CN110133249B

CN110133249B - Immunoadsorbent and immunoaffinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone

Info

Publication number: CN110133249B
Application number: CN201910360692.3A
Authority: CN
Inventors: 李培武; 张奇; 白艺珍; 李慧; 张文
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2022-09-02
Anticipated expiration: 2039-04-30
Also published as: CN110133249A

Abstract

The invention provides an immunoadsorbent for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone, an immunoaffinity column and application. The immunoadsorbent for aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone comprises a solid phase carrier, and an anti-aflatoxin monoclonal antibody, an anti-cyclopiazonic acid monoclonal antibody, an anti-ochratoxin A monoclonal antibody and an anti-zearalenone monoclonal antibody which are coupled with the solid phase carrier, wherein the cyclopiazonic acid monoclonal antibody is a monoclonal antibody produced by secretion of a hybridoma cell strain YTT-2 with the preservation number of CCTCC NO. C201871. The immunoaffinity column provided by the invention can be used for purification-detection pretreatment of samples simultaneously containing aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone, the four samples have no mutual interference influence and have stable performance, and an economical, quick, accurate and safe high performance liquid chromatography-mass spectrometer detection method is established by using the immunoaffinity column.

Description

Immunoadsorbent and immunoaffinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone

Technical Field

The invention relates to an immunoadsorbent for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone and an immunoaffinity column thereof.

Background

Mycotoxins are toxic secondary metabolites produced by fungi during their growth, and over 300 structurally distinct mycotoxins have been discovered. Fungi can be widely distributed in various agricultural products and food, wherein a part of strains can secrete and produce mycotoxin, so that acute or chronic mycotoxicosis symptoms of people or livestock are caused, and the health of people and animals is seriously threatened. In view of the hazards of mycotoxins, the contents of mycotoxins are strictly limited in countries around the world. With the improvement of food safety requirements of people, the detection of mycotoxin in agricultural products needs to be enhanced, a safe and reliable detection technology is developed, and the food safety level in China is improved.

Aflatoxins are a group of secondary metabolites with similar structures produced by aspergillus flavus, aspergillus parasiticus and the like, are a group of compounds taking dicranostigma as a basic structure, and widely exist in grain, feed and processed products thereof. Aflatoxins have been found in more than 20 species, mainly including aflatoxin B1(AFB1), B2(AFB2), AFG and M1(AFM1), etc. Among them, AFB1 has the highest toxicity, which is 10 times that of potassium cyanide and 68 times that of arsenic, and is classified as a class I carcinogen by cancer research institutes of the world health organization. The aflatoxin has the effects of inducing mutation, inhibiting immunity and causing cancer, mainly acts on the liver, and the liver of a person eating food containing low-level aflatoxin is damaged after long-term consumption.

Cyclopianianic acid is mainly a secondary metabolite secreted by aspergillus flavus and aspergillus parasiticus, and is a natural toxic compound capable of causing various damages to human and livestock. Cyclopianianic acid is chemically stable and cannot be destroyed under ordinary storage conditions and processing, and even if pasteurized, can be almost completely destroyed. Cyclopianianic acid is widely contained in agricultural products such as corn, peanut, feed and the like and food such as cheese and the like, and can directly or indirectly enter a food chain after polluting the food and the feed, thereby threatening the health and life safety of people and livestock. Therefore, the detection, especially the quick detection, of the cyclopimaric acid in the agricultural products and the food is enhanced, so as to know and master the health information of the food and the feed in time.

Ochratoxins are mycotoxins produced by ochratoxins and pure penicillium viridis, are widely present in agricultural products such as wheat, corn, peanut, corn and the like, and have the characteristics of highest toxicity, widest distribution and highest toxin production amount, namely ochratoxin A, and have high contentChemical stabilityAnd thermal stability. Ochratoxin A has teratogenic, carcinogenic, mutagenic and teratogenic effectsImmunosuppressive effectAfter the ochratoxin A is taken, the poisoning of the liver and the kidney can be caused, and the symptoms of renal tubular epithelium injury, intestinal lymph gland necrosis, diarrhea, anorexia, dehydration and the like can be caused.

Zearalenone is a mycotoxin produced by fusarium, has immunotoxicity, hepatotoxicity, genetic toxicity, potential carcinogenicity, a toxic effect similar to estrogen, has a certain influence on tumorigenesis, and has potential harm to the health of people and animals. Zearalenone has an effect of promoting animal growth, has been used as a growth promoter for animals by adding it to feed to promote weight gain of livestock, and has been prohibited from being used as a growth promoter in accordance with the study of toxicity. Zearalenone is widely present in crops such as corn, sorghum, wheat and the like and milk, and enters animals and human bodies through polluted agricultural products, so that threats are caused to animal husbandry and human health.

