution, add it dropwise to the sample pad of the rapid DON immunochromatographic test strip for detection, which serves as the test strip; take an equal volume of the test sample sustained-release solution as the negative control solution, and add it dropwise to the sample pad of another rapid DON immunochromatographic test strip, which serves as the control test strip. After 15-20 minutes, perform color development comparison between the test strip and the control test strip. When the color of the test line on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the DON content in the sample solution is less than 5 ng / mL; when the color is lighter than the corresponding test line on the control test strip, it indicates that the DON content in the sample solution is equal to or higher than 5 ng / mL but less than 50 ng / mL; when no color develops, it indicates that the DON content in the sample solution is equal to or higher than 50 ng / mL; when the control line does not develop color, the test strip is considered invalid regardless of whether the test line on the test strip develops color; finally, the DON content in the sample can be calculated.

[0187] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A vomiting toxin anti-idiotype nanobody, characterized in that, The amino acid sequence of the nanobody is selected from any of the following: (a) The amino acid sequence as shown in SEQ ID NO:1; (b) An amino acid sequence formed by adding a tag sequence, restriction site sequence or signal sequence to one or both ends of the sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the vomiting toxin anti-idiotypic nanobody as described in claim 1.

3. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. Transform or transfect the host bacteria of the expression vector as described in claim 3.

5. A kit for detecting vomitoxin, characterized in that, Including the anti-idiotype nanobody against vomiting toxin as described in claim 1.

6. The method for preparing the anti-idiotypic nanobody against vomitoxin as described in claim 1, characterized in that, The vomitoxin anti-idiotype nanobody is produced using the host bacteria of claim 4.

7. The application of the anti-idiotypic nanobody for vomitoxin as described in claim 1 in the immunoassay detection of vomitoxin for non-diagnostic purposes.

8. The application according to claim 7, characterized in that, The immunoassays include enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, colloidal gold immunochromatography, time-resolved fluorescence immunochromatography, electrochemical immunosensing, bioimmunosensing, nanozyme immunoassay, nanoimmunosensing, immunodot hybridization, and latex immunoturbidimetry.

9. The application according to claim 7 or 8, characterized in that, The vomitoxin anti-idiotypic nanobody, as an alternative to vomitoxin, is used in immunoassays for non-diagnostic purposes in the form of a competitive antigen, coating antigen, calibrator, quality control, or standard.

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