Preparation and application of anti-isotenuazonic acid nano antibody immunoadsorbent and immunoaffinity column

Abstract

The invention discloses preparation and application of an anti-isotenuazonic acid nano antibody immunoadsorbent and an immunoaffinity column. The immunoadsorbent provided by the invention comprises a solid-phase carrier containing a protein cross-linking agent, and an anti-isotenuazonic acid nano antibody which is directionally coupled with the solid-phase carrier and is provided with a cysteine label at the C terminal. The nano antibody and the cysteine tag are subjected to fusion expression, directional coupling with a solid phase carrier is realized, the problem of antigen binding site occupation in traditional coupling is solved, the adsorption capacity of the immunoaffinity column is improved, the column content of the prepared immunoaffinity column reaches 905.06 ng, and the column adsorption capacity of the immunoaffinity column is improved by 202.40 ng compared with that of a non-directional coupling immunoaffinity column. And the repeated use frequency is also increased. Meanwhile, the method for detecting the isotenuazonic acid through combination with mass spectrometry has good accuracy and precision, the detection limit and the quantitation limit are lower, the sample pretreatment process is simplified, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and more specifically, relates to the preparation and application of an anti-alternaria acid nano-antibody immunoadsorbent and an immunoaffinity column. Background Art

[0002] Alternaria acid is a secondary metabolite produced by fungi such as Alternaria alternata. It has a wide range of pollution and potential health hazards. It is detected at a high rate in grains, fruits, vegetables and their products, posing certain risks to food safety. Affected by its structural characteristics, Alternaria acid is difficult to separate during sample pretreatment and chromatographic separation. The currently applicable chromatographic methods cannot achieve a good separation effect, and the mainstream pretreatment methods also lack specificity and are prone to co-extraction of impurities. They are only suitable for samples with simple matrices and require quantification with expensive internal standards.

[0003] The immunoaffinity column is a pretreatment method that utilizes the specific reversible binding reaction of antigen and antibody to enrich the target. It can accurately extract the target from a complex matrix and has significant advantages over other methods. The high selectivity of the immunoaffinity column not only improves the purity of the eluted product, but also simplifies the pretreatment process, making it widely used in the fields of food, medicine, environment, etc., especially suitable for the enrichment of fungal toxins in samples. The immunoaffinity column is obtained by filling an immunoadsorbent with antibodies as the core element. The quality of the antibodies in the immunoadsorbent will directly affect the use effect of the immunoadsorbent. Traditional preparation of immunoadsorbents mostly uses polyclonal antibodies or monoclonal antibodies to couple with solid phase carriers, but the acquisition of polyclonal antibodies or monoclonal antibodies requires a large number of animals and cumbersome purification steps, which increases the production cost. The prepared immunoadsorbents also have problems such as rapid activity degradation and fewer reusable times.

[0004] In recent years, nano-antibody immunoadsorbents have been developed for the purification of endotoxins, hormones, enzymes, antibodies and other objects, and some commercial affinity ligands have also begun to appear. Nano-antibodies are a new type of genetically engineered antibodies, which are obtained by cloning the variable region of heavy chain antibodies that naturally lack light chains in camelids. Due to their unique structure, nano-antibodies have better solubility, higher affinity and stronger stability. At the same time, nano-antibodies can be expressed and modified at a high level by means of genetic engineering, greatly reducing production costs; for example, existing studies have disclosed that anti-aflatoxin B1 and anti-ochratoxin A nano-antibody immunoaffinity columns show superior performance than monoclonal antibody immunoaffinity columns. At present, there has been no research on immunoaffinity columns for the purification of isothiocyanate, and after coupling antibodies to solid phase carriers using traditional methods, there is still a problem of antigen binding site occupancy, resulting in loss of antibody activity and affecting the adsorption efficiency of immunoaffinity columns. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the problem of antigen binding site occupancy in existing immunosorbent couplings and the shortcomings of anti-alternarianic acid immunosorbents, and to provide an anti-alternarianic acid nanoantibody immunosorbent and the preparation and application of an immunoaffinity column.

[0006] The first object of the present invention is to provide an anti-alternaria acid nano-antibody immunoadsorbent.

[0007] The second object of the present invention is to provide a method for preparing a nanobody immunoadsorbent.

[0008] The third object of the present invention is to provide the use of nano antibody immunoadsorbent.

[0009] The fourth object of the present invention is to provide an immunoaffinity column.

[0010] The fifth object of the present invention is to provide a method for pre-treating a sample of Alternaria japonica acid.

[0011] The sixth object of the present invention is to provide a method for detecting isoalternarianic acid.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The invention provides an anti-alternarianic acid nano antibody immunosorbent, which comprises: a solid phase carrier containing a protein cross-linking agent, and an anti-alternarianic acid nano antibody which is directionally coupled to the solid phase carrier and has a cysteine ​​tag at the C-terminus.

