WO2021093887A1 - 净化伏马毒素b1、蛇形毒素、t-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱 - Google Patents

净化伏马毒素b1、蛇形毒素、t-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱 Download PDF

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WO2021093887A1
WO2021093887A1 PCT/CN2020/129075 CN2020129075W WO2021093887A1 WO 2021093887 A1 WO2021093887 A1 WO 2021093887A1 CN 2020129075 W CN2020129075 W CN 2020129075W WO 2021093887 A1 WO2021093887 A1 WO 2021093887A1
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toxin
monoclonal antibody
zearalenone
fumonisin
affinity column
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PCT/CN2020/129075
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English (en)
French (fr)
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张奇
白艺珍
印南日
马飞
李培武
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中国农业科学院油料作物研究所
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    • G—PHYSICS
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    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • B01D15/08—Selective adsorption, e.g. chromatography
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    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
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    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3225—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product
    • B01J20/3227—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product by end-capping, i.e. with or after the introduction of functional or ligand groups
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
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Definitions

  • the invention relates to an immunoadsorbent for purifying fumonisin B1, snake-shaped toxin, T-2 toxin, zearalenone, vomitin and a composite affinity column.
  • Fumonisin B1 is a water-soluble metabolite produced by Fusarium under a certain temperature and humidity. It is a kind of diester compound composed of different polyhydric alcohols and glycerin. Fumonisin B1 can interfere with the normal physiological functions of plants in a relatively low concentration range. It is a non-enzymatic compound that is toxic to plant metabolism and belongs to mycotoxins and non-host specialized toxins. It is mainly distributed on crops such as corn, sorghum, and wheat, which can cause agricultural economic losses such as wilting of seedlings, rot of roots, stems, and seeds.
  • Fumonisin B1 can cause various specific toxicological effects on livestock, poultry and laboratory animals, such as horse and rabbit leukomalacia, which is manifested as: neurotoxicity, disturbance of consciousness, blindness and dyskinesia, etc. In severe cases, it may even cause death. It can also cause pulmonary edema and hydrothorax in pigs, liver and esophagus damage. Fumonisin B1 can also cause atherosclerosis in primates, liver cell apoptosis and nephrotoxicity in rats, lambs, and calves, as well as Liver toxicity and carcinogenic effects have brought serious economic losses to the animal husbandry industry.
  • DAS Fusarium diacetate
  • Fusarium toxin has a high level of pollution and a more harmful trichothecenes, which is more acutely toxic.
  • Snake-shaped toxin is a colorless crystal, hardly soluble in water, and soluble in polar solvents (such as methanol, etc.). The substance is very stable and will not be destroyed during cooking.
  • the snake-shaped toxin has an LDS of 0.75 mg/kg for rats. , Thermal stability is also strong.
  • snake toxins in grains and feed has only been investigated in a small amount in the United States, Germany, Italy, and India, and its content is between 0.05 and 31.5 mg/kg.
  • Snaketoxin has strong toxicity and is a fat-soluble toxin.
  • the poisoning symptoms it causes are similar to T-2 toxin, but more serious.
  • the clinical manifestations include severe dermatitis, nausea, vomiting, bloody diarrhea, bone marrow hematopoietic system damage, Nervous system disorders, anorexia and death.
  • T-2 toxin is the most toxic of the trichothecenes produced by Fusarium. It was discovered and reported by Bamburg et al. in 1968.
  • the fungi that produce T-2 toxin are mainly parasitic on the grains in the field, and most of them belong to the genus Fusarium, such as Fusarium pseudomycosporum, Fusarium pyris and Fusarium triline.
  • the most suitable environment of Cladosporium toxin-producing is 40%-50% humidity and 3-7°C; it has the strongest toxin-producing ability in corn and rye, followed by barley, rice and wheat. It can cause poisoning in animals such as chickens, pigs, rabbits, cats, rats, mice, monkeys and pins.
  • T-2 toxin may be associated with four known human diseases: one is food toxic leukopenia (ATA); the other is bone disease-Kashin-Beck disease (KBD), cartilage damage, etc. ; The third is the damage to the reproductive development system; the fourth is the DNA damage of the peripheral blood lymphocytes.
  • Zearalenone also known as F-2 toxin, was first isolated from corn with head blight.
  • the toxin-producing bacteria of zearalenone are mainly Fusarium species, such as Fusarium graminearum and Fusarium triline.
  • Zearalenone mainly pollutes corn, wheat, rice, barley, millet and oats and other grains.
  • the positive detection rate of corn is 45%, and the highest toxic content can reach 2909mg/kg; the detection rate of wheat is 20% The toxic content is 0.364 ⁇ 11.05mg/kg.
  • Zearalenone has strong heat resistance, and it is completely destroyed after treatment at 110°C for 1h.