At present, the existing detection method of cyclopiazonic acid comprises thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. Wherein, the thin layer chromatography does not need special instruments and equipment, but can not accurately quantify, and has great harm to the experimenters and the surrounding environment. High-performance liquid chromatography, liquid chromatography-mass spectrometry combined method and other precision instrument analysis methods are high in sensitivity and accuracy, and the sample pretreatment process is complicated. An immunoaffinity chromatographic column is a novel sample pretreatment technology, combines an immunoreaction with a chromatographic analysis method, utilizes the high specificity and affinity of antigen-antibody combination, combines a specific antibody to a chromatographic adsorbent by a chemical coupling bonding method, and realizes the separation, enrichment and purification of the effect of a target substance in a complex sample based on immunological reversible combination. At present, no report related to an immunoadsorbent and an immunoaffinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone is available.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an immunoadsorbent for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone, a composite affinity column and application.

In order to achieve the purpose, the invention adopts the technical scheme that:

the immunoadsorbent comprises a solid phase carrier, and an aflatoxin monoclonal antibody, a cyclopiazonic acid monoclonal antibody, an ochratoxin A monoclonal antibody and a zearalenone monoclonal antibody which are coupled with the solid phase carrier, wherein the anti-cyclopiazonic acid monoclonal antibody is generated by secreting a hybridoma cell strain YTT-2 with the preservation number of CCTCC NO. C201871.

According to the scheme, the solid phase carrier is agarose gel.

According to the scheme, the anti-aflatoxin monoclonal antibody is a general-purpose monoclonal antibody for aflatoxin and is secreted and generated by a hybridoma cell line 1C11 with the preservation number of CCTCC NO. C201013, the anti-ochratoxin A monoclonal antibody is secreted and generated by a hybridoma cell line 1H2 with the preservation number of CCTCC NO. C201329, and the anti-zearalenone monoclonal antibody is secreted and generated by a hybridoma cell line 2D3 with the preservation number of CCTCC NO. C201328.

A composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone is loaded with immune adsorbents for aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone.

The preparation of the composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone comprises the following steps:

a) solid support matrix treatment

Washing CNBr-activated sepharose matrix powder with HCl under the condition of pH2-3 to remove impurities; CNBr activated sepharose is provided in lyophilized form.

b) Ligand coupling

Dissolving an aflatoxin monoclonal antibody, a cyclopiazonic acid monoclonal antibody, an ochratoxin A monoclonal antibody and a zearalenone monoclonal antibody to be coupled by using a coupling buffer solution to obtain antibody solutions, and quickly transferring the activated sepharose gel matrix in the step a) into the antibody solutions for coupling; c) ligand blocking

Blocking all residual reactive groups;

d) removing excess ligand that is not coupled after coupling;

e) and (5) column packing.

According to the scheme, the concentration of HCl used for washing in the step a) is 1mmol/L, and the washing time is 15 min.

According to the above scheme, the coupling buffer in step b) is 0.2mol/L NaHCO ₃ pH8.3, and the concentration of each antibody solution is 10-15 mg/mL.

According to the above scheme, the coupling conditions in step b) are: the mixture is fully mixed for 2 to 4 hours at room temperature (20 to 25 ℃).

According to the scheme, the ligand blocking process in the step c) comprises the following steps: transferring the agarose gel matrix treated by the step b) into 0.1mol/LTris-HCl buffer solution, and standing for 2-4h at room temperature.

According to the scheme, the step d) is as follows: washing the agarose gel matrix treated by the step c) by using buffer solutions with pH value of 4 and pH value of 8 in sequence for at least 3 cycles; the buffer solution with pH value of 4 and pH value of 8 can be respectively 0.1mol/L acetic acid/sodium acetate buffer solution and 0.1mol/L Tris-HCl buffer solution.

Following the protocol described above, the gel treated in step d) was washed with 5 agarose gel volumes of 0.01% NaN ₃ PBS wash and use 0.01% NaN ₃ PBS and then column packed.

On the basis, the invention establishes a method for detecting the contents of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone by an immunoaffinity column purification high performance liquid chromatography-mass spectrometer, when a sample containing aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone is passed through an immunoaffinity column, the immunoadsorbent can specifically adsorb the aflatoxin, the cyclopiazonic acid, the ochratoxin A and the zearalenone, and other impurities flow out of the immunoaffinity column, then the affinity column is eluted by chromatographic grade methanol at the eluting flow rate of 1-2 mL/min, the aflatoxin, the cyclopiazonic acid, the ochratoxin A and the zearalenone are eluted from the column, a sample is well purified, and the collected eluent is used for detection of a high performance liquid chromatography-mass spectrometer.

