Zearalenone single-chain antibody and application thereof in preparation of immunoadsorbent and affinity column

Abstract

The invention discloses a zearalenone single-chain antibody and application of the zearalenone single-chain antibody in preparation of an immunoadsorbent and an affinity column. The invention firstly provides a zearalenone single-chain antibody with good specificity, high sensitivity and high affinity, and then the zearalenone immunoaffinity column with high affinity is formed after a carrier is coupled with the single-chain antibody and the single-chain antibody is packed into a column. The zearalenone immunoadsorbent can be used for high performance liquid chromatography-mass spectrometry detection of zearalenone, the affinity and sensitivity of the immunoadsorbent are high, and the affinity column is stable in performance, strong in tolerance, high in specificity and good in renewability. The sample to be detected can be purified after being simply treated by the immunoaffinity column, the operation steps are simple, and the specificity is strong. The method can be directly used for high performance liquid chromatography on-machine detection without subsequent purification treatment, more time and cost are saved for operators, and the method has the potential of large-scale production and application. The possibility is provided for establishing a sensitive, stable, environment-friendly and economical detection method.

Description

Technical Field

[0001] The present invention relates to a zearalenone single-chain antibody and application thereof in the preparation of an immunosorbent and an affinity column, belonging to the technical field of food safety detection. Background Art

[0002] Zearalenone (ZEN) is a meta-phenylene lactone with the molecular formula C 18 H 22 O5. ZEN, also known as F-2 toxin, is a toxic metabolite synthesized by Fusarium species such as Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum, and Fusarium nivalis through the polyketide pathway. ZEN appears as white crystals with low polarity and is virtually insoluble in water and carbon tetrachloride solutions. It has a high melting point (161°C-163°C) and is highly thermally stable. ZEN content remains virtually unchanged when food is heated at boiling or retorting temperatures. Because ZEN shares a chemical structure with natural estrogens, it exhibits estrogen-like effects in mammals and can interfere with the normal development of animal or human germ cells and embryos. In addition to reproductive organs, the liver is a major target organ for ZEN toxicity. Studies have shown that ZEN-treated rats exhibit significantly reduced glutathione peroxidase and superoxide dismutase activities in their serum and liver, significantly impairing their ability to resist liver damage and carcinogenesis. ZEN also exhibits immunotoxicity, genotoxicity, and carcinogenicity, posing a serious threat to human health. At present, the main ZEN detection methods include thin layer chromatography, immunoaffinity chromatography-high performance liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, and immunochromatography.

[0003] A complete antibody is composed of two heavy chains and two light chains. The heavy and light chain variable regions are linked to form a recombinant gene using a synthetic linker peptide gene. The antibody expressed from this recombinant gene is called a single-chain antibody (scFv). scFvs possess antigen-binding properties, a small molecular weight (20-30 kd), ease of expression in prokaryotic cell systems, and ease of genetic engineering. Therefore, the use of single-chain antibodies as the core component of the immunoadsorbent in the ZEN immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry assay offers significant advantages.

[0004] Immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis combines immune responses with chromatography-mass spectrometry. Leveraging the high specificity and affinity of antigen-antibody binding, specific antibodies are chemically coupled to immunosorbents. This allows for efficient enrichment and purification of ZEN from complex matrices based on immunologically reversible binding. Therefore, the development of an efficient, stable, and reproducible ZEN immunoaffinity column is crucial for developing efficient, stable, and accurate HPLC-MS detection methods for ZEN.