  • Zearalenone has an estrogenic effect, which mainly acts on the reproductive system and can cause hyperestrogen in livestock, poultry and experimental mice. Animals (including humans) during pregnancy can cause zearalenone-containing foods. Miscarriage, stillbirth and teratogenesis. Eating various pasta made from wheat flour containing head blight can also cause symptoms of central nervous system poisoning, such as nausea, chills, headache, mental depression and ataxia.
  • Deoxynivalenol is a metabolite produced by the parasitic grains of Fusarium nivalis and Fusarium avenae, which often pollute wheat.
  • Deoxynivalenol also known as vomiting toxin (DON)
  • DON is a secondary metabolite of Fusarium mold.
  • DON is mostly slowly produced in cereal crops during low temperature, humidity and harvest seasons. It mainly pollutes crops such as corn and wheat. Generally, it contains high concentrations in wheat, barley, oats, and corn, and in rye, sorghum, and rice. The concentration is low, and it also pollutes food products, such as bread, biscuits, wheat melons, etc.
  • DON residues are found in animal milk and eggs. DON is less toxic, but it is the most likely to appear, so it has the highest incidence in agricultural products. The toxic effects of DON mainly affect the immune system and gastrointestinal tract of animals.
  • these mycotoxins detection methods mainly include thin layer chromatography, enzyme-linked immunoassay (ELISA), immunoaffinity chromatography-liquid chromatography, immunoaffinity chromatography-fluorescence spectrometry, etc.
  • Thin layer chromatography requires exposure to a large number of standards, which is not conducive to the health of the experimenter, and its sensitivity is very low.
  • Enzyme-linked immunosorbent assay is only suitable for qualitative testing, and false positives and false negatives are prone to occur.
  • Immunoaffinity chromatography-liquid chromatography is a combination of immune response and chromatographic analysis method, using the high specificity and affinity of antigen and antibody binding, and using chemical coupling bonding method to bind specific antibodies to chromatography adsorbents , Based on immunological reversible binding to achieve effective separation and enrichment purification of target substances in complex samples. This can specifically separate the mycotoxins in the sample, and avoid the use of toxic solvents such as chloroform and dichloromethane.
  • the preparation of a stable performance purification immunoaffinity column is to establish an economical, fast, accurate and safe fumonisin B1, snake toxin, T-2 toxin, zearalenone, and vomiting toxin multi-toxin mixed pollution solution Prerequisite for the detection method of gas chromatography.
  • the present invention provides a purification fumonisin B1, snake toxin, T-2 toxin, zearalenone, vomiting toxin immunosorbent and composite affinity column and its preparation method and application .
  • the antibody (anti-fusarium diacetate monoclonal antibody) is a monoclonal antibody secreted by the hybridoma cell line DAS5G11E7 with the deposit number CCTCC NO: C201881.
  • the hybridoma cell line DAS5G11E7 has been deposited in the China Type Culture Collection (CCTCC) on April 3, 2018, the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO.C2018
  • the solid phase carrier is agarose gel.
  • the agarose gel matrix powder activated by CNBr is washed with HCl under the condition of pH 2-3 to remove impurities;
  • CNBr activated agarose gel is provided in lyophilized form.
  • the concentration of washing HCl in step a) is 1 mmol/L, and the washing time is 15 min.
  • the coupling buffer in step b) is 0.2mol/L Na 2 HCO 3 , pH 8.3.
  • the concentration of each antibody solution in step b) is 10-15 mg/mL.
  • the coupling conditions of the step b) are: fully mixing the above mixture at room temperature (20-25° C.) for 2-4 hours.
  • the ligand blocking process of step c) is: transfer the agarose gel matrix treated in step b) to 0.1 mol/LTris-HCl buffer, and let it stand at room temperature for 2-4 hours.
  • step d) is: sequentially washing the agarose gel matrix treated in step c) with a buffer with a pH value of 4 and a pH value of 8 for at least 3 cycles;
  • the buffers with pH 4 and pH 8 can be 0.1mol/L acetic acid/sodium acetate buffer and 0.1mol/L Tris-HCl buffer respectively.
  • step e) is an agarose gel was washed 5 times the volume with 0.01% NaN 3 -PBS, using 0.01% NaN 3 -PBS save, and then packed.
  • the IC50 of the anti-deoxynivalenol (vomitin) monoclonal antibody is less than or equal to 15 ppb; the IC50 of the anti-T-2 toxin monoclonal antibody is less than or equal to 2 ppb;
  • the fumonisin B1 antibody can be selected by The monoclonal antibody secreted by the hybridoma cell line Fm7A11 with the deposit number of CCTCC NO.C201636;
  • the zearalenone antibody may be the monoclonal antibody secreted by the hybridoma cell line 2D3 with the deposit number of CCTCC NO.C201328.
