An immunoadsorbent and affinity column for regeneratively purifying aflatoxin B1

Abstract

The present application relates to a kind of reproducible aflatoxin B1 immunoadsorbent and affinity column for purifying.The immunoadsorbent includes solid phase carrier and the aflatoxin B1 nanobody coupled on the solid phase carrier, the aflatoxin B1 nanobody is the aflatoxin B1 nanobody of amino acid sequence as shown in SEQ ID NO:1, or it is as shown in SEQ ID NO:3 Self-assembly multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6.The aflatoxin B1 nanobody in the immunoadsorbent of the present application is high in sensitivity, high in specificity, can be used for the specific adsorption of aflatoxin B1, is used as reproducible purification aflatoxin B1 immune affinity column after packing column, for the high performance liquid chromatography-mass spectrometry detection of aflatoxin B1.The preparation step of the immune affinity column of the present application is simple, low in cost, stable in performance, strong in tolerance, high in specificity, good in reproducibility.Provide the possibility for establishing an environmentally friendly, economic, sensitive, fast detection method.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a renewable immunosorbent and affinity column for purifying aflatoxin B1. Background Technology

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. AFB1 contamination is widespread, occurring throughout the entire supply chain from production and processing to transportation and storage. It primarily contaminates grains, nuts, and oilseeds, severely impacting international trade and sustainable development in the food industry. AFB1 is the most potent carcinogen known, exhibiting hepatotoxicity, nephrotoxicity, immunotoxicity, mutagenicity, and teratogenicity, posing a serious threat to human health. Currently, the main methods for AFB1 detection include thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), immunoaffinity chromatography-liquid chromatography (ILC), and immunoaffinity chromatography-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0003] In 1993, scientists first discovered small heavy-chain antibodies lacking light chains in camel blood; these antibodies are called nanobodies (Nb) or VHH single-domain structures. Nanobodies are uniquely attractive in the field of rapid immunoassays due to their small size (15-17 kDa), high stability, strong tolerability, ease of production, and excellent affinity and specificity. Therefore, using nanobodies as the core component of the immunoadsorbent in the detection of AFB1 by immunoaffinity chromatography-high performance liquid chromatography-tandem mass spectrometry has significant advantages.

[0004] Immunoaffinity chromatography-high performance liquid chromatography-tandem mass spectrometry (AHPLC-MS / MS) combines immunoreaction with chromatographic and mass spectrometric analysis. Utilizing the high specificity and affinity of antigen-antibody binding, specific antibodies are chemically coupled to an immunosorbent. Based on reversible immunological binding, this method achieves efficient enrichment, separation, and purification of AFB1 in complex matrices. Therefore, developing high-performance, stable, and reproducible AFB1 immunoaffinity columns is a prerequisite for developing efficient, stable, economical, and accurate AFB1 HPLC-MS / MS detection methods. Summary of the Invention

[0005] The purpose of this invention is to provide a renewable immunosorbent and affinity column for purifying aflatoxin B1, as well as its preparation method and application, in order to address the shortcomings of existing technologies.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] An immunoadsorbent for the regenerative purification of aflatoxin B1 is provided. The immunoadsorbent comprises a solid-phase support and aflatoxin B1 nanobody coupled to the solid-phase support. The aflatoxin B1 nanobody is an aflatoxin B1 nanobody with the amino acid sequence shown in SEQ ID NO:1, or a self-assembled multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6 as shown in SEQ ID NO:3. The aflatoxin B1 nanobody in the immunoadsorbent of this invention exhibits high sensitivity and specificity, enabling the specific adsorption of aflatoxin B1. After packing, it can be used as a regenerative immunoaffinity column for the purification of aflatoxin B1.

[0008] According to the above scheme, the solid support is agarose gel.

[0009] An affinity column is provided, loaded with an immunosorbent that can regenerate and purify aflatoxin B1.

[0010] A method for preparing a regenerable aflatoxin B1 affinity column is provided, comprising the following steps:

[0011] a) Substrate treatment

[0012] First, the agarose gel matrix (sepharose 4B) activated with hydrogen bromide (CNBr) was swollen in hydrochloric acid solution, and then washed with hydrochloric acid to remove impurities. The CNBr-activated agarose gel was provided in lyophilized form.

[0013] b) Ligand coupling

[0014] After rinsing the above matrix with conjugation buffer, the aflatoxin B1 nanobody solution was rapidly transferred into the rinsed matrix for conjugation.

[0015] c) Ligand blocking

[0016] To seal excess active groups on the gel surface;

[0017] d) Remove uncoupled redundant ligands;

[0018] e) Pack the column.