[0005] Chinese patent CN116064562A, "Methods for preparing and using eukaryotically expressed single-chain antibodies and full-length antibodies against zearalenone," discloses a single-chain antibody against zearalenone. However, these antibodies exhibit poor stability, with significant reductions in their recognition of ZEN-BSA after storage at 4°C for five weeks. Furthermore, the full-length antibody exhibits high cross-reactivity rates for α-zearalenol and β-zearalenol, at 17.54% and 20.05%, respectively, while the single-chain antibody exhibits a high cross-reactivity rate for α-zearalenol, at 27.71%. Therefore, there is a need for a single-chain antibody against zearalenone with improved stability and specificity for use in immunoadsorption. Summary of the Invention

[0006] In order to solve the problems in the prior art, the present invention provides a single-chain antibody to zearalenone, and uses the single-chain antibody to prepare an immunosorbent and affinity column that can regenerate and purify zearalenone.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] The first object of the present invention is to provide a single-chain antibody against zearalenone, which is composed of a heavy chain variable region and a light chain variable region connected by a linker, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0009] In one embodiment of the present invention, the linker is (Gly4Ser)3.

[0010] In one embodiment of the present invention, the amino acid sequence of the zearalenone single-chain antibody is shown as SEQ ID NO.1.

[0011] In one embodiment of the present invention, the nucleotide sequence of the zearalenone single-chain antibody is shown as SEQ ID NO.2.

[0012] In the present invention, the zearalenone single-chain antibody can be prepared by genetic engineering methods, which has the advantages of short preparation cycle and low cost. The specific steps are as follows:

[0013] a) obtaining a bacterial solution containing the zearalenone single-chain antibody gene and streaking and culturing it on a plate;

[0014] b) Pick a single colony from the plate and shake it in small amounts;

[0015] c) Inoculate the bacterial solution into a shake flask and shake the bacteria in large quantities;

[0016] d) Isopropylthiogalactoside (IPTG) induced high-efficiency expression of single-chain antibody at room temperature;

[0017] e) Bacterial lysis, supernatant was filtered through a 0.22 μm filter membrane and then purified using a nickel column;

[0018] f) Dialysis, centrifugation and ultrafiltration, concentration, and 50% glycerol storage at -20°C for later use.

[0019] In the present invention, the zearalenone single-chain antibody can be expressed in E. coli. From the perspective of production time and cost, prokaryotic expression of the single-chain antibody in the present invention is faster and less expensive than eukaryotic expression. Compared to Expi293F cells, antibody production in E. coli is also simpler, less susceptible to contamination, and has a higher success rate.

[0020] The second object of the present invention is to provide an immunoadsorbent prepared using the zearalenone single-chain antibody, wherein the immunoadsorbent comprises a solid phase carrier and the zearalenone single-chain antibody coupled to the solid phase carrier.

[0021] The zearalenone single-chain antibody in the immunosorbent of the present invention has high sensitivity and specificity, can be used for the specific adsorption of zearalenone, and can be used as a regenerable immunoaffinity column for purifying zearalenone after being loaded into a column.

[0022] In one embodiment of the present invention, the solid phase carrier is agarose gel.

[0023] The third object of the present invention is to provide a regenerable affinity column for purifying zearalenone, wherein the affinity column is loaded with the zearalenone single-chain antibody or the immunosorbent.

[0024] A fourth object of the present invention is to provide a method for preparing the regenerable zearalenone purification affinity column, comprising the following steps:

[0025] a) Matrix treatment: The agarose gel microspheres activated with cyanogen bromide were swollen in a hydrochloric acid solution and then repeatedly washed with hydrochloric acid solution to remove impurities;

[0026] b) Ligand coupling: After washing the matrix with coupling buffer by filtration, the zearalenone single-chain antibody solution is quickly transferred to the washed matrix for coupling reaction;

[0027] c) Ligand blocking: blocking excess active groups on the surface of agarose gel;

[0028] d) removing uncoupled excess ligand;

[0029] e) Pack the column.

[0030] In one embodiment of the present invention, in step (a), the concentration of hydrochloric acid is 0.5-1.5 mM, the swelling time is 20-40 min, and washing is performed 2-4 times.

[0031] In one embodiment of the present invention, the coupling buffer in step (b) is 0.08-0.12 M NaHCO 3 containing 0.4-0.6 M NaCl, pH=8.0-8.5.