Based on the method for detecting the content of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone by using the composite affinity column, when a sample containing aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone passes through the immunoaffinity column, the immunoadsorbent can specifically adsorb aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone, other impurities flow out of the immunoaffinity column, then the affinity column is eluted by chromatographic grade methanol, and the eluate, namely the purified and concentrated sample, is collected and detected by using a high performance liquid chromatography-mass spectrometer to obtain the content of each toxin.

Detailed Description

Example 1: the anti-cyclopiazonic acid monoclonal antibody is produced by a hybridoma cell strain YTT-2 with the preservation number of CCTCC NO. C201871. The method comprises the following specific steps:

injecting the cyclopiazonic acid monoclonal antibody hybridoma cell strain YTT-2 into a BALB/c mouse body which is treated by Freund incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the concrete operation is as follows: filtering ascites of mice by using double-layer filter paper, centrifuging the filtered ascites at 4 ℃ and 12000r/min for more than 15min, sucking supernatant, mixing the supernatant with 4 times of acetate buffer solution, slowly adding n-octanoic acid while stirring, wherein the volume of the n-octanoic acid required by each milliliter of ascites is 30-35 mu L, mixing at room temperature for 30-60 min, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding precipitates, filtering the obtained supernatant by using the double-layer filter paper, adding 1/10 phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration is 0.277g/mL, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding the supernatant, resuspending the obtained precipitate with 0.01mol/L phosphate buffer solution with original ascites volume of 1/10, placing into a dialysis bag, dialyzing with pure water, freezing the fully dialyzed protein solution in a refrigerator at-70 ℃, then freeze-drying with a freeze vacuum drier, collecting the freeze-dried powder to obtain a purified anti-cyclopiazonic acid monoclonal antibody, and placing the antibody in a refrigerator at-20 ℃ for later use;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 0.9g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate.

The subtype of the anti-cyclopiazonic acid monoclonal antibody secreted by the hybridoma cell strain YTT-2 is identified to be IgG2a type by using a commercial subtype identification kit.

The titer of the mouse ascites antibody of YTT-2 can reach 1.2 multiplied by 10 measured by the conventional indirect non-joint competitive enzyme-linked immunosorbent assay (ELISA) ⁵ I.e. dilution of murine ascites antibody by 1.2X 10 ⁵ The results of the solution assay at time of doubling were positive. Sensitivity (IC) of cyclopiazonic acid identified by conventional indirect competitive ELISA method ₅₀ ) The concentration is 0.84ng/mL, and the cross reaction rate to aflatoxin B1, B2, G1, G2, M1 and variegated aflatoxin is less than 0.1%.

Screening hybridoma cell strain YTT-2:

1. antigen synthesis and animal immunization

The method is characterized in that a commercially available cyclopiazonic acid standard is purchased for complete antigen synthesis, and the specific synthesis steps are as follows: dissolve 1mg CPA in 1mL 0.05M NaHCO ₃ 50% aqueous methanol solution; adding 2mg of hemocyanin (KLH) into 0.4mL of 3M sodium acetate, dropwise adding 0.2mL of formaldehyde within 1min under the condition of stirring at room temperature, and continuously stirring for 10 min; CPA was slowly added dropwise to KLH with constant stirring at room temperature for more than 16 h. The final reaction product CPA-KLH was placed in a dialysis bag of appropriate size and dialyzed in PBS at 4 ℃ for three days. The same method is used for synthesizing the detection reagentCPA-OVA。

6 female Balb/c mice at 6 weeks of age were purchased and immunized against the self-synthesized cyclopiazonic acid complete antigen CPA-KLH at a dose of 100. mu.g/mouse. The complete antigen CPA-KLH is mixed and emulsified with Freund's complete adjuvant for the first immunization, and the immunization is carried out on the back by subcutaneous multipoint injection. The priming was performed 3 weeks apart, followed by 2 weeks apart, and immunization was performed using Freund's incomplete adjuvant emulsion. And (3) taking blood from the tail vein one week after the third immunization, separating serum, monitoring the antibody titer of the serum of the mouse by adopting an indirect ELISA method, measuring the sensitivity of the serum of the mouse by adopting an indirect competitive ELISA method, selecting the mouse corresponding to the serum with relatively high titer and sensitivity for the last sprint immunization, and taking 100 mu g of immunogen to be dissolved in 200 mu LPBS for direct injection into the abdominal cavity 3 days before fusion. Freund's adjuvant was purchased from Sigma-Aldrich.