[0019] According to the above scheme, in step (a), the concentration of HCl in both the swelling solution and the washing solution is 1 mM, the swelling time is 15 min, and the rinsing is performed 5 times.

[0020] According to the above scheme, the coupling buffer in step (b) is 0.1M NaHCO3 and 0.5M NaCl, pH=8.3.

[0021] According to the above scheme, the coupling conditions in step (b) are: reacting in a shaker at room temperature for 3 hours.

[0022] According to the above scheme, the sealing process in step (c) is as follows: first rinse with 5 times the volume of 0.1M Tris-HCl solution, and then seal the reaction with 0.1M Tris-HCl solution for 2 hours.

[0023] According to the above scheme, step (d) is as follows: the matrix treated in step (c) is rinsed sequentially with buffer A (0.1M CH3COONa / CH3COOH and 0.5NaCl, pH=4) and buffer B (0.1M Tris-HCl, pH=8), and the washing is performed at least 3 times.

[0024] The present invention also provides a method for detecting aflatoxin B1 content using the above-mentioned affinity column. When the sample to be tested is passed through the immunoaffinity column, the immunoadsorbent will specifically adsorb aflatoxin B1, while other impurities will not be adsorbed by the immunoaffinity column and will elute. Then, chromatographic grade methanol is used for elution, and the eluent is collected as the purified and concentrated sample. The content of aflatoxin B1 in the eluent is detected by high performance liquid chromatography-mass spectrometry.

[0025] The highly resistant aflatoxin B1 nanobody of this invention can be produced using genetic engineering methods, featuring a short preparation cycle, low cost, and a simple preparation method. The specific steps are as follows:

[0026] a) Obtain bacterial culture containing aflatoxin B1 nanobody gene on streak plates;

[0027] b) Select single-clonal colonies of small-scale shaking bacteria;

[0028] c) Large-scale transfer of vaccinations;

[0029] d) Low-temperature induction of high-efficiency expression of nanobodies by isopropyl thiogalactoside (IPTG);

[0030] e) Bacterial lysis, supernatant dialysis, and nickel column purification;

[0031] f) Concentrate by ultrafiltration, add an equal volume of 50% glycerol, and store at -20°C for later use.

[0032] This invention first provides aflatoxin B1 nanobody with high specificity, high sensitivity, and high affinity. Then, by coupling the nanobody with a carrier and packing it onto a column, a high-affinity aflatoxin B1 immunoaffinity column is formed. This column can be used for high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) detection of aflatoxin B1. The immunoadsorbent exhibits high affinity and sensitivity, and the affinity column is stable, robust, highly specific, and regenerable. The sample to be tested can be purified simply by passing it through the immunoaffinity column. The operation is simple and highly specific, eliminating the need for further purification before direct HPLC detection, thus saving operator time and costs and possessing the potential for large-scale production and application. This provides a possibility for establishing an environmentally friendly, economical, sensitive, and rapid detection method.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] The aflatoxin B1 nanobody used in the immunosorbent of this invention has good specificity, high sensitivity, strong tolerance (resistant to high temperature, high concentration of organic reagents, and acids and alkalis), and high affinity. Based on this, the provided immunosorbent has high affinity, specificity, and sensitivity, and can be used for the specific adsorption of aflatoxin B1. After packing, it can be used as a regenerable aflatoxin B1 purification immunoaffinity column. The affinity column has stable performance, strong tolerance, high specificity, simple operation, high purification efficiency, and minimal interference from other toxins.

[0035] The aflatoxin B1 nanobody in the immunoadsorbent of this invention has high affinity and strong binding, which helps to obtain immunoaffinity columns with high recovery rate and column capacity.

[0036] The immunoaffinity column of this invention exhibits excellent regenerative properties. This invention utilizes an immunoaffinity column composed of a self-assembled multivalent aflatoxin B1 fused with the nanobody NbAF-EGFP-H6 for regenerative purification of aflatoxin B1. It can be reused 200-300 times, and the recovery rate remains above 84.43% even after 150 consecutive uses. Attached Figure Description

[0037] Figure 1 This is a diagram showing the expression and purification results of recombinant protein NbAF-EGFP-H6. Lane M: Protein Marker; Lane 1: Total Periplasmic Protein; Lane 2: Flow-through Buffer; Lane 3: 20 mmol / L Imidazole Eluent; Lane 4: 40 mmol / L Imidazole Eluent; Lane 5: 300 mmol / L Imidazole Eluent.

[0038] Figure 2 These are field emission scanning electron microscope (FESEM) images of purified self-assembled nanobodies. The scale bars in the images are all 100 nm.