[0032] In one embodiment of the present invention, the coupling conditions in step (b) are: mixing and reacting at 20-30° C. for 2-4 hours.

[0033] In one embodiment of the present invention, the blocking process in step (c) is to first wash with 3 to 5 volumes of coupling buffer and deionized water respectively, and then block with ethanolamine solution for 1 to 3 hours.

[0034] In one embodiment of the present invention, step (d) is: sequentially rinsing the substrate treated in step (c) with buffer A (0.1 M CH3COONa buffer containing 0.5 M NaCl, pH = 4) and buffer B (0.1 M Tris-HCl buffer containing 0.5 M NaCl, pH = 8), and washing at least 4 times.

[0035] The fifth object of the present invention is to provide a use of the zearalenone single-chain antibody, the immunosorbent or the affinity column in detecting the zearalenone content.

[0036] In the present invention, when the sample to be tested passes through the immunoaffinity column, the immunoadsorbent will specifically adsorb zearalenone, and other impurities are not adsorbed by the immunoaffinity column and flow out. The sample is then eluted with chromatographic grade methanol, and the eluate, i.e., the purified and concentrated sample, is collected. The eluate is then used to detect the content of zearalenone by high performance liquid chromatography-mass spectrometry.

[0037] The present invention first provides a single-chain antibody to zearalenone with good specificity, high sensitivity, and high affinity. Then, a high-affinity zearalenone immunoaffinity column is formed by coupling the single-chain antibody with a carrier and loading it onto a column. The immunoaffinity column can be used for high-performance liquid chromatography-mass spectrometry detection of zearalenone. The immunoadsorbent has high affinity and sensitivity, and the affinity column has stable performance, strong tolerance, high specificity, and good reproducibility. The sample to be tested can be purified after simple treatment on the immunoaffinity column, with simple operation steps and high specificity. Subsequent purification treatment is unnecessary and the sample can be directly used for high-performance liquid chromatography detection, further saving operator time and expenses and having the potential for large-scale production and application. This provides the possibility of establishing a sensitive, stable, environmentally friendly, and economical detection method.

[0038] Beneficial effects of the present invention:

[0039] The affinity of the zearalenone single-chain antibody of the present invention is 5.43×10 -3 , sensitivity IC 50 The single-chain antibody of the present invention has a cross-reactivity rate of less than 10% for both α-zearalenol and β-zearalenol, and has high specificity for zearalenone. The single-chain antibody of the present invention has almost no change in its recognition effect on ZEN-BSA after storage at 4°C for 3 months, and has good stability. In summary, the single-chain antibody of zearalenone prepared in the present invention is more suitable for preparing immunosorbents and affinity columns with excellent performance, stability, and lower cost.

[0040] In addition, the immunoaffinity column prepared by the present invention has good regeneration properties and can be used for the purification of zearalenone. It can be reused for more than 50 times, and the recovery rate can still reach more than 80% when the number of consecutive uses reaches 40 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is a map of the constructed plasmid containing the sequence of the single-chain antibody against zearalenone;

[0043] Figure 2 This is a diagram showing the expression and purification results of the recombinant protein ZEN-scFv. Lane M: protein marker; Lane 1: flow-through; Lane 2: wash solution; Lane 3: 250 mM imidazole eluate; Lane 4: 300 mM imidazole eluate; Lane 5: 400 mM imidazole eluate.

[0044] Figure 3 This is the affinity identification curve of the zearalenone single-chain antibody;

[0045] Figure 4 This is the sensitivity standard curve of the zearalenone single-chain antibody ELISA method;

[0046] Figure 5 This is a schematic diagram of the structure of the zearalenone immunoaffinity column;

[0047] Figure 6 This is a schematic diagram of the reproducibility verification of the zearalenone immunoaffinity column;

[0048] Figure 7This is a standard curve diagram of zearalenone detected by high performance liquid chromatography-mass spectrometry;

[0049] Figure 8 It is the peak shape diagram of the actual sample detected by high performance liquid chromatography-mass spectrometry. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.