[0014] The present invention successfully constructs a prokaryotic expression vector pET-32a-B3G3-Cys by using genetic engineering technology, and transfers it to a host bacterium, obtains a high-purity recombinant protein B3G3-Cys by Ni-NTA affinity chromatography; the recombinant protein B3G3-Cys is directional coupled with an activated agarose gel containing a protein cross-linking agent (SMCC), and an anti-iso-alternaria acid nano-antibody immunosorbent is successfully prepared. The present invention fuses the nano-antibody with a cysteine ​​tag for expression, realizes directional coupling with a solid phase carrier, and the prepared anti-iso-alternaria acid nano-antibody immunosorbent avoids the problem of antigen binding site occupancy in traditional coupling, and improves the adsorption capacity. The column content of the prepared immunoaffinity column is further 905.06 ng, which is 202.40 ng higher than the column content of the non-directional coupled immunoaffinity column, and the number of times it is reused is also increased. At the same time, the immunosorbent or immunoaffinity column of the present invention is used for the pretreatment of the sample of isothiocyanate and combined with mass spectrometry. The constructed detection method has good accuracy and precision, the detection limit is 0.61 μg / kg, the quantitative limit is 2.51 μg / kg, the recovery rate is between 90.11% and 106.74%, the RSD is less than 10%, and it has good application value for food sample pretreatment.

[0015] Preferably, the nucleic acid sequence of the anti-alternaria acid nanobody with a cysteine ​​tag at the C-terminus is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. In particular, the present invention can also be prepared using other anti-alternaria acid antibodies with cysteine, and the same effect can be achieved.

[0016] Preferably, the solid phase carrier is agarose gel, or a solid phase carrier commonly used in existing immunosorbents.

[0017] Preferably, the protein cross-linking agent is a maleimide cross-linking agent; the existing maleimide cross-linking agents used in the art can be directed coupled with the cysteine ​​tag, react specifically with the sulfhydryl group under near-neutral conditions to form a stable thioether bond.

[0018] More preferably, the protein cross-linking agent is sulfo-SMCC.

[0019] The present invention provides a method for preparing the above-mentioned nano antibody immunoadsorbent, comprising the following steps:

[0020] (1) Construction of pET-32a-B3G3-Cys expression vector: Using pET-32a plasmid carrying anti-alternaria nicotinic acid nanoantibody B3G3 as a template, using primers containing cysteine ​​tags, PCR amplification, and then digestion, cleaning, recovery, recombination and cyclization, and then transformed into DH5α competent cells, and identified and obtained pET-32a-B3G3-Cys plasmid; the primer sequences containing cysteine ​​tags are shown in SEQ ID NOs: 3 to 4;

[0021] (2) Expression and purification of B3G3-Cys: After transformation with the pET-32a-B3G3-Cys plasmid, the expression was induced, the cells were collected by centrifugation, the periplasmic soluble protein solution was obtained by osmotic pressure method, purified by Ni-NTA affinity column, and the dimer was reduced by TCEP to identify B3G3-Cys;

[0022] (3) activating agarose gel to obtain amino agarose gel;

[0023] (4) Directed coupling: Take the activated agarose gel, add a protein cross-linking agent, synthesize a maleimide-based gel, wash away the reaction solution, add an equal volume of B3G3-Cys, react, and complete the coupling;

[0024] (5) Blocking the active sites of the gel: Wash away the reaction solution, add cysteine, react and block the sites, and successfully prepare the immunosorbent.

[0025] As the most preferred embodiment, the present invention provides a more specific method for preparing the immunosorbent:

[0026] (1) Construction of pET-32a-B3G3-Cys expression vector: Using pET-32a-B3G3 plasmid as template and Cys-F and Cys-R as primers (primer sequences are shown in SEQ ID NOs: 3-4), PCR amplification was performed, and the amplified product was verified by agarose gel electrophoresis. After digestion, cleaning, recovery, and recombination and cyclization, it was transformed into DH5α competent cells, and positive single clones were selected for sequencing to obtain pET-32a-B3G3-Cys plasmid;

[0027] (2) Expression and purification of B3G3-Cys: The pET-32a-B3G3-Cys plasmid was transformed into OverExpress C43 (DE3) competent cells, and positive single clones were selected and inoculated into LB medium. 600=0.6-0.8, add 0.5mM IPTG, place in a 25°C shaker at 220rpm to induce expression for 16h, then collect the bacteria by centrifugation, obtain the periplasmic soluble protein solution by osmotic pressure method, purify by Ni-NTA affinity column, and reduce the dimer with TCEP, identify by SDS-PAGE, and obtain B3G3-Cys;

[0028] (3) Activation of agarose gel: The agarose gel was washed and dried, and an epoxidation reaction solution (30% 0.8 M NaOH, 40% DMSO, 30% ECH, v / v / v) was added at a ratio of 6 mL / g, and the mixture was placed on a shaker at 30°C and 180 rpm for reaction for 3.5 h. The reaction solution was washed off with a large amount of ultrapure water and dried to obtain an epoxidized agarose gel. An amination reaction solution (3 M ethylenediamine) was added at a ratio of 6 mL / g, and the mixture was placed on a shaker at 30°C and 180 rpm for reaction for 12 h. The reaction solution was washed off with a large amount of ultrapure water and dried to obtain an amination agarose gel.