2. Cell fusion

After 3 days of the last sprint immunization, 50 percent (weight percentage) of polyethylene glycol (PEG) (molecular weight 1450) is adopted as a fusion agent, and cell fusion is carried out according to a conventional method, which comprises the following steps:

the immunized mice were sacrificed by cervical dislocation, the spleen was harvested under aseptic conditions, the spleen cells were ground and isolated, mixing with murine myeloma cell SP2/0 at a ratio of 5:1, washing the mixed cells with RPMI-1640 basic culture solution, fusing with 50% PEG for 1min, then adding RPMI-1640 basic culture solution, centrifuging, removing supernatant, re-suspending the fusion cells formed by mouse splenocytes and mouse myeloma cells SP2/0 with 72mLRPMI-1640 basic culture solution, dripping the re-suspended cells into 96-well cell culture plates, culturing at 2 drops/well in a carbon dioxide incubator at 37 ℃, the RPMI-1640 basic culture solution contains 20% (volume percentage) of fetal bovine serum, 2% (weight percentage) of growth factor and 1% (weight percentage) of hypoxanthine-aminopterin-thymidine, namely HAT. The SP2/0 was purchased from Shanghai Panko Biotech Co., Ltd; RPMI-1640 basal medium was purchased from Hyclone; 1% hypoxanthine-aminopterin-thymidine, HAT, was purchased from Sigma-Aldrich.

3. Screening and cloning of cell lines

To be fusedAbout day 12 later, the cell colonies grow to the size of 1/2 area at the bottom of the well, and the culture solution turns yellow, and then antibody detection can be carried out. Screening culture holes with hybridoma cells growing by adopting an ELISA method, wherein the screening is carried out in two steps, and in the first step, positive holes which resist cyclopiazonic acid but not resist carrier protein KLH are screened out by adopting an indirect non-competitive ELISA method; and a second step of detecting the positive holes screened in the first step by adopting an indirect competitive ELISA method, taking cyclopiazonic acid as a competitive antigen, and selecting holes with higher light absorption value and sensitivity (the higher light absorption value means that the final measured value of the holes with the competitive antigen of 0, namely the positive control holes, is higher, and the higher sensitivity means that the competitive antigen concentration when the inhibition rate is 50%, namely the IC ₅₀ Smaller value), cloning by a limiting dilution method, detecting by the same two-step method about 10 days after cloning, repeating cloning for 2-3 times to obtain a hybridoma cell strain YTT-2 which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO. C201871 in Wuhan university, Wuhan, China.

Determination of sequence of variable region of anti-cyclopiazonic acid monoclonal antibody hybridoma cell line YTT-2 antibody

(1) Extracting total RNA: total RNA extraction kit of Tiangen company is adopted and total RNA capable of generating hybridoma cell strain YTT-2 is extracted according to the instruction.

(2) Synthesis of cDNA: oligo (dT) using the total RNA obtained in step 1 as a template ₁₅ As primers, according to SuperScript ^TM -2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT) ₁₅ Purchased from Invitrogen;

(3) cloning of variable region genes by PCR: designing a primer according to a conserved site of a mouse antibody gene sequence in GENBANK, and amplifying antibody heavy chain and light chain variable region genes by using CDNA as a template. The PCR procedure was: amplification is carried out for 30 cycles at 94 ℃ for 30s, 50s at 55 ℃ for 1min at 72 ℃ and finally for 10min at 72 ℃. After 1% (weight percentage) agarose gel electrophoresis separation of the PCR product, using the kit to purify and recover DNA fragment, connecting the DNA fragment to the vector pMD18-T, transforming Escherichia coli DH5 alpha competent cell, selecting positive clone, and sending to Suzhou Hongxi biotechnology Limited for sequencing. Wherein the sequences of the primers are respectively as follows: heavy chain variable region primers were 5 '-AGG TSM ARC TGC AGS AGT CWG G-3' (22mer) and 5'-TGA GGA GACGGT GAC CGT GGT CCC TTG GCC CC-3' (32mer) where S, M, R and W are degenerate bases, M ═ a/C, R ═ a/G, S ═ C/G, W ═ a/T, and light chain variable region primers were 5'-GAC ATT GAG CTCACC CAG CTT GGT GCC-3' (24mer) and 5'-CCG TTT CAG CTC CAG CTT GGT CCC-3' (24 mer).

Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 360bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 120 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence has the length of 322bp and is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced to consist of 107 amino acids according to the obtained gene sequence, and the sequence is shown as SEQ ID NO. 4.