[0051] Example 1: Obtaining Zearalenone Single-chain Antibody

[0052] (1) Obtaining the single-chain antibody ZEN-scFv against zearalenone

[0053] The antibody format of the present invention is a single-chain antibody, composed of a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3 (MAQVKLQESGGGLVQPGGSLKLSCAASGFTFYNYGMSWV RQTPDKRLEFVANIRGNGGKTYYPGSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRVAFDGYYDDFWGQGTTVTVSS), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4 (DIEL TQSPATLSVTPGDRVSLSCRASQYISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSG SDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK). The heavy chain variable region and the light chain variable region are connected by a linker ((Gly4Ser)3); to facilitate purification, a six-histidine tag (His×6) was designed at the C-terminus of the single-chain antibody. The target gene was inserted into the universal vector pET-22b. The single-chain antibody gene was transformed into an expression strain, and after induced expression, purification, and identification, a single-chain antibody ZEN-scFv with excellent protein performance was obtained. The amino acid sequence of the zearalenone single-chain antibody is shown in SEQ ID NO: 1 (MAQVKLQESGGGLVQPGGSLKLSCAASGFTFYNYGMSWVRQTPDK RLEFVANIRGNGGKTYYPGSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRVAFDGYYDDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPATLSVTPGDRVSLSCRASQYISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK).The nucleotide sequence is as shown in SEQ ID NO:2 (ATGGCCCAAGTGAAGCTCCAA GAGAGCGGCGGGGGCCTCGTGCAGCCCGGCGGGTCCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCTACAACTACGGCATGAGCTGGGTGAGACAGACCCCCGACAAGAGACTGGAGTTCGTGGCCAACATCCGCGGCAACGGCGGCAAGACCTACTACCCCGGCAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCGCCATGTACTACTGCGTGAGAGTGGCCTTCGACGGCTACTACGACGACTTCTGGGGCCAAGGCACCACCGTGACCGTGAGCAGCGGAGGAGGAGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGAAGCGACATCGAGCTGACACAGAGCCCCGCCACCCTGAGCGTGACCCCCGGCGACAGAGTGAGCCTGAGCTGCAGAGCTAGCCAAtacATCAGCGACTACCTGCACTGGTATCAGCAGAAGAGCCACGAGAGCCCTAGACTGCTGATCAAGTACGCTAGCCAAAGCATCAGCGGCATCCCTAGCAGATTCAGCGGCAGCGGCAGCGGCAGCGACTTCACCCTGAGCATCAACAGCGTGGAGCCCGAGGACGTGGGCGTGTACTACTGTCAGAACGGCCACAGCTTCCCCCCCACCTTCGGCGGGGGCACCAAGCTGGAGATCAAG).,

[0054] The expression and purification steps of the above zearalenone single-chain antibody are specifically as follows:

[0055] Inoculate TOP10 glycerol stock containing the zearalenone single-chain antibody gene onto an ampicillin-resistant plate and incubate inverted at 37°C overnight. Select a single colony from the plate and inoculate it into 10 mL of ampicillin-resistant LB medium. Incubate at 37°C with shaking at 220 rpm for 8-12 hours. After 12 hours, centrifuge the culture at 12,000 rpm for 10 minutes, remove the supernatant, and resuspend the culture in 1 mL of ultrapure water to extract the plasmid. Add 1 μg of the purified plasmid to 100 μL of thawed Escherichia coli BL21 competent cells and incubate on ice for 30 minutes. After 30 minutes, remove the centrifuge tube and place it in a 42°C water bath for 90 seconds. Immediately place it on ice for 2 minutes. Add 700 μL of LB medium without ampicillin to the heat-shocked tube, gently pipette to mix, and incubate at 37°C with shaking at 150 rpm for 1 hour. After the incubation, pipette to mix the system, take out 200 μL and spread it on LB / Amp plate, and culture at 37℃ overnight. Inoculate a single colony that has been successfully transformed into LB / Amp medium and culture at 37℃, 220r / min for 12h. Take out 1mL of culture and add it to LB / Amp medium and continue to culture until the bacterial solution OD is 0. 600 When the pH value is about 0.6, 0.2 mM IPTG is added to the system and induced at 30 ° C, 220 rpm for 10 hours. After the induction is completed, the culture is centrifuged at 12000 rpm for 15 minutes, and the precipitated bacteria are redissolved in PBS solution (0.01M, pH 7.4). Ultrasonic disruption is performed, and the product is centrifuged and the supernatant is collected for subsequent nickel column purification. The purified zearalenone single-chain antibody is dialyzed with PBS buffer at pH 7.4 at 4 ° C for 3 days, and the dialyzate is replaced every 4 hours. It is then filtered and concentrated using an ultrafiltration tube. The concentrated protein is added with an equal volume of glycerol and stored in a -20 ° C refrigerator.