[0039] Figure 3This is an affinity identification curve of the recombinant multivalent nanobody NbAF-EGFP-H6;

[0040] Figure 4 This is an affinity curve for the nanobody NbAF;

[0041] Figure 5 This is a sensitivity standard curve of the recombinant nanobody ELISA method;

[0042] Figure 6 This is the competitive inhibition curve of recombinant nanobody against aflatoxin analogue.

[0043] Figure 7 Schematic diagram of the structure of the aflatoxin B1 immunoaffinity column.

[0044] A: Inlet plug; B: Inlet; C: Column tube; D: Upper sieve plate; E: Immunosorbent; F: Lower sieve plate; G: Outlet; H: Outlet plug.

[0045] Figure 8 Schematic diagram of the reproducibility verification of aflatoxin B1 immunoaffinity column. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The embodiments are merely examples and do not constitute any limitation on the scope of the present invention.

[0047] Part 1: Obtaining Aflatoxin B1 Nanobodies

[0048] (1) Obtaining aflatoxin B1 nanobody NbAF

[0049] This invention uses aflatoxin B1 as a hapten conjugated with bovine serum albumin (BSA) to immunize alpacas. A phage display nanobody library is constructed by extracting total RNA from alpacas leukocytes. Using AFB1-BSA as the target, four rounds of affinity enrichment panning are employed, progressively reducing the coating concentration and competitive elution concentration to obtain specific aflatoxin B1-positive phage nanobodies with good specificity. The aflatoxin B1 nanobody gene is transformed into an expression strain, and after induction, purification, and identification, a high-performance protein-form nanobody, NbAF, is obtained. The amino acid sequence of the aflatoxin B1 nanobody is shown in SEQ ID NO:1, and the nucleotide sequence of the NbAF nanobody fragment (VHH) is shown in SEQ ID NO:2.

[0050] Affinity, sensitivity, and specificity analyses of the aflatoxin B1 nanobody NbAF showed that its affinity was 0.47 × 10⁻⁶. -3The sensitivity IC50 was 39.6 ng / mL, and the cross-reactivity rates to other aflatoxin analogs aflatoxin B2, G1, G2, and M1 were: AFB2: 0.92%, AFG1: 2.2%, AFG2: ND, and AFM1: 8.5%.

[0051] The specific steps for the expression and purification of the aflatoxin B1 nanobody are as follows:

[0052] Bacterial culture containing the aflatoxin B1 nanobody gene was streaked overnight in LB solid medium containing ampicillin (AMP) resistance at 37°C. The next day, single colonies were picked and inoculated into 5 mL of LB liquid medium and cultured overnight. The following day, the overnight bacteria were transferred to AMP-resistant LB liquid medium at a 1:100 inoculum and cultured until the logarithmic OD phase. 600 IPTG was added to a final concentration of 1 mmol / L when the pH was 0.6–0.8. After induction at 16°C for 16–18 h, the cells were collected by centrifugation (8000 rpm, 15 min). Using cell lysis buffer, the cells were centrifuged again (8000 rpm, 15 min) to collect the supernatant, which was E. coli periplasmic protein. The extracted periplasmic protein was dialyzed three times with 0.01 M PBS buffer, with the dialysate changed every 3–4 h. Then, the cells were centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was purified by nickel column affinity chromatography. The purified aflatoxin B1 nanobody was dialyzed five times with PBS buffer (pH 7.4) at 4°C, with the dialysate changed every four hours. The protein was then concentrated by ultrafiltration. The concentrated protein was added with an equal volume of glycerol and stored at -20°C.

[0053] (2) Obtaining the self-assembled multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6

[0054] Example 1

[0055] Example: Construction of the recombinant plasmid pET-22b-VHH-linker-EGFP-H6 vector

[0056] 1. Design of NbAF amplification primer sequences

[0057]

[0058] Note: Underlined text indicates Linker: GGGGS

[0059] 2. Amplification of the NbAF gene

[0060] Polymerase chain reaction (PCR) amplification was performed using the VHH gene fragment plasmid as a template. The reaction system (50 μL) is as follows:

[0061] 2*Taq Master Mix 25μL 10 μM upstream primer VHH-F 2μL 10μM downstream primer VHH-R 2μL Template DNA 1μL <![CDATA[ddH2O]]> Up to 50μL

[0062] The PCR amplification reaction procedure is as follows:

[0063]

[0064]

[0065] 3. PCR product validation and gel recovery

[0066] Weigh 1% agarose (W / V) and dissolve it in 1*TAE solution by heating. Pour the solution into a gel casting tank and cool to form a gel. PCR products are spotted and electrophoresed. First, 4 μL of the product is used for verification to obtain a gene fragment containing the linker sequence with a size of 430 bp. After successful verification, the product is purified and recovered according to the gel extraction kit manual. Finally, 1 μL of DNA is taken and its concentration is determined by Nanodrop. Store at -20℃ for later use.