Example 2: obtaining of aflatoxin universal monoclonal antibody

The universal monoclonal antibody for resisting aflatoxin is secreted and generated by a hybridoma cell line 1C11 with the preservation number of CCTCC NO. C201013, and is prepared in advance according to a method reported in a patent with the application number of 201010245095.5, and the preparation method comprises the following steps: and injecting the obtained hybridoma cell strain 1C11 into a BALB/C mouse body which is treated by Freund incomplete adjuvant in an abdominal cavity, collecting ascites of the mouse, and purifying to obtain the anti-aflatoxin universal monoclonal antibody. Wherein, the purification method is an octanoic acid-ammonium sulfate method, and the specific operations are as follows: the ascites was thawed by taking out from a freezer at-20 ℃. Filtering ascites of mice by using double-layer filter paper, centrifuging the filtered ascites at 4 ℃ and 12000r/min for more than 15min, sucking supernatant, mixing the supernatant with 4 times of acetate buffer solution, slowly adding n-octanoic acid while stirring, wherein the volume of the n-octanoic acid required by each milliliter of ascites is 30-35 mu L, mixing at room temperature for 30-60 min, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding precipitates, filtering the obtained supernatant by using the double-layer filter paper, adding 1/10 phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration is 0.277g/mL, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding the supernatant, resuspending the obtained precipitate with 0.01mol/L phosphate buffer solution with original ascites volume of 1/10, placing into a dialysis bag, dialyzing with pure water, freezing the fully dialyzed protein solution in a refrigerator at-70 ℃, then freeze-drying with a freeze vacuum drier, collecting the freeze-dried powder to obtain a purified universal monoclonal antibody against aflatoxin, and placing the antibody in a refrigerator at-20 ℃ for later use;

Example 3: obtaining of ochratoxin A monoclonal antibody

The ochratoxin A monoclonal antibody is secreted and generated by a hybridoma cell strain 1H2 with the preservation number of CCTCC NO. C201329, and is specifically prepared in advance according to a method reported in a patent with the application number of 201310115921.8, and the preparation method comprises the following steps: injecting the hybridoma cell strain 1H2 into BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the specific operation is as follows: filtering ascites of mice with double-layer filter paper, centrifuging for 15min at 4 ℃ and 12000r/min, sucking supernatant, mixing the obtained ascites supernatant with acetate buffer solution with 4 times of volume, slowly adding n-octanoic acid under stirring, mixing 30min at room temperature with the volume of the n-octanoic acid required by each milliliter of ascites being 33 mu L, standing for 2h at 4 ℃, then centrifuging for 30min at 4 ℃ and 12000r/min, discarding precipitate, filtering the obtained supernatant with double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH value of 7.4 of 1/10 filtrate volume, adjusting the pH value of the mixed solution to 7.4 with 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration of the ammonium sulfate is 0.277g/mL, standing for 2h at 4 ℃, centrifuging for 30min at 4 ℃ and 12000r/min, discarding supernatant, and using the obtained precipitate with the original volume of 1/10 of the mixed solution, Resuspending phosphate buffer solution with the pH value of 7.4, putting into a dialysis bag, dialyzing pure water, freezing the fully dialyzed protein solution in a refrigerator at the temperature of-70 ℃, then freeze-drying by using a freeze dryer, collecting freeze-dried powder to obtain a purified anti-ochratoxin A monoclonal antibody, and placing the antibody in the refrigerator at the temperature of-20 ℃ for later use;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate; the 0.01mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate.

Example 4: obtaining monoclonal antibody of zearalenone

The zearalenone monoclonal antibody is secreted and produced by a hybridoma cell strain 2D3 with the preservation number of CCTCC NO. C201328, and is prepared in advance according to a method reported in a patent with the application number of 201310115825.3, and the preparation method comprises the following steps: injecting the hybridoma cell strain 2D3 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the specific operation steps are as follows: filtering ascites of mice with double-layer filter paper, centrifuging for 15min at 4 ℃ and 12000r/min, sucking supernatant, mixing the obtained ascites supernatant with acetate buffer solution with 4 times of volume, slowly adding n-octanoic acid under stirring, mixing 30min at room temperature with the volume of the n-octanoic acid required by each milliliter of ascites being 33 mu L, standing for 2h at 4 ℃, then centrifuging for 30min at 4 ℃ and 12000r/min, discarding precipitate, filtering the obtained supernatant with double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH value of 7.4 of 1/10 filtrate volume, adjusting the pH value of the mixed solution to 7.4 with 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration of the ammonium sulfate is 0.277g/mL, standing for 2h at 4 ℃, centrifuging for 30min at 4 ℃ and 12000r/min, discarding supernatant, and using the obtained precipitate with the original volume of 1/10 of the mixed solution, Resuspending phosphate buffer solution with pH value of 7.4, putting into a dialysis bag, dialyzing pure water, freezing the fully dialyzed protein solution in a refrigerator at-70 ℃, freeze-drying by a freeze dryer, collecting freeze-dried powder to obtain a purified monoclonal antibody against zearalenone, and placing the antibody in the refrigerator at-20 ℃ for later use;

Example 5: preparation of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone composite immunoaffinity column

1. Preparation of the substrate

The desired 1g of Sepharose lyophilized matrix powder (which forms a 3.5mL final volume of swollen matrix per gram of lyophilized matrix powder) was weighed out and dissolved in 1mmol/L HCl. The matrix will immediately swell and then be placed on a sintered glass filter and washed with 1mmol/L HCl for 15 min.