[0056] like Figure 2 As shown, the recombinant protein ZEN-scFv was successfully purified and eluted by imidazole. The actual molecular weight of the recombinant protein was consistent with the theoretical size of 33 kDa. After purification and concentration, the concentration of ZEN-scFv was 1 mg / mL.

[0057] (2) Identification of affinity of single-chain antibody against zearalenone

[0058] The affinity of ZEN-scFv was determined by indirect non-competitive ELISA. ZEN-BSA was coated on the microplate at concentrations of 1.0, 2.0, and 4.0 μg / mL, 100 μL / well, and incubated at 37°C for 2 h. After blocking with 3% skim milk powder / PBS for 2 h, the microplate was washed with PBS.

[0059] The gradient diluted ZEN-scFv was added to the corresponding ELISA plate, incubated at 37°C for 1 hour, washed 3 times, and HRP-labeled anti-His tag mouse monoclonal antibody diluted 1:2000 with PBST was added, and incubated at 37°C for 0.5 hours; after washing the ELISA plate 3 times, the substrate colorimetric solution was added, and the color reaction was carried out at 37°C for 15 minutes, and concentrated sulfuric acid was added to terminate the reaction. The OD 450 The logarithm of the ZEN-scFv concentration was plotted on the ordinate and the logarithm of the ZEN-scFv concentration on the abscissa. Three sigmoidal curves were drawn for each of the three concentrations. The affinity constant of the antibody was calculated using the formula Ka = (n-1) / 2(n[Ab']t - [Ab]t), where [Ag] represents the antigen concentration and n represents [Ag] / [Ag'], the ratio of the different antigen concentrations. [Ab']t and [Ab]t are the antibody concentrations corresponding to half the maximum absorption value in the affinity curves of the two antibodies in each group. The average Ka value was calculated to obtain the affinity constant of the ZEN-scFv.

[0060] The "S" curve was fitted using Origin 2024 software. Figure 3 As shown in Figure 2, the antibody concentrations corresponding to the 50% ODmax values ​​of each curve at the three coating concentrations were calculated to be 236.9 ng / mL, 319.7 ng / mL, and 583.5 ng / mL, respectively. The average of the three Ka values ​​was calculated according to the formula Ka = (n-1) / 2(n[Ab']t-[Ab]t), and the final affinity of ZEN-scFv was 5.43×10 -3 .

[0061] (3) ZEN-scFv sensitivity identification

[0062] A 96-well ELISA plate was coated with 0.5 μg / mL ZEN-BSA overnight at 4°C. The plate was washed three times and blocked with 3% skim milk powder / PBS for 2 h. The plate was washed three times and the purified ZEN-scFv was diluted with PBS to different concentrations, added to the ELISA plate, and incubated at 37°C for 1 h. The remaining steps were the same as the indirect non-competitive ELISA method. The OD values ​​were measured. 450 Value.OD 450 The ZEN-scFv dilution factor when the value is around 1.0 is used as the antibody working concentration.