[0029] (4) Directed coupling of B3G3-Cys and agarose gel: 1 g of activated gel was taken, equilibrated with 5 column volumes of coupling buffer (0.01 M PBS), and transferred to 6 mL of PBS. 40 μL of sulfo-SMCC (10 mg / mL) was added, and the mixture was shaken at 25°C and 150 rpm for 1.5 h to synthesize maleimide-based gel. The reaction solution was washed with 20 column volumes of PBS and transferred to 3 mL of PBS. An equal volume of B3G3-Cys (1 mg / mL) was added, and the mixture was shaken at 25°C and 150 rpm for 3 h to achieve coupling.

[0030] (5) Blocking of active sites on the gel: The reaction solution was washed with 12 mL of PBS, and 5 column volumes of blocking buffer (0.01 M cysteine, pH 7.4) were added for equilibration. The mixture was then transferred to 6 mL of 0.01 M cysteine ​​and placed on a shaker at 25°C and 150 rpm for 1 h to achieve blocking and prepare an immunosorbent.

[0031] The invention provides application of an anti-alternarianic acid nano antibody immunoadsorbent in detecting alternarianic acid.

[0032] The invention provides application of an anti-alternarianic acid nano antibody immunosorbent in preparing an alternarianic acid detection product.

[0033] The invention provides an immunoaffinity column loaded with an anti-alternarianic acid nano antibody immunoadsorbent.

[0034] Furthermore, the present invention provides a method for preparing an immunoaffinity column: using 5 times the column volume of 0.01M cysteine, ultrapure water and 20% ethanol to sequentially wash the above-prepared immunosorbent, when the 20% ethanol flows to about 1 / 2 of the column body, install the upper sieve plate, compact the gel, block the liquid outlet with the lower cover, install the upper cover, and store at 4°C.

[0035] The invention provides an anti-alternarianic acid nano antibody immunoadsorbent or an immunoaffinity column for use in enriching alternarianic acid.

[0036] The invention provides a method for pre-treating a sample of isoalternaria acid, which adopts an anti-isoalternaria acid nano antibody immunoadsorbent or the above-mentioned immunoaffinity column to perform sample pre-treating.

[0037] The present invention also provides a method for detecting isoalternaria acid, which uses an anti-isoalternaria acid nanoantibody immunoadsorbent or the above-mentioned immunoaffinity column to perform sample pretreatment, and then performs ultra-high performance liquid chromatography-tandem mass spectrometry detection. The specific detection conditions are:

[0038] Liquid chromatography conditions: chromatographic column is T3 column (100 mm×2.1 mm×1.8 μm); column temperature is 30-50° C.; injection volume is 1-10 μL; mobile phase A is 10 mM ammonium acetate aqueous solution (pH 9.0), mobile phase B is 0.02% formic acid-methanol solution, and the gradient elution program is:

[0039]

[0040] Mass spectrometry conditions: The ion source was an electrospray ion source (ESI - ), 140~160℃; desolvation gas is nitrogen, 980~1000L / h, 400~600℃; cone gas is nitrogen, 140~160L / h; collision gas is argon, 0.1~0.2mL / min; capillary voltage is 1~3kV; detection method is multiple reaction monitoring (MRM), specific parameters are:

[0041]

[0042] Note: a-qualifier ion; b-quantifier ion.

[0043] The present invention has the following beneficial effects:

[0044] The invention expresses an anti-alternaria acid nano antibody by fusion with a cysteine ​​tag, realizes directional coupling with a solid phase carrier, and the prepared anti-alternaria acid nano antibody immunoadsorbent avoids the problem of antigen binding site occupancy in traditional coupling, and improves the adsorption capacity of an immunoaffinity column; the column content of the prepared immunoaffinity column is 905.06 ng, which is 202.40 ng higher than the column content of a non-directionally coupled immunoaffinity column, and the number of repeated uses is also increased, thereby reducing the detection cost, and is of great significance for the detection of sodium alternaria acid.

[0045] At the same time, the nano antibody immunoaffinity column provided by the present invention can achieve efficient enrichment of isoalternaria acid, simplify the sample pretreatment process, and is used for the detection of isoalternaria acid after being combined with mass spectrometry, with good accuracy and precision, a detection limit of 0.61 μg / kg, a quantitative limit of 2.51 μg / kg, a recovery rate between 90.11% and 106.74%, an RSD of less than 10%, and good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the agarose gel electrophoresis diagram of the pET-32a-B3G3-Cys plasmid after amplification (in the figure, lane M: DNA molecular weight standard; lane 1: pET-32a-B3G3-Cys plasmid).

[0047] Figure 2 This is the SDS-PAGE electrophoresis diagram after the expression and purification of B3G3-Cys (in the figure, lane M: protein molecular weight standard; lane 1: B3G3-Cys).

[0048] Figure 3 This is the parent ion and daughter ion chromatogram of the standard series working solution of Alternaria japonica acid (Figure a is the parent ion, Figure b is the quantitative daughter ion, and Figure c is the qualitative daughter ion).

[0049] Figure 4 is the standard curve of Alternaria japonica acid.

[0050] Figure 5 This is an investigation of the number of times the immunoaffinity column was used (n=3) (Figure a is a directional coupling immunoaffinity column, and Figure b is a non-directional coupling immunoaffinity column).