2. Ligand (antibody) conjugation

a use of coupling buffer 0.2mol/L NaHCO ₃ And (3) dissolving the universal aflatoxin monoclonal antibody, the cyclopiazonic acid monoclonal antibody, the ochratoxin A monoclonal antibody and the zearalenone monoclonal antibody to be coupled at the pH of 8.3, wherein the concentration of each monoclonal antibody is 12.5mg/mL, and temporarily storing the dissolved antibodies in an ice bath. The above antibody-containing coupling buffer was added to a hermetically sealable container with a lid. CNBr activated Sepharose was quickly transferred to the antibody solution. The mixture is fully mixed for 2 to 4 hours at room temperature (20 to 25 ℃).

b calculation of coupling ratio: centrifugation at 2,000rpm, sepharose centrifugation to the bottom of the tube, transfer of the supernatant to a new centrifuge tube, and determination of the protein content value of the supernatant. The coupling ratio was calculated to be 98.5% (indicating that the coupling was successful). Sepharose centrifuged to the bottom of the tube was washed with a coupling buffer to remove excess ligand.

c, sealing: transfer the substrate to 0.1mol/L Tris-HCl buffer. Standing for 2-4h at room temperature, and blocking all residual active groups.

d for removing the excessive ligand which is not coupled after coupling, washing the substrate by using buffers with pH values of 4 and 8, namely 0.1mol/L acetic acid/sodium acetate buffer and 0.1mol/L Tris-HCl buffer, and washing for at least 3 cycles, wherein the usage amount of each buffer is at least 5 times of the volume of the substrate. Each washing cycle step: the washing was performed with 0.1mol/L acetic acid/sodium acetate buffer, followed by 0.1mol/L Tris-HCl buffer.

e 0.01% NaN with 5 gel volumes ₃ PBS wash and preservation with 0.01% NaN 3-PBS.

3. The column was filled with a slurry prepared using a binding buffer and mixed at a ratio of 75% sedimentation matrix and 25% phosphate buffer (pH 7.0). The slurry was poured into the column in a continuous operation. The use of a glass rod that is inclined against the inside wall of the column to perform the column packing operation will help reduce the generation of bubbles. After the column is packed, the opening at the lower end of the affinity column is closed and the top member of the affinity column is removed. Carefully, PBS buffer, pH7.0, was added to the remainder of the packed affinity column to form an upward meniscus at the top of the column. The top frit was inserted into the affinity column at an angle to ensure that there was no air under the frit. The sieve plate is locked in place on the substrate surface, the opening below the affinity column is opened, and sterile filtered 0.01% NaN is applied in 5 bed volumes ₃ Column chromatography in PBS and use of 0.01% NaN ₃ And (4) storing in PBS, wherein the aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone affinity columns are filled and balanced, and can be directly used.

Example 6: detection of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone in corn

1. Detection of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone in corn

Corn addition recovery experiment: grinding negative corn sample, respectively adding 500. mu.g/kg, 1000. mu.g/kg, 2000. mu.g/kg concentration gradient cyclopiazonic acid and 10. mu.g/kg, 20. mu.g/kg, 50. mu.g/kg concentration gradient aflatoxin B ₁ Aflatoxins B ₂ Aflatoxin G ₁ Aflatoxin G ₁ Ochratoxin A and zearalenone. Five parallel runs were made for each experiment.

Three gradients:

addition amount of experiment 1: 500. mu.g/kg cyclopiazonic acid, 10. mu.g/kg aflatoxin B ₁ 10 ug/kg aflatoxin B ₂ 10 ug/kg aflatoxin G ₁ 10 ug/kg aflatoxin G ₂ 10 mug/kg ochratoxin A and 10 mug/kg zearalenone.

Addition amount of experiment 2: 1000 mug/kg cyclopiazonic acid, 20 mug/kg aflatoxin B ₁ 20 ug/kg aflatoxin B ₂ 20 ug/kg aflatoxin G ₁ 20 ug/kg aflatoxin G ₂ 20 mug/kg ochratoxin A and 20 mug/kg zearalenone.

Addition amount of experiment 3: 2000. mu.g/kg cyclopiazonic acid, 50. mu.g/kg aflatoxin B ₁ 50 ug/kg aflatoxin B ₂ 50 ug/kg aflatoxin G ₁ 50 ug/kg aflatoxin G ₂ 50 mug/kg ochratoxin A and 50 mug/kg zearalenone.