[0063] Coat a 96-well ELISA plate with 0.5 μg / mL ZEN-BSA overnight at 4°C; wash the plate three times and block with 3% skim milk powder / PBS for 2 hours; wash the plate three times, dilute ZEN in 10% methanol / PBS, add 50 μL to the plate, add 50 μL of ZEN-scFv diluted to the working concentration, and react at 37°C for 1 hour; wash the plate three times with PBST, add 100 μL of HRP-labeled anti-His tag mouse monoclonal antibody to each well, and continue incubation at 37°C for 0.5 hours; wash the plate three times, add substrate colorimetric solution, develop the color at 37°C for 15 minutes, and then add 50 μL of concentrated sulfuric acid to terminate the reaction. Determine the OD value. 450 The ELISA standard curve was drawn.

[0064] The standard curve was drawn with ZEN concentration as the horizontal axis and B / B0 as the vertical axis. The four-parameter fitting function in the origin2024 drawing software was used to fit the data and draw an "S" curve. The results are shown in the figure. Figure 4 As shown, the IC of the standard curve 50 The value is 34.6ng / mL.

[0065] ZEN-scFv specificity identification

[0066] The specificity of an antibody can be evaluated using the cross-reactivity rate. An indirect competitive ELISA method was used to determine the specificity of zearalenone, α-zearalanol, β-zearalanol, α-zearalenol, β-zearalenol, zearalanol, deoxynivalenol, ochratoxin A, and aflatoxin B1. These solutions were prepared in a series of concentrations, along with an equal volume of a zearalenone single-chain antibody (SCAb), and added to an ELISA plate. The plate was incubated for 1 hour, with all other steps being the same as the indirect competitive ELISA method. The sensitivity of the SCAb corresponding to a 50% inhibition rate for each toxin was calculated and compared with the sensitivity of ZEN. The cross-reactivity rate was calculated. The results are shown in Table 1. The results demonstrate the high specificity of the expressed zearalenone SCAb.

[0067] Table 1 Specificity of single-chain antibody against zearalenone

[0068]

[0069] Stability characterization of ZEN-scFv

[0070] The optimal concentration of a single-chain antibody against zearalenone, determined by ELISA, was stored at 4°C for three months and then used in an ELISA. The antibody's recognition of the ZEN-BSA antigen remained unchanged after three months. The OD450 values ​​measured by ELISA were 1.20±0.12 for the freshly prepared single-chain antibody and 1.05±0.08 for the single-chain antibody stored at 4°C for three months, respectively.

[0071] The zearalenone single-chain antibody can be used to prepare a regenerable zearalenone purification immunoadsorbent and further be prepared into an immunoaffinity column.

[0072] Example 2: Preparation of Zearalenone Immunoadsorbent and Immunoaffinity Column

[0073] 1. Preparation of Matrix

[0074] 1g CNBr activated agarose gel (Sepharose 4B) freeze-dried matrix powder (per gram of freeze-dried matrix powder can be

[0075] Dissolve the matrix (to a final volume of 3.5 mL) in 30 mL of 1 mM hydrochloric acid, mix thoroughly, and soak at 4°C for 30 min. The matrix will swell immediately. Mix thoroughly, filter, and wash three times with 20 mL of 1 mM hydrochloric acid.

[0076] 2. Conjugation of Zearalenone Single-chain Antibody

[0077] a) Wash the matrix with a coupling buffer containing 0.5M NaCl in 0.1M NaHCO₃, pH 8.3. Dissolve the zearalenone single-chain antibody to be coupled in the coupling buffer to a concentration of 1 mg. Store the dissolved antibody in an ice bath. Add the coupling buffer containing the zearalenone single-chain antibody to a sealed container and quickly transfer the CNBr-activated agarose gel to the antibody solution. Incubate the reaction at 25°C for 3 h.

[0078] b) Calculation of coupling efficiency: Centrifuge at 3000 rpm to remove the agarose gel to the bottom of the tube. Transfer the supernatant to a new centrifuge tube and measure the protein content of the supernatant. The coupling efficiency was calculated to be 95.1%.