[0067] 4. NbAF gene was introduced into pET22b-EGFP-H6 plasmid (one-step cloning method)

[0068] (1) Preparation of linear vector: The vector was prepared by double digestion. The unique sites BspMI and BamHI were selected for digestion. In NEBuffer r2.1, BspMI and BamHI enzymes were used to digest the vector at 37°C for 4 hours. The vector was then verified by gel electrophoresis. The vector was purified and recovered using the kit and stored at -20°C for later use.

[0069] The linearization double enzyme digestion reaction system for pET22b-EGFP-H6 plasmid is as follows:

[0070] pET22b-EGFP-H6 plasmid 10μL NEBuffer r2.1 5μL BspMI 2μL BamHI 2μL <![CDATA[ddH2O]]> Up to 50μL

[0071] (2) The recombination reaction is carried out, and the recombination reaction system is as follows:

[0072]

[0073] (3) Use a pipette to gently aspirate and mix, and then centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0074] (4) React at 37℃ for 30 min, then cool to 4℃ or immediately place on ice to cool;

[0075] (5) The recombinant plasmid pET-22b-VHH-linker-EGFP-H6 can be stored at -20℃ for one week and transformed in time.

[0076] 5. Transformation of recombinant plasmids

[0077] Place competent E. coli DH5α cells on ice. Add 10 μL of recombinant product to 100 μL of competent cells, gently tap the tube to mix, and incubate on ice for 30 min. After heat shock in a 42°C water bath for 45 s, immediately cool on ice for 2–3 min. Add 900 μL of antibiotic-free LB medium and incubate at 37°C for 1 h. Centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, and resuspend the remaining supernatant on LB agar plates containing the antibiotic AMP. Incubate overnight at 37°C upside down. The next day, pick positive single colonies for colony PCR identification. The primers for detection and amplification are designed as follows:

[0078] JC-F 5'-ACCATACCCACGCCGAAACA-3' JC-R 5'-CCGGACACGCTGAACTTGTG-3'

[0079] The PCR reaction procedure was the same as in step 2. The colony PCR produced a band size of 756 bp, consistent with the theoretical value, and the sequencing results also showed complete consistency with the expected nucleotide sequence. The recombinant plasmid pET-22b-VHH-linker-EGFP-H6 was successfully constructed.

[0080] Example 2

[0081] Expression and purification of recombinant protein from recombinant plasmid pET-22b-VHH-linker-EGFP-H6

[0082] The recombinant plasmid pET-22b-VHH-linker-EGFP-H6 was transformed into competent E. coli BL2(DE3) cells, following the same method as described in Example 1, Section 5. Positive single colonies were screened by colony PCR, and the positive bacterial suspensions were stored at -80°C with 50% glycerol for later use. The positive NbAF-EGFP-H6 / BL21 bacterial suspensions were streaked overnight in LB solid medium containing ampicillin (AMP) resistance. Single colonies were picked and inoculated into 5 mL of LB liquid medium. The next day, the overnight colonies were transferred to AMP-resistant LB liquid medium at a 1:100 inoculation rate and cultured until the logarithmic OD phase. 600 IPTG was added to a final concentration of 1 mmol / L when the pH was between 0.6 and 0.8. After induction at 37°C for approximately 12 hours, bacterial cells were collected by centrifugation. Cell lysis buffer was used, and the supernatant was collected by centrifugation to obtain E. coli periplasmic protein. The recombinant nanobody was purified by nickel column affinity chromatography. The collected samples before and after purification, as well as the percolation buffer, were added to the loading buffer and boiled in water for 5 min. SDS-PAGE electrophoresis was performed on a stacking gel at 80V and a separating gel at 120V. After electrophoresis, the sample was stained with Coomassie Brilliant Blue and then destained with destaining solution. The purified recombinant protein NbAF-EGFP-H6 was dialyzed five times at 4°C with PBS buffer at pH 7.4, with the dialysate changed every four hours. Then, the protein was concentrated by ultrafiltration and stored at -20°C.

[0083] Result: As Figure 1 As shown, the recombinant protein NbAF-EGFP-H6 was successfully purified and eluted with 300mM imidazole. The actual molecular weight of the recombinant protein was consistent with the theoretical size of 43.3kDa. The concentration of NbAF-EGFP-H6 after purification and concentration was 2.726mg / mL.