[0051] Figure 6 This is the chromatogram of the spiked tomato sauce sample after cleanup with an immunoaffinity column. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0053] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0054] Example 1 Preparation of anti-alternaria acid nanobody B3G3-Cys

[0055] 1. Construction of pET-32a-B3G3-Cys expression vector

[0056] (1) Primer design

[0057] Using the pET-32a-B3G3 plasmid (pET-32a plasmid with anti-alternaria acid nanobody B3G3) prepared in the laboratory as a template, a pair of primers Cys-F and Cys-R (primer sequences are shown in SEQ ID NOs: 3-4) were designed with reference to the "Rapid Site-Directed Mutagenesis Kit", and the site to be mutated was placed in its reverse complementary region to achieve the addition of a cysteine ​​tag at the C-terminus of the nanobody B3G3 for directional coupling. The primer sequences are shown in Table 1.

[0058] Table 1 Site-directed mutagenesis primer sequences

[0059]

[0060] (2) Plasmid amplification

[0061] Subsequently, according to the reaction system shown in Table 2 and the reaction conditions shown in Table 3, the pET-32a-B3G3 plasmid was subjected to inverse PCR amplification.

[0062] Table 2 PCR reaction system

[0063]

[0064] Table 3 PCR reaction conditions

[0065]

[0066] (3) Identification by agarose gel electrophoresis

[0067] After the reaction was completed, the amplified product was verified by agarose gel electrophoresis. Figure 1As shown, the molecular weight of the pET-32a-B3G3-Cys plasmid should be 6235bp. According to the molecular weight corresponding to the DNA molecular weight standard band, the sample lane has an obvious high-purity pET-32a-B3G3-Cys band, which identifies the successful amplification of the pET-32a-B3G3-Cys plasmid.

[0068] (4) Digestion of amplified products

[0069] Because the amplified product contains the original template plasmid, in order to prevent it from forming false positive transformants after transformation, it must be digested before recombination circularization to remove the methylated template plasmid. Add 1 μL of Dpn I enzyme to the amplified product and place it in a 37°C water bath for 1.5 hours.

[0070] (5) Clean recycling

[0071] Use the Universal DNA Purification and Recovery Kit to clean and recover the amplified product.

[0072] (6) Recombination cyclization

[0073] Prepare the homologous recombination reaction system shown in Table 4 on ice, and circularize the linear DNA in vitro under the catalysis of Exnase II. Use a pipette to gently mix, centrifuge briefly, and place in a 37°C enzyme chain analyzer for reaction for 30 minutes.

[0074] Table 4 Homologous recombination reaction system

[0075]

[0076] (7) Transformation Identification

[0077] Take out the DH5α competent cells from the -80℃ refrigerator and insert them into ice. After thawing, add 10μL of homologous recombination products, transform, plate, and culture, and do negative controls at the same time. The next day, if the number of clones on the recombination reaction transformation plate is significantly higher than the negative control, randomly pick 3 single clones and inoculate them into 10mL LB-Amp liquid culture medium, and place them in a 37℃ shaker at 250rpm for 8h. Finally, the samples were sent for sequencing to verify that the nucleic acid sequence of B3G3-Cys was as expected, as shown in SEQ ID NO:1, indicating that the cysteine ​​tag was successfully inserted. According to the codon table, the amino acid sequence of B3G3-Cys was determined as shown in SEQ ID NO:2.

[0078] 2. Expression and purification of B3G3-Cys

[0079] (1) Plasmid transformation

[0080] Take out 1 OverExpress C43 (DE3) competent cell from -80℃ freezer and insert it into ice. After thawing, add 1μL pET-32a-B3G3-Cys plasmid, flick the tube wall to mix, let it stand on ice for 30min, transfer it to 42℃ water bath for heat shock for 90s, and then quickly insert it back into ice to cool for 3min. Add 700μLL LB liquid culture medium in the clean bench and place it in a 37℃ shaker at 250rpm for 1h. Centrifuge at 5000rpm for 1min, discard 550μL upper culture medium, use the remaining culture medium to blow and resuspend the bacteria, take 100μL from it and evenly spread it on the LB-Amp plate, and invert it in a 37℃ incubator for 12h.

[0081] (2) Expanding training

[0082] In the clean bench, pick a single clone and inoculate it into 1mL LB-Amp liquid culture medium, and place it in a 37℃ shaker at 250rpm for 12h. Inoculate the overnight bacteria into 10mL LB-Amp liquid culture medium at a ratio of 1:100, and place it in a 37℃ shaker at 250rpm for 3h. Inoculate the bacterial solution into 750mL LB-Amp liquid culture medium at a ratio of 1:100, and place it in a 37℃ shaker at 250rpm to the logarithmic phase (measure the bacterial solution OD with a UV spectrophotometer). 600 =0.6~0.8).

[0083] (3) Inducible expression

[0084] Add 375 μL 1 M IPTG and culture at 25°C in a shaker at 220 rpm for 16 h.

[0085] (4) Bacteria fragmentation

[0086] Centrifuge the bacterial solution at 12000rpm for 5min, discard the upper culture medium, add 5mL 5×TES to resuspend the bacteria, and freeze at -80℃ for 3h. After thawing, add 20mL ultrapure water and mix at 220rpm at 25℃ for 1h. Centrifuge at 15000rpm for 40min, and keep the supernatant as the periplasmic soluble protein solution.