[0079] c) Blocking: The agarose gel that has been centrifuged to the bottom of the tube was washed with 4 volumes of coupling buffer and deionized water, respectively. The gel was then blocked with ethanolamine solution for 2 hours to block excess active groups on the gel surface.

[0080] d) To remove excess uncoupled ligand after coupling, wash the substrate at least four times, sequentially with 0.1 M CH3COONa buffer containing 0.5 M NaCl, pH 4, and then with 0.1 M Tris-HCl buffer containing 0.5 M NaCl, pH 8. Each buffer should be used in an amount equal to three times the volume of the substrate. Each wash cycle begins with a wash in 0.1 M CH3COONa buffer containing 0.5 M NaCl, followed by a wash in 0.1 M Tris-HCl buffer containing 0.5 M NaCl. Finally, equilibrate with three volumes of 0.01 M PBS and store in 20% ethanol at 4°C.

[0081] 3. Column Packing

[0082] The structure of the zearalenone immunoaffinity column is as follows Figure 5 As shown, the column packing steps are as follows:

[0083] a) Add 3 mL of PBS buffer to the conjugated immunosorbent and mix.

[0084] b) Take a 3 mL stoppered column tube, add the lower frit, add 1 mL of PBS buffer, and let it settle naturally.

[0085] c) Cover the sample outlet with a cap, add 1 mL of treated immunosorbent into the column tube, and let it stand for five minutes to allow the matrix to settle naturally.

[0086] d) Open the cover of the sample outlet and slowly add 3 mL of PBS buffer to the sample inlet to equilibrate the column, allowing the liquid to slowly flow out.

[0087] e) Cover the sample outlet with a cap, store in 20% ethanol, cover the sample inlet cap, and store the prepared zearalenone immunoaffinity column at 4°C.

[0088] Example 3: Verification of the reproducibility of the zearalenone immunoaffinity column

[0089] The prepared zearalenone single-chain antibody immunoaffinity column was first equilibrated with 5 column volumes of PBS solution, followed by the addition of 400 ng of zearalenone dissolved in 10% methanol / PBS solution. Non-specific adsorption was then washed out with PBS solution, and finally eluted with 100% methanol solution. The eluted zearalenone solution was then analyzed for zearalenone content by high-performance liquid chromatography-mass spectrometry. The used immunoaffinity column was immediately equilibrated with 5 column volumes of PBS buffer to regenerate the immunoaffinity column. 400 ng of zearalenone solution was then added, and the elution step was repeated. The recovery rate of the regenerated affinity column was calculated. The regeneration process was repeated, and the immunoaffinity column was reused in a cycle. The recovery rate of the immunoaffinity column after every 5 uses was calculated.

[0090] The results are as follows Figure 6 As shown, a blank sample was spiked using the affinity column through a regeneration cycle and the eluate was passed through a high

[0091] HPLC-MS, calculate the recovery rate after every 5 uses, from Figure 6 It can be seen that the recovery rate shows a slowly decreasing trend with the increase in the number of uses. When the number of continuous uses reaches 40 times, the recovery rate can still reach more than 80%, indicating that this affinity column not only has good affinity, but also has strong reproducibility.

[0092] Example 4: Purification and detection of zearalenone in corn samples using a renewable zearalenone immunoaffinity column

[0093] In this example, a quantitative amount of zearalenone standard was added to a normal corn sample (a blank corn sample not containing zearalenone), and then purified using an immunoaffinity column. The purified test sample was detected by high performance liquid chromatography-mass spectrometry to determine the recovery rate.

[0094] 1. Sample Extraction

[0095] First, grind the corn sample into powder, weigh 5g of corn sample into a 50mL centrifuge tube, add 20mL of 70% methanol aqueous solution, and ultrasonically extract for 30min. The extract is centrifuged at 12000rpm for 15min. The supernatant is filtered with double-layer filter paper, diluted 5 times with PBS, mixed and filtered for later use.