[0084] The amino acid sequence of NbAF-EGFP-H6 is shown in SEQ ID NO: 3, the nucleotide sequence of the NbAF-EGFP-H6 encoding gene is shown in SEQ ID NO: 6, the amino acid sequence of EGFP is shown in SEQ ID NO: 4, the nucleotide sequence is shown in SEQ ID NO: 5, the amino acid sequence of the linker is shown in SEQ ID NO: 7, the nucleotide sequence is shown in SEQ ID NO: 8, and the nucleotide sequence of H6 is shown in SEQ ID NO: 9.

[0085] Example 3: Characterization of the self-assembled multivalent recombinant protein NbAF-EGFP-H6

[0086] 1. Identification of the physical structure of NbAF-EGFP-H6

[0087] Under near-natural conditions, the ultrastructural morphology (size and shape) of the multivalent NbAF-EGFP-H6 nanobody was measured using field emission scanning electron microscopy (FESEM). Five μL of nanoparticles resuspended in buffer solution were deposited directly onto a silicon wafer for 2 minutes. Excess liquid was aspirated and air-dried, and the nanoparticles were immediately observed in a Fesem Merlin (Zeiss) microscope equipped with an in-lens auxiliary electron detector at 1 kV. Representative images of the multivalent NbAF-EGFP-H6 nanobody nanostructure were captured.

[0088] The structure and morphology of multivalent nanobodies were captured by field emission scanning electron microscopy of the purified protein products, such as... Figure 2 As shown, the diameter of the green fluorescent protein (EGFP) monomer is approximately 2.4 nm, the diameter of the aflatoxin B1 nanobody monomer is approximately 2.5 nm, and the theoretical diameter of the monovalent recombinant nanobody is around 10 nm. From... Figure 2 As can be seen, the protein building blocks oligomerize to form regular pseudo-spherical nanoparticles with a size of 50-100 nm, proving that the protein building blocks constructed by genetic engineering in this invention form a self-assembled multivalent aflatoxin B1 fusion nanobody with green fluorescence.

[0089] 3. Identification of NbAF-EGFP-H6 affinity

[0090] The affinity of NbAF-EGFP-H6 was determined using an indirect, non-competitive ELISA. Microplates were coated with AFB1-BSA at concentrations of 1.0, 0.5, 0.25, and 0.125 μg / mL (100 μL / well) and incubated at 37°C for 2 h. After blocking with 4% skim milk powder / PBST for 1 h, serially diluted NbAF-EGFP-H6 nanobodies were added to the corresponding microplates and incubated at 37°C for 1 h. After washing three times, HRP-labeled anti-His-tagged mouse monoclonal antibody diluted 1:5000 with PBST was added, and incubation continued at 37°C for 1 h. After washing the microplates six times, substrate chromogenic solution was added, and the reaction was carried out at 37°C for 15 min. The reaction was then terminated by adding concentrated sulfuric acid. Four S-shaped curves were plotted with the measured OD450 value on the ordinate and the logarithm of the NbAF-EGFP-H6 nanobodies concentration on the abscissa for the four concentrations. Find the maximum OD value (ODmax) at the top of each S-curve, and then find the antibody concentration corresponding to 50% ODmax for each curve. Pair the four concentrations arbitrarily, and calculate the antibody affinity constant using the formula Ka = (n-1) / 2(n[Ab']t - [Ab]t), where [Ab']t and [Ab]t are the fusion nanoantibody concentrations corresponding to the two 50% maximum OD values ​​in each pair, and n is a multiple of the coating antigen concentration in each pair (including ratios of 1:2, 1:4, and 1:8). This yields six Ka values. Average the six Ka values ​​to obtain the affinity of the NbAF-EGFP-H6 nanoantibody using enzyme-linked immunosorbent assay (ELISA).

[0091] An S-shaped curve for affinity testing was plotted using Prism 9 software, as shown below. Figure 3 As shown, the antibody concentrations corresponding to the 50% ODmax values ​​of each curve for the four coating concentrations were calculated to be 36.46 ng / mL, 43.84 ng / mL, 45.94 ng / mL, and 77.64 ng / mL, respectively. The average of the six Ka values ​​was calculated using the formula Ka = (n-1) / 2(n[Ab']t - [Ab]t), ultimately yielding an affinity of 7.75 × 10⁻⁶ for the NbAF-EGFP-H6 recombinant nanobody in ELISA. -3 The same method was used to determine the affinity of the aflatoxin B1 nanobody NbAF before gene fusion. Figure 4 The calculation method is the same as above, yielding an affinity of 0.47 × 10⁻⁶ for NbAF nanobody ELISA. -3 The affinity of the self-assembled multivalent NbAF-EGFP-H6 fusion nanobody after gene recombination was greatly improved by 16.5 times compared with that of the NbAF nanobody.