[0087] (5) Ni-NTA column purification

[0088] Shake the Ni-NTA agarose gel and storage solution, pipette and load into an empty chromatography column, and wait until it is drained to obtain a 4mL His tag purification column. Add 5 times the column volume of ultrapure water to wash the storage solution, and then add 5 times the column volume of TBS equilibrium buffer system. Add the periplasmic soluble protein solution for loading, and repeat twice. Add 20mM imidazole-TBS to elute the impurities, add 5mL at a time, and collect the last drop of flow-through each time. Use Nanodrop to measure its protein concentration. If it is less than 0.1mg / mL, it is completely eluted. Add 200mM imidazole-TBS in the same way to elute the nanoantibody, and the elution is complete when the protein concentration of the flow-through is less than 0.1mg / mL.

[0089] (6) Desalination by dialysis

[0090] Take an appropriate amount of 5kD dialysis bag, place it in ultrapure water and boil it, clamp the lower end with a clamp, add 200mM imidazole-TBS eluent, clamp the upper end, place it in 4L TBS, and dialyze at 4℃. Replace TBS every 12h until 6 dialysis cycles are completed.

[0091] (7) Dimer reduction

[0092] Since cysteine ​​is added to the C-terminus of the antibody, it is easy to form disulfide bonds between antibody molecules, produce dimers, and lead to the loss of terminal thiol groups of the antibody, which affects the directional coupling effect. Therefore, the dimer needs to be reduced. Add the antibody solution to a 5kD ultrafiltration tube and centrifuge at 4500rpm until the protein concentration of the upper layer of the ultrafiltration tube is 1mg / mL measured by Nanodrop. Add 250mM TCEP·HCl at a ratio of 10μL / mL, place it at 4℃ for 20min of rotation and oscillation, add 75mM 4-azidobenzoic acid at a ratio of 137.5μL / mL, and stand on ice for 10min to bind excess TCEP and terminate the reaction. The reduction product is dialyzed with PBS to remove the reaction reagent to avoid errors in concentration determination and prevent side reactions during coupling.

[0093] (8) SDS-PAGE electrophoresis identification

[0094] The antibody solution was verified by SDS-PAGE electrophoresis. Figure 2 As shown, the molecular weight of B3G3-Cys should be 17.7kDa. According to the molecular weight corresponding to the protein molecular weight standard band, the sample lane has an obvious high-purity B3G3-Cys band, which identifies the successful expression and purification of B3G3-Cys. The antibody solution was ultrafiltered and concentrated to a protein concentration of more than 1mg / mL measured by Nanodrop. After calculation, the expression yield of B3G3-Cys was 44.0mg / L bacterial solution.

[0095] Example 2 Preparation of Directed Coupling Anti-Alternaria Acid Nanobody Immunoaffinity Column

[0096] 1. Activation of agarose gel

[0097] (1) Epoxidation of agarose gel

[0098] The agarose gel and the storage solution were shaken, a certain amount was drawn and loaded into an empty chromatography column, the solution was drained, and 5 times the column volume of ultrapure water was added to clean the storage solution, vacuum-drained with a solid phase extractor, transferred to a conical flask, and the epoxidation reaction solution (30% 0.8M NaOH, 40% DMSO, 30% ECH, v / v / v) was added at a ratio of 6mL / g, and placed in a 30°C shaker at 180rpm for 3.5h. After the reaction was completed, the gel was transferred to an empty chromatography column, the solution was drained, and a large amount of ultrapure water was added to wash away the residual reaction solution, 100mL was added at a time, and the last 1mL of the flow-through liquid was collected each time, 3mL 1.3M Na2S2O3 and 2 drops of phenolphthalein were added, and the mixture was allowed to stand for 30s. If the solution did not turn red after standing for 30s, it indicated that the cleaning was complete. Subsequently, the solid phase extractor was vacuum-drained, ultrapure water was added, and the volume was fixed at a ratio of 4mL / g. 1 g of activated gel (i.e. 4 mL of gel preservation solution) was taken and placed in an empty chromatography column and vacuum-dried using a solid phase extractor. The amount of epoxy groups formed on the gel was determined using the same method. At the same time, ultrapure water was used instead of Na2S2O3 in the blank control group, and 0.01 M HCl was used to titrate the OH groups generated by the reaction. - The end point of titration is when the red color fades. Based on the volume of HCl used, the amount of epoxy groups formed after activation is calculated to be 61.3±1.6μmol / g.

[0099] (2) Amination of agarose gel

[0100] The epoxidized gel and the storage solution were shaken evenly, and a certain amount was taken and placed in an empty chromatography column. The solution was drained, and vacuum-drained with a solid phase extractor. The mixture was transferred to a conical flask, and the amination reaction solution (3M ethylenediamine) was added at a ratio of 6 mL / g. The mixture was placed on a 30°C shaker at 180 rpm for reaction for 12 h. The residual epoxy content on the gel was determined by the above method, and the amount of amino group formation was indicated by the reduction in the amount of epoxy group. If the volume of HCl used for titration in the Na2S2O3 group was basically the same as that in the blank control group, it indicated that the amination reaction was complete. The gel was transferred to an empty chromatography column, the solution was drained, and a large amount of ultrapure water was added to wash away the residual reaction solution. 50 mL was added at a time, and the last 1 mL of the flow-through liquid was collected each time. Two drops of phenolphthalein were added, and the mixture was mixed and allowed to stand. If the solution did not turn red after standing for 30 seconds, it indicated that the cleaning was complete. The mixture was then vacuum-drained with a solid phase extractor, and 20% ethanol was added. The volume was fixed at a ratio of 4 mL / g, and the mixture was stored at 4°C.