[0096] 2. Immunoaffinity Column Cleanup

[0097] A regenerable zearalenone immunoaffinity column was first equilibrated with 5 mL of PBS buffer. 10 mL of the sample dilution was then slowly loaded onto the column. The column was then washed with 5 mL of PBS to remove nonspecifically adsorbed impurities. Finally, the column was eluted with 100% methanol, and the eluate was analyzed for zearalenone content by HPLC-MS.

[0098] 3. HPLC-MS Conditions

[0099] a) Mobile phase: A: acetonitrile solution, B: ultrapure water

[0100] b) Gradient elution: 0 min, 25% A, 75% B; 5 min, 70% A, 30% B; 6 min, 70% A, 30% B; 9 min, 25% A, 75% B;

[0101] c) Chromatographic column: C 18 column

[0102] d) Flow rate: 0.2 mL / min

[0103] e) Column temperature: 40℃

[0104] f) Injection volume: 2 μL

[0105] g) Detection method: Multiple reaction monitoring (MRM)

[0106] 4. Quantification

[0107] The injector draws different concentrations of zearalenone (10, 20, 50, 100, 200, 500, 1000 ng / mL) standard working solution and injects it into the high performance liquid chromatography-mass spectrometer. Under the above conditions, the peak area of ​​the standard solution is measured and the standard curve is drawn. The standard curve is as follows: Figure 7 Then calculate the content of zearalenone. The result is as shown in Figure 8 As shown in Table 2, the recoveries of three concentrations of zearalenone purified by the immunoaffinity column ranged from 95.60% to 102.23%, and the coefficients of variation were all less than 10%.

[0108] Table 2 Recovery results of spiked zearalenone in corn samples.

[0109]

[0110] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A single-chain antibody against zearalenone, characterized in that The zearalenone single-chain antibody is composed of a heavy chain variable region and a light chain variable region connected by a linker. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. The zearalenone single-chain antibody according to claim 1, characterized in that The linker is (Gly4Ser)3.

3. The zearalenone single-chain antibody according to claim 1 or 2, characterized in that The amino acid sequence of the zearalenone single-chain antibody is shown in SEQ ID NO.

1.

4. An immunosorbent prepared using the single-chain antibody against zearalenone according to any one of claims 1 to 3, characterized in that: The immunosorbent comprises a solid phase carrier and a zearalenone single-chain antibody coupled to the solid phase carrier.

5. The immunoadsorbent according to claim 4, characterized in that The solid phase carrier is agarose gel.

6. A regenerable affinity column for purifying zearalenone, characterized in that: The affinity column is loaded with the zearalenone single-chain antibody according to any one of claims 1 to 3 or the immunoadsorbent according to any one of claims 4 to 5.

7. A method for preparing a regenerable affinity column for purifying zearalenone according to claim 6, characterized in that: The steps include: a) Matrix treatment: The agarose gel microspheres activated with cyanogen bromide were swollen in a hydrochloric acid solution and then repeatedly washed with hydrochloric acid solution to remove impurities; b) Ligand coupling: After washing the matrix with coupling buffer by filtration, the zearalenone single-chain antibody solution is quickly transferred to the washed matrix for coupling reaction; c) Ligand blocking: blocking excess active groups on the surface of agarose gel; d) removing uncoupled excess ligand; e) Pack the column.

8. The preparation method according to claim 7, characterized in that The coupling buffer in step (b) is 0.08-0.12 M NaHCO 3 containing 0.4-0.6 M NaCl, pH=8.0-8.

5.

9. The preparation method according to claim 7, characterized in that The blocking process in step (c) is to first wash with 3 to 5 volumes of coupling buffer and deionized water respectively, and then block with ethanolamine solution for 1 to 3 hours.

10. Use of the single-chain antibody against zearalenone according to any one of claims 1 to 3, the immunosorbent according to any one of claims 4 to 5, or the affinity column according to claim 6 in detecting the content of zearalenone.

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