Labeling aflatoxins B in maize ₁ Aflatoxins B ₂ Aflatoxin G ₁ Aflatoxin G ₁ And (3) extracting cyclopiazonic acid, ochratoxin A and zearalenone:

weighing 5g of the standard-added corn sample, adding 20mL of 70% methanol aqueous solution by volume concentration, performing vortex oscillation extraction for 30 minutes, centrifuging to obtain a supernatant, and diluting the supernatant by 10 times with pH7.4 PBS to obtain a sample solution to be detected. The immunoaffinity column was attached under a 10.0mL glass syringe. Accurately transferring 10.0mL of sample extracting solution, injecting the sample extracting solution into a glass syringe, connecting an air pressure pump with the glass syringe, and regulating the pressure to enable the solution to slowly pass through the composite immunoaffinity column at the flow speed of about 6mL/min until 2-3 mL of air passes through the column. The column was rinsed 2 times with 10.0mL of water, all effluent discarded, and 2-3 mL of air was passed through the column. Accurately adding 1.0mL of chromatographic grade methanol for elution at the flow rate of 1 mL/min-2 mL/min, and collecting all the eluates in a glass test tube for detection.

2. Quantification of

Absorbing aflatoxin B with different concentrations by using a sample injector ₁ Aflatoxins B ₂ Aflatoxin G ₁ Aflatoxin G ₁ And injecting the standard working solution of cyclopiazonic acid, ochratoxin A and zearalenone into a high performance liquid chromatography-mass spectrometer to obtain a high performance liquid chromatography-mass spectrometer graph of each toxin and the peak area of each toxin standard solution, drawing a standard curve of each toxin, and measuring the content of each toxin by using an external standard method.

3. Results

The standard recovery rate of the corn is 87.8-103.3%, and the RSD is less than 10%. The result shows that the method completely meets the requirement of aflatoxin B in corn ₁ Aflatoxins B ₂ Aflatoxin G ₁ Aflatoxin G ₁ And analysis requirements of detection of cyclopiazonic acid, ochratoxin A and zearalenone. The results are shown in tables 1 to 7, respectively.

TABLE 1 aflatoxins B in maize ₁ Results of addition recovery

TABLE 2 aflatoxins B in maize ₂ Results of addition recovery

TABLE 3 aflatoxins G in maize ₁ Results of addition recovery

TABLE 4 aflatoxin G in maize ₂ Results of addition recovery

TABLE 5 Cyclopiazonic acid addition recovery results in corn

TABLE 6 results of recovery of ochratoxin A addition from corn

TABLE 7 zearalenone addition recovery results in corn

<110> institute of oil crop of academy of agricultural sciences of China

<120> purification aflatoxin, cyclopiazonic acid immunoadsorbent and composite affinity column

<160> 4

<210> 1

<211> 360bp

<212> DNA

<213> mice

<400> 1

gagatccagc tgcagcagtc tggacctgac ctgatgaagc ctggggcttc 50

agtgaagata tcctgcaagg cttctggtta ctcattcact acctactaca 100

tgcactgggt gaagcagagc catggaaaga gccttgagtg gattggatat 150

attgatcctt tcaatggtga tactaggtac aacccgaaat tcaaggccaa 200

ggccacattg actgtagaca aatcttccag cacagcctac atgcagctca 250

gcagcctgac atctgaggac tctgcagtct attactgtgc aagagtttat 300

tactacggta gtagctggtt tgcttactgg ggccaaggga ctctggtcac 350

tgtctctgca 360

<210> 2

<211> 322bp

<212> DNA

<213> mice

<400> 2

gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga 50

cacagtcacc atcacttgcc atgcaagtca gggcattagc agtaatatag 100

ggtggttgca gcagaaacca gggaaatcat ttaagggcct gatctatcaa 150

ggaagcaact tggaagatgg agttccatca aggttcagtg gcagtggatc 200

tggagcagat tattctctca ccatcagcag cctggaatat gaagattttg 250

cagactatta ctgtgtacag tttgctcagt ttcctcccac gttcggtgct 300

gggaccaagc tggagctgaa ac 322

<210> 3

<211> 120

<212> PRT

<213> mice

<400> 3

Glu Ile Gln Leu Gln Gln Ser Gly Pro Asp Leu Met Lys Pro Gly

1 5 10 15

Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr

20 25 30

Thr Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu

35 40 45

Glu Trp Ile Gly Tyr Ile Asp Pro Phe Asn Gly Asp Thr Arg Tyr

50 55 60

Asn Pro Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser

65 70 75

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp

80 85 90

Ser Ala Val Tyr Tyr Cys Ala Arg Val Tyr Tyr Tyr Gly Ser Ser

95 100 105

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

110 115 120

<210> 4

<211> 107

<212> PRT

<213> mice

<400> 4

Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu

1 5 10 15

Gly Asp Thr Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser

20 25 30

Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys

35 40 45

Gly Leu Ile Tyr Gln Gly Ser Asn Leu Glu Asp Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile

65 70 75

Ser Ser Leu Glu Tyr Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln

80 85 90

Phe Ala Gln Phe Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu

95 100 105

Leu Lys

107

Claims

1. The immune adsorbent for aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone is characterized in that: the immunoadsorbent comprises a solid phase carrier and an anti-aflatoxin monoclonal antibody, an anti-cyclopiazonic acid monoclonal antibody, an anti-ochratoxin A monoclonal antibody and an anti-zearalenone monoclonal antibody which are coupled with the solid phase carrier, wherein the anti-cyclopiazonic acid monoclonal antibody is secreted and generated by a hybridoma cell strain YTT-2 with the preservation number of CCTCC NO. C201871, the heavy chain variable region sequence of the anti-cyclopiazonic acid monoclonal antibody is shown as SEQ ID NO. 3, and the light chain variable region sequence is shown as SEQ ID NO. 4.

2. The immunoadsorbent of aflatoxin, cyclopiazonic acid, ochratoxin a, and zearalenone of claim 1, wherein: the anti-aflatoxin monoclonal antibody is an anti-aflatoxin universal monoclonal antibody and is secreted and generated by a hybridoma cell strain 1C11 with the preservation number of CCTCC NO. C201013; the anti-ochratoxin A monoclonal antibody is secreted and generated by a hybridoma cell strain 1H2 with the preservation number of CCTCC NO. C201329; the zearalenone resistant monoclonal antibody is secreted and generated by a hybridoma cell strain 2D3 with the preservation number of CCTCC NO. C201328.

3. A purified aflatoxin, cyclopiazonic acid, ochratoxin a and zearalenone composite affinity column loaded with the immunoadsorbent of aflatoxin, cyclopiazonic acid, ochratoxin a and zearalenone of claim 1.

4. The method for preparing the composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 3, which is characterized in that: the method comprises the following steps:

a) solid support matrix treatment

Washing CNBr-activated sepharose matrix powder with HCl under the condition of pH2-3 to remove impurities;

b) ligand coupling

Dissolving an anti-aflatoxin monoclonal antibody, an anti-cyclopiazonic acid monoclonal antibody, an anti-ochratoxin A monoclonal antibody and an anti-zearalenone monoclonal antibody to be coupled by using a coupling buffer solution to obtain antibody solutions, and quickly transferring the agarose gel substrate treated in the step a) into the antibody solutions for coupling;

c) ligand blocking

Blocking all residual reactive groups;

d) removing excess ligand that is not coupled after coupling;

e) and (5) column packing.

5. The method for preparing the composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 4, which is characterized in that: the concentration of HCl for washing in the step a) is 1mmol/L, and the washing time is 15 min; the coupling buffer in step b) was 0.2mol/L NaHCO ₃ ，pH8.3。

6. The method for preparing the affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 4, which is characterized in that: the concentration of each antibody solution in the step b) is 10-15 mg/mL; the coupling conditions were: mixing at room temperature of 20-25 deg.C for 2-4 hr.

7. The method for preparing the composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 4, which is characterized by comprising the following steps: the ligand sealing process of the step c) is as follows: transferring the agarose gel matrix treated by the step b) into 0.1mol/LTris-HCl buffer solution, and standing for 2-4h at room temperature.

8. The method for preparing the composite affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 4, which is characterized by comprising the following steps: the step d) is: washing the agarose gel matrix treated by the step c) by using buffer solutions with pH value of 4 and pH value of 8 in sequence for at least 3 cycles; the buffer solution with pH value of 4 and pH value of 8 is selected from 0.1mol/L acetic acid/sodium acetate buffer solution and 0.1mol/L Tris-HCl buffer solution respectively.

9. The method for preparing the affinity column for purifying aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone according to claim 4, which is characterized in that: after the treatment of step d), 0.01% NaN was applied in a volume of 5 times the volume of the agarose gel ₃ PBS wash and use 0.01% NaN ₃ PBS and then column packed.

10. The method for detecting the content of aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone on the basis of the purification aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone composite affinity column as claimed in claim 3, is characterized in that: when a sample containing aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone passes through the composite affinity column, the immunoadsorbent can specifically adsorb the aflatoxin, the cyclopiazonic acid, the ochratoxin A and the zearalenone, other impurities flow out of the composite affinity column, then chromatographic grade methanol is used for eluting the affinity column, eluent is collected, namely the purified and concentrated sample is detected by a high performance liquid chromatography-mass spectrometer, and the content of each toxin in the sample is analyzed.