[0101] 2. Directed coupling of B3G3-Cys and agarose gel

[0102] 1g of activated gel was taken and placed in an empty chromatography column, the solution was drained, 5 column volumes of ultrapure water were added to wash the storage solution, and then 5 column volumes of coupling buffer (0.01M PBS) were added to balance the buffer system. 6mL of PBS was added and the gel was transferred to a conical flask, 40μL of sulfo-SMCC (10mg / mL) was added, and the reaction was carried out on a 25℃ shaker at 150rpm for 1.5h to synthesize maleimide-based gel. The gel was then transferred to an empty chromatography column, the solution was drained, 20 column volumes of PBS were added to wash off the residual reaction solution, and then 5 column volumes of PBS were added to balance the buffer system. 3mL of PBS was added and the gel was transferred to a conical flask, an equal volume of B3G3-Cys (1mg / mL) was added, and the reaction was carried out on a 25℃ shaker at 150rpm for 3h to allow the antibody's thiol group to bind to the gel's maleimide group to achieve coupling. After the reaction was completed, the gel was transferred to an empty chromatography column, the solution was drained, 12 mL of PBS was added to wash away the unstable coupled antibody twice, the coupled flow-through and the two washing solutions were collected, and the protein concentration was determined by Nanodrop. The coupled amount of B3G3-Cys was calculated to be 1.57 ± 0.05 mg / g.

[0103] 3. Blocking of active sites of gel

[0104] Five column volumes of blocking buffer (0.01 M cysteine, pH 7.4) were added to balance the buffer system, and then 6 mL of 0.01 M cysteine ​​was added and transferred to a conical flask, which was placed in a shaker at 25 °C and 150 rpm for 1 h to allow the thiol group of cysteine ​​to bind to the maleimide group of the gel to achieve blocking and avoid nonspecific adsorption of the gel to the sample matrix and drugs.

[0105] 4. Column Packing and Storage

[0106] Transfer the gel into an empty chromatography column, drain the solution, add 5 times the column volume of 0.01M cysteine, ultrapure water and 20% ethanol to wash in sequence. When 20% ethanol flows to about 1 / 2 of the column body, install the upper sieve plate horizontally, compact the gel, block the liquid outlet with the lower cover, install the upper cover, and store at 4°C.

[0107] Example 3 Performance Evaluation of Immunoaffinity Column

[0108] 1. Preparation and extraction of test samples

[0109] Commercially purchased beer, tomato sauce and flour samples were crushed and homogenized as needed, and 5 g was weighed into a 50 mL centrifuge tube. 20 mL of 70% methanol aqueous solution (v / v) was added and vortexed, and then placed on a shaker for 20 min. Centrifuged at 6000 rpm for 10 min, 4 mL of supernatant was taken, and 46 mL of loading buffer (5 mM ammonium acetate aqueous solution, pH 8.0) was added for dilution, and after mixing, it was filtered through a 0.22 μm polyethersulfone filter.

[0110] 2. Immunoaffinity column purification

[0111] Take out the immunoaffinity column prepared in Example 2, return to room temperature, drain the solution, add 5 times the column volume of ultrapure water to wash, and then add 5 times the column volume of loading buffer to balance. Add 50mL of sample diluent, control the sample solution to drip steadily at a rate of 1-3mL / min, so that the antibody binds to the drug in the sample. Add 10mL of loading buffer, drip naturally at a stable flow rate, remove other impurities in the sample that are non-specifically adsorbed on the affinity column, and vacuum dry with a solid phase extractor after draining. Disconnect from the vacuum system and place a new collection tube under the affinity column. Add 1.5mL of elution buffer (50% methanol-50mM ammonium acetate aqueous solution, v / v, pH 2.0), drip steadily at a rate of 1-3mL / min to destroy the binding of the antibody and the drug, and vacuum dry with a solid phase extractor after draining. Filter the eluted product with a 0.22μm polytetrafluoroethylene filter and place it in a sample injection bottle for standby.

[0112] 3. Ultra-high performance liquid chromatography-tandem mass spectrometry detection

[0113] A Waters Xevo TQ-S ultra-high performance liquid chromatography-tandem mass spectrometer was used to develop a detection method for the acid of Alternaria serrata. Liquid chromatography conditions: the chromatographic column was a T3 column (100 mm × 2.1 mm × 1.8 μm); the column temperature was 40 °C; the injection volume was 10 μL; the mobile phase A was a 10 mM ammonium acetate aqueous solution (pH 9.0), the mobile phase B was a 0.02% formic acid-methanol solution, and the gradient elution program was shown in Table 5. Mass spectrometry conditions: the ion source was an electrospray ionization source (ESI - ), 150°C; the desolvation gas is nitrogen, 990L / h, 500°C; the cone gas is nitrogen, 149L / h; the collision gas is argon, 0.14mL / min; the capillary voltage is 2kV; the detection method is multiple reaction monitoring (MRM), and the parameters are shown in Table 6.