[0092] 4. Sensitivity identification of NbAF-EGFP-H6

[0093] Coat a 96-well ELISA plate with 0.5 μg / mL AFB1-BSA overnight at 4°C; wash the plate three times, block with 4% skim milk / PBST for 1 h; wash the plate three times, then serially dilute the purified NbAF-EGFP-H6 nanobody to different concentrations with PBS, add to the ELISA plate, and incubate at 37°C for 1 h; the remaining steps are the same as the indirect non-competitive ELISA method, and measure OD. 450nm Value. OD 450nm The dilution factor of the NbAF-EGFP-H6 nanobody with a value of around 1.0 is used as the working concentration of the antibody.

[0094] Coat a 96-well ELISA plate with 1 ppm AFB1-BSA overnight at 4°C; wash the plate three times, block with 4% skim milk / PBST for 1 h; wash the plate three times, serially dilute AFB1 with 10% methanol / PBS and add 50 μL to the plate, then add 50 μL of NbAF-EGFP-H6 nanobody diluted to the working concentration to the well, and incubate at 37°C for 1 h; wash the plate three times with PBST, add 100 μL of HRP-labeled anti-His-tagged mouse monoclonal antibody to each well, and continue incubation at 37°C for 1 h; wash the plate six times, add substrate chromogenic solution, and perform chromogenic reaction at 37°C for 15 min, then add 50 μL of concentrated sulfuric acid to terminate the reaction. Measure the OD450nm value and plot the ELISA standard curve.

[0095] Using Prism 9 software, the absorbance ratio Bx / B0 as a function of AFB1 (log 10) concentration was plotted using four-parameter logistic regression. Figure 5 As shown, the fitting formula is Y = 100 / (1 + (1.504 / X)^-0.8038), where Y represents the absorbance ratio Bx / B0, X represents the AFB1 concentration, and the sensitivity IC50 (50% inhibition concentration against AFB1) is 1.5 ng / mL. The sensitivity of the nanobody NbAF was also detected using the same method. Figure 5 Its sensitivity IC50 is 39.6 ng / mL. The sensitivity of the recombinant multivalent nanobody NbAF-EGFP-H6 is greatly improved compared with that of the nanobody NbAF, by 26.4 times.

[0096] 5. Specificity identification of NbAF-EGFP-H6

[0097] The specificity of an antibody can be evaluated using cross-reactivity. An indirect competitive ELISA method was used for the assay. Standard solutions of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and aflatoxin M1 (AFM1) were prepared at a series of concentrations. These solutions were then added to an equal volume of NbAF-EGFP-H6 nanobody nanoparticles and incubated for 1 hour. Other steps were the same as in the indirect competitive ELISA method. A competition inhibition curve was plotted with the concentration of the toxin standards on the x-axis and the OD value (B / B0) at 450 nm measured by the ELISA reader on the y-axis. The cross-reactivity was determined by calculating the ratio of the IC50 values ​​with other toxins. The calculation formula is: CR% = (IC50DAS / IC50 other toxins) × 100%.

[0098] Specificity analysis was performed on recombinant sodium antibody NbAF-EGFP-H6, and cross-reactivity was determined using aflatoxin B1, B2, G1, G2, and M1. Results are shown in [Figure number missing]. Figure 6 The sensitivity corresponding to a 50% inhibition rate of the recombinant nanoantibody against each toxin was calculated and compared with the sensitivity of AFB1. The cross-reactivity rate was calculated, and the results are shown in Table 1. The results showed that the self-assembled recombinant aflatoxin B1 fusion nanoantibody had no cross-reactivity with other aflatoxin analogs. Compared with the cross-reactivity rate of the unmodified NbAF nanoantibody (AFB2: 0.92%, AFG1: 2.2%, AFG2: ND, AFM1: 8.5%), the specificity of NbAF-EGFP-H6 was enhanced.

[0099] Table 1 Cross-reactivity of recombinant nanobody NbAF-EGFP-H6 with Aspergillus analogues

[0100]

[0101] a ND, not detectable

[0102] Part Two: Renewable Immunoaffinity Columns for Purifying Aflatoxin B1 and Their Applications

[0103] The aflatoxin B1 nanobody NbAF or the self-assembled multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6 in the first part can be used to prepare an immunoadsorbent for the regenerative purification of aflatoxin B1 and to further prepare it into an immunoaffinity column.