[0114] Table 5 Liquid chromatography mobile phase gradient elution program

[0115]

[0116] Table 6 Mass spectrometry MRM parameters

[0117]

[0118] Note: a-qualifier ion; b-quantifier ion.

[0119] The standard series working solutions (1, 2, 5, 10, 20, 50, 100 ng / mL) were injected in sequence, and the data were collected using MassLynx software and analyzed using TargetLynx software. The peak time of isoalternariae oxadiazole acid was determined to be 2.13 min. The results are as follows: Figure 3 As shown. The peak area of ​​the quantitative ion peak is plotted against the concentration, and the standard curve obtained by fitting is as follows Figure 4 As shown, the curve regression equation is Y = 201.319X + 27.1899, R 2 =0.9993, in the range of 1-100 ng / mL, the linear relationship of the curve is good, and the content of iso-alternaria oxadione in the eluate can be calculated by the external standard method. Combined with the sample weight and dilution multiple, the detection limit of the method is 0.6 μg / kg, and the quantification limit is 2.5 μg / kg, which is lower than the derivatization method reported by Asam et al. (detection limit is 3 μg / kg, quantification limit is 9 μg / kg).

[0120] 4. Performance evaluation of immunoaffinity columns

[0121] At the same time, the directional coupled immunoaffinity column prepared in Example 2 and the non-directional coupled immunoaffinity column prepared in Comparative Example 1 were used for detection and comparison, and the method was the same as above.

[0122] (1) Column content

[0123] A blank tomato sauce sample was selected, and an excess amount (2000 ng) of isoalternarianic acid was added. The extraction, purification and detection were carried out according to the above method. According to the content of isoalternarianic acid in the eluate, the contents of the prepared directional coupling and non-directional coupling immunoaffinity columns were calculated to be 905.06±62.12 ng and 702.66±40.35 ng, respectively.

[0124] (2) Number of times used

[0125] A blank tomato sauce sample was selected, and 250 ng of isoalternaria acid was added. The extraction, purification and detection were performed according to the above method. The purification was repeated 10 times using the same immunoaffinity column. The recovery rate was calculated based on the content of isoalternaria acid in the eluate. The results are shown in Figure 2. Figure 5 As shown in FIG. 1 , with a recovery rate greater than 80%, it was determined that the prepared directional coupling and non-directional coupling immunoaffinity columns can be reused 8 times and 7 times, respectively. The chromatogram of one of the directional coupling immunoaffinity columns is shown in FIG. Figure 6As shown, according to the response intensity of the chromatographic peak, it can be seen that the interference of the sample matrix has been removed, showing the excellent purification performance of the immunoaffinity column.

[0126] At the same time, the immunoaffinity column was stored at 4°C for one week and then taken out for the above-mentioned addition recovery experiment. The recovery rates of the two immunoaffinity columns were both greater than 80%, indicating that the directional coupling immunoaffinity column and the traditional anti-alternaria acid nanoantibody immunoaffinity column have consistent stability.

[0127] Example 4 Validation and Application of Immunoaffinity Column Purification-Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Detection Method

[0128] The directional coupled immunoaffinity column was combined with ultra-performance liquid chromatography-tandem mass spectrometry to verify the method and apply it to the detection of actual samples.

[0129] (1) Accuracy and precision

[0130] Blank beer, tomato sauce and flour samples were selected, and three levels (10, 50 and 100 μg / kg) of isoalternaria acid were added. Extraction, purification and detection were carried out according to the method of Example 3 above. Each level was measured in parallel 6 times, and the recovery rate was calculated based on the content of isoalternaria acid in the eluate.

[0131] The test results are shown in Table 7. The recoveries of the three samples were between 90.11% and 106.74%, and the RSD was less than 10%, indicating that the constructed method has good accuracy and precision and can be used to detect the content of tricholoma oxadione in samples.

[0132] Table 7 Actual sample addition recovery experiment (n=6)

[0133]

[0134] (2) Actual sample testing

[0135] 5 portions of beer, tomato sauce and flour of different brands were purchased from a supermarket (another sample was weighed randomly and 250 ng of isoalternarianic acid was added as a positive control), and the extraction, purification and detection were carried out according to the method of Example 3 above.

[0136] The test results are shown in Table 8. Thirteen positive samples were detected in 15 samples, among which the detection amounts of beer, tomato sauce and flour samples were 2.17-11.14, 26.11-92.35, and 10.25-33.08 μg / kg, respectively, indicating that the method has good application potential.

[0137] Table 8 Actual sample detection (n=3)

[0138]

[0139]

[0140] Note: *-between the limit of detection and the limit of quantification; ND-not detected.

[0141] Comparative Example 1 Preparation of non-directional coupling anti-isoalternaria acid nanoantibody immunoaffinity column

[0142] The pET-32a-B3G3 plasmid was transformed according to the method of Example 1, and B3G3 was obtained by expression and purification. The epoxidized gel prepared in Example 2 was used as a carrier and a non-directional coupled immunoaffinity column was prepared by a random covalent coupling method to compare the performance.

[0143] 1. Random covalent coupling of B3G3 and agarose gel

[0144] 1g of activated gel was taken and placed in an empty chromatography column, the solution was drained, 5 column volumes of ultrapure water were added to wash the storage solution, and then 5 column volumes of coupling buffer (0.1M NaHCO3, containing 0.5M NaCl) were added to balance the buffer system. 3mL of coupling buffer was added and the gel was transferred to a conical flask, an equal volume of B3G3 (1mg / mL) was added, and the reaction was placed in a 25℃ shaker at 150rpm for 1h to allow the amino group of the antibody to combine with the epoxy group of the gel to achieve coupling. After the reaction is completed, the gel was transferred to an empty chromatography column, the solution was drained, 12mL of coupling buffer was added twice to wash away the unstabilized coupled antibody, the coupling flow-through liquid and the two washing liquids were collected, and the protein concentration was determined by Nanodrop. The coupling amount of B3G3 was calculated to be 1.47±0.05mg / g.

[0145] 2. Blocking of active sites of gel

[0146] Five column volumes of blocking buffer (0.01 M Tris-HCl, pH 8.3) were added to balance the buffer system, and then 6 mL of 0.01 M Tris-HCl was added and transferred to a conical flask, which was placed on a shaker at 25 °C and 150 rpm for 1 h to allow the amino group of cysteine ​​to combine with the epoxy group of the gel to achieve blocking and avoid nonspecific adsorption of the gel to the sample matrix and drugs.

[0147] 3. Column storage

[0148] Transfer the gel into an empty chromatography column, drain the solution, add 5 times the column volume of 0.01M Tris-HCl, ultrapure water and 20% ethanol to wash in sequence. When 20% ethanol flows to about 1 / 2 of the column body, install the upper sieve plate horizontally, compact the gel, block the liquid outlet with the lower cover, install the upper cover, and store at 4°C.

[0149] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An anti-alternaria acid nanoantibody immunoadsorbent, characterized in that: The immunosorbent comprises: a solid phase carrier containing a protein cross-linking agent, and an anti-alternarianic acid nano antibody which is directionally coupled to the solid phase carrier and has a cysteine ​​tag at the C-terminus.

2. The nanobody immunoadsorbent according to claim 1, characterized in that: The nucleic acid sequence of the anti-alternaria acid nanobody with a cysteine ​​tag at the C-terminus is shown in SEQ ID NO:1, and the amino acid sequence thereof is shown in SEQ ID NO:

2.

3. The nanobody immunoadsorbent according to claim 1, characterized in that: The solid phase carrier is agarose gel.

4. The nanobody immunoadsorbent according to claim 1, characterized in that: The protein cross-linking agent is a maleimide cross-linking agent.

5. The method for preparing the nanobody immunoadsorbent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Construction of pET-32a-B3G3-Cys expression vector: Using pET-32a plasmid carrying anti-alternaria nicotinic acid nanoantibody B3G3 as a template, using primers containing cysteine ​​tags, PCR amplification, and then digestion, cleaning, recovery, recombination and cyclization, and then transformed into DH5α competent cells, and identified and obtained pET-32a-B3G3-Cys plasmid; the primer sequences containing cysteine ​​tags are shown in SEQ ID NOs: 3 to 4; (2) Expression and purification of B3G3-Cys: After transformation with the pET-32a-B3G3-Cys plasmid, the expression was induced, the cells were collected by centrifugation, the periplasmic soluble protein solution was obtained by osmotic pressure method, purified by Ni-NTA affinity column, and the dimer was reduced by TCEP to identify B3G3-Cys; (3) activating agarose gel to obtain amino agarose gel; (4) Directed coupling: Take the activated agarose gel, add a protein cross-linking agent, synthesize a maleimide-based gel, wash away the reaction solution, add an equal volume of B3G3-Cys, react, and complete the coupling; (5) Blocking the active sites of the gel: Wash away the reaction solution, add cysteine, react and block the sites, and successfully prepare the immunosorbent.

6. Use of the anti-alternarianic acid nanoantibody immunosorbent according to any one of claims 1 to 4 in detecting alternarianic acid or in preparing an alternarianic acid detection product.

7. An immunoaffinity column, characterized in that The invention is loaded with the anti-alternaria acid nanoantibody immunoadsorbent according to any one of claims 1 to 4.

8. Use of the anti-alternarianic acid nanobody immunoadsorbent according to any one of claims 1 to 4, or the immunoaffinity column according to claim 6 in enriching alternarianic acid.

9. A method for pre-treating a sample of isoalternaria acid, characterized in that: The anti-alternarianic acid nanoantibody immunosorbent according to any one of claims 1 to 4, or the immunoaffinity column according to claim 6 is used for sample pretreatment.

10. A method for detecting isoalternaria acid, characterized in that: After the sample is pre-treated using the anti-alternarianic acid nanoantibody immunosorbent according to any one of claims 1 to 4 or the immunoaffinity column according to claim 6, ultra-performance liquid chromatography-tandem mass spectrometry detection is performed.

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