[0104] The specific preparation method for the immunosorbent and affinity column made from the self-assembled multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6 is as follows:

[0105] Example 1: Preparation of a regenerable immunosorbent and affinity column for purifying aflatoxin B1

[0106] 1. Preparation of the matrix

[0107] Dissolve 1 g of CNBr-activated agarose gel (Sepharose 4B) lyophilized matrix powder (each gram of lyophilized matrix powder forms a final volume of 3.5 mL of swollen matrix) in 40 mL of 1 mM HCl and keep for 15 min. The matrix will swell immediately. Then place it in a sintered glass filter and rinse with 1 mM HCl for 15 min.

[0108] 2. Conjugation of aflatoxin B1 nanobodies

[0109] a) Dissolve the aflatoxin B1 nanobody to be conjugated in 0.2 mol / L NaHCO3 pH 8.3 conjugation buffer. Add 2 mg of antibody and temporarily store the dissolved antibody in an ice bath. Add the conjugation buffer containing the aflatoxin B1 nanobody to a sealable container. Quickly transfer CNBr-activated Sepharose 4B to the antibody solution. Incubate at room temperature (20-25°C) on a shaker for 3 hours.

[0110] b) Calculation of coupling rate: Centrifuge at 2,000 rpm until the Sepharose 4B reaches the bottom of the tube. Transfer the supernatant to a new centrifuge tube and determine the protein content of the supernatant. The calculated coupling rate is 98.8%. Take the Sepharose 4B that has reached the bottom of the tube and wash it with 0.1M Tris-HCl solution to remove any unbound aflatoxin B1 nanobody.

[0111] c) Blocking: The matrix was dissolved in 0.1 mol / L Tris-HCl buffer and allowed to stand at room temperature for 2 h to block excess active groups on the gel surface.

[0112] d) To remove excess ligands from uncoupled ligands after coupling, the matrix was washed sequentially with 0.1 mol / L acetate / sodium acetate buffer (pH=4) and 0.1 mol / L Tris-HCl buffer (pH=8) for 5 cycles, using 5 times the matrix volume of each buffer. Each washing cycle consisted of: first washing with 0.1 mol / L acetate / sodium acetate buffer, followed by washing with 0.1 mol / L Tris-HCl buffer. Finally, equilibration was performed with 5 times the gel volume of 0.01 M PBS, and the equilibrated matrix was stored at 4°C in 0.01% NaN3-PBS.

[0113] 3. Column mounting

[0114] The aflatoxin B1 immunoaffinity column structure is composed of, for example, Figure 7 As shown, the column packing steps are as follows:

[0115] a) Mix the conjugated immunosorbent with PBS buffer at a ratio of 3:1.

[0116] b) Take a 5ml stoppered column tube, add it to the sieve plate below, add 1mL of PBS buffer, and let it drain naturally.

[0117] c) Cover the sample outlet with a cap, add 1 mL of the treated immunosorbent to the column tube, and let it stand for five minutes to allow the matrix to settle naturally.

[0118] d) Add the upper sieve plate and press it over the substrate, ensuring there is no residual air underneath.

[0119] e) Open the cap of the sample outlet and slowly add 5 mL of PBS buffer to the inlet to equilibrate the column, allowing the liquid to flow out slowly.

[0120] f) Plug the outlet with a cap, store it with PBS buffer, put the inlet cap back on, and store the prepared aflatoxin B1 immunoaffinity column at 4°C.

[0121] Example 2: Verification of the reproducibility of aflatoxin B1 immunoaffinity column

[0122] The prepared aflatoxin B1 nanobody immunoaffinity column was taken out, and 200 ng of aflatoxin B1 solution was added. Non-specific adsorption was first washed with 5% methanol / PBS solution, followed by elution with 100% methanol solution. The aflatoxin B1 content in the eluted aflatoxin B1 solution was detected by high-performance liquid chromatography-mass spectrometry. The used immunoaffinity column was immediately equilibrated with 5 column volumes of PBS buffer to regenerate it. Another 200 ng of aflatoxin B1 solution was added, and the above elution steps were repeated. The recovery rate after regeneration was calculated. The regeneration process was repeated sequentially, and the immunoaffinity column was recycled. The recovery rate after every ten uses was calculated.

[0123] The results are as follows Figure 8 As shown, blank samples were spiked using an affinity column through regeneration and cycling. The eluent was then passed through high-performance liquid chromatography (HPLC), and the recovery rate was calculated after every ten uses. Figure 8 It can be seen that the recovery rate slowly decreases with the increase of the number of uses. When the number of consecutive uses reaches 150, the recovery rate can still reach more than 84.43%, which indicates that this affinity column not only has good affinity, but also has super renewability.

[0124] Example 3: Purification and detection of aflatoxin B1 in maize samples using a regenerable aflatoxin B1 immunoaffinity column

[0125] In this embodiment, a quantitative amount of aflatoxin B1 standard was added to a normal corn sample (a blank corn sample without aflatoxin B1), and then purified using an immunoaffinity column. The purified sample was then detected by high performance liquid chromatography-mass spectrometry to determine the recovery rate.

[0126] 1. Sample extraction

[0127] First, grind the corn sample into powder, weigh 10g of corn sample into a 50mL centrifuge tube, add 20mL of 70% methanol aqueous solution, shake vigorously for 30min, filter the solution with double-layer filter paper, dilute it 5 times with PBS, mix well and pass it through a membrane for later use.

[0128] 2. Immune affinity column purification

[0129] A regenerable aflatoxin B1 immunoaffinity column was used. The column was first equilibrated with 15 mL of PBS buffer. 10 mL of sample dilution was then slowly loaded onto the column. The column was then washed with 15 mL of 5% methanol / PBS solution to remove non-specifically adsorbed impurities. Finally, elution was performed with 100% methanol. The aflatoxin B1 content in the eluent was determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0130] 3. High Performance Liquid Chromatography-Mass Spectrometry Conditions

[0131] a) Mobile phase: A, 5 mmol / L ammonium acetate solution; B, 50% methanol / acetonitrile solution

[0132] b) Gradient elution: 0-0.5 min, 32% B; 3-4 min, 45% B; 4.2-4.8 min, 100% B; 5-7 min, 32% B;

[0133] c) Chromatographic column: C 18 Column (100mm length, 2.1mm inner diameter, 1.7μm packing diameter)

[0134] d) Flow rate: 300 μL / min

[0135] e) Column temperature: 40℃

[0136] f) Injection volume: 10 μL

[0137] g) Detection method: Multiple ion reaction detection (MRM)

[0138] 4. Quantitative

[0139] Different concentrations of aflatoxin B1 standard working solutions (0.1, 0.5, 1.0, 2.0, 5.0, 8.0, and 10 ng / mL) were aspirated into the syringe and injected into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. The peak areas of the standard solutions were measured under the above conditions, and standard curves for each toxin were plotted. The content of each toxin was then calculated using the external standard method. The results are shown in Table 1. The recoveries of three concentrations of aflatoxin B1 purified using this immunoaffinity column for intra-day and inter-day spiking ranged from 94.97% to 102.19%, with coefficients of variation all less than 10%.

[0140] Table 1. Recovery rate of aflatoxin B1 in corn samples.

[0141]

[0142]

[0143] The above embodiments are preferred embodiments of the present invention. Any modifications or substitutions to the details and form of the present invention without departing from the spirit and scope of the present invention are also within the protection scope of the present invention.

Claims

1. A renewable immunosorbent for purifying aflatoxin B1, characterized in that: The immunoadsorbent comprises a solid-phase carrier and an aflatoxin B1 nanobody coupled to the solid-phase carrier. The aflatoxin B1 nanobody is an aflatoxin B1 nanobody with an amino acid sequence as shown in SEQ ID NO:1, or a self-assembled multivalent aflatoxin B1 fusion nanobody NbAF-EGFP-H6 as shown in SEQ ID NO:

3.

2. The regenerable immunosorbent for purifying aflatoxin B1 according to claim 1, characterized in that: The solid support is agarose gel.

3. An affinity column loaded with the regenerable immunosorbent for purifying aflatoxin B1 as described in claim 1.

4. The method for preparing the affinity column according to claim 3, characterized in that: Includes the following steps: a) Matrix treatment First, the agarose gel matrix activated with hydrogen bromide is swollen in hydrochloric acid solution, and then washed with hydrochloric acid to remove impurities; b) Ligand coupling After rinsing the above matrix with conjugation buffer, the aflatoxin B1 nanobody solution was rapidly transferred into the rinsed matrix for conjugation. c) Ligand blocking To seal excess active groups on the gel surface; d) Remove uncoupled redundant ligands; e) Pack the column.

5. The method for detecting aflatoxin B1 content based on the affinity column of claim 3, characterized in that: When the sample to be tested is passed through the affinity column described in claim 3, the immunoadsorbent will specifically adsorb aflatoxin B1, while other impurities will not be adsorbed by the immunoaffinity column and will elute. Then, chromatographic grade methanol is used for elution, and the eluent is collected as the purified and concentrated sample. The content of aflatoxin B1 in the eluent is detected by high performance liquid chromatography-mass spectrometry.

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