  • the present invention establishes a method for detecting fumonisin B1, snake toxin, T-2 toxin, zearalenone and vomiting toxin by immunoaffinity column purification-liquid mass spectrometry, when the sample to be tested passes
  • the immunoadsorbent will specifically adsorb fumonisins B1, snake toxin, T-2 toxin, zearalenone, and vomiting toxin, and other impurities will flow out of the immunoaffinity column, and then use the chromatography Grade methanol elution affinity column, the elution flow rate is 1mL/min ⁇ 2mL/min, the fumonisin B1, snake toxin, T-2 toxin, zearalenone, and vomitin are eluted from the column, and the sample That is, a good purification is obtained, and the eluate collected from this is used for detection by a high performance liquid chromatography-mass spectrometer;
  • the immunoadsorbent will be specific The adsorbed fumonisin B1, snake toxin, T-2 toxin, zearalenone, vomiting toxin, and other impurities flow out of the immunoaffinity column, and then the affinity column is eluted with chromatographic grade methanol, and the eluate is collected That is, the purified and concentrated sample is used for detection by high performance liquid chromatography-mass spectrometry, which contains more toxins;
  • A 0.05% formic acid/water solution
  • B 0.05% formic acid/acetonitrile solution
  • the specific quantitative method can adopt the following method: use the sampler to draw different concentrations of fumonisin B1, snake toxin, T-2 toxin, zearalenone and vomiting toxin standard working solution into high performance liquid chromatography-mass spectrometry Measure the peak area of the standard solution under the above conditions, draw the standard curve of various toxins, and then use the external standard method to calculate the content of each toxin.
  • the elution flow rate is 1mL/min ⁇ 2mL/min.
  • the affinity column prepared by the present invention can be used for the high performance liquid chromatography-mass spectrometry detection of fumonisin B1, snake toxin, T-2 toxin, zearalenone, and vomitin, and its performance is stable . Furthermore, the present invention establishes an economical, fast, accurate and safe detection method based on the affinity column, which can be used for the purification and detection of these several toxin samples at the same time, and there is no mutual interference between the five kinds of toxins.
  • Figure 1 is the data of the affinity determination of Fusarium diacetate monoclonal antibody provided by the present invention.
  • Figure 2 (a) is the cross-reaction result of the fusarium diacetate monoclonal antibody provided by the present invention and other mycotoxins; (b) the second establishment of the fusarium diacetate monoclonal antibody provided by the present invention The standard curve of Fusarium enol ELISA method for Pterodactylaceae.
  • the monoclonal antibody against diacetyl fusarium enol is secreted and produced by the hybridoma cell line DAS5G11E7 with the deposit number CCTCC NO.C201881, and the preparation method is as follows:
  • the hybridoma cell line DAS5G11E7 was injected into BALB/c mice pretreated with incomplete Freund’s adjuvant, and the ascites of the mice were collected.
  • the antibody was purified by the caprylic acid-ammonium sulfate method. Rat ascites, 4°C, 12000r/min centrifugation for more than 15 minutes, aspirate the supernatant, mix the resulting ascites serum with 4 times the volume of acetate buffer, slowly add n-octanoic acid under stirring, the volume of n-octanoic acid required per ml of ascites It is 30-35 ⁇ L, mixed at room temperature for 30-60min, and allowed to stand at 4°C for more than 2h.
  • the acetate buffer solution is 0.29g sodium acetate, 0.141mL acetic acid is added to 100mL with water;
  • the 0.01mol/L phosphate buffer solution is 0.8g sodium chloride, 0.29g dodecahydrate hydrogen phosphate Disodium, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, dilute to 100mL with water;
  • the 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate , 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, add water to make up to 100mL.
  • a commercially available subtype identification kit was used to identify the subtype of the monoclonal antibody against fusarium diacetate secreted by the hybridoma cell line DAS5G11E7 as IgG2b.
  • the antibody titer obtained from the purification of mouse ascites can reach 3.2 ⁇ 10 5 , that is, the solution test result is positive when the antibody is diluted 3.2 ⁇ 10 5 times.
  • the sensitivity to fusarium diacetate was 3.08ng/mL by conventional indirect competitive ELISA.
  • the cross-reactions with other mycotoxins, T2 toxin, HT2 toxin, vomiting toxin, 3-acetyldeoxyfenacillin, ochratoxin, and fumonisin are all less than 0.01% (Table 1; Figure 2).
  • the specificity of the antibody can be evaluated by the cross-reaction rate.
  • the DAS5G11E7 monoclonal antibody was determined by the indirect competitive ELISA method.
  • DAS, T2 toxin, HT2 toxin, DON, 3-ACDON, OTA, FB 1 were prepared with a series of standard solutions of concentration, and the same volume of antibody was added to the plate and incubated 1h, other steps are the same as indirect competitive ELISA method.
  • concentration of the above toxin standard substance as the abscissa
  • the OD value B/B0 at 450 nm measured by the microplate reader as the ordinate
  • draw a competition inhibition curve and determine the cross-reaction rate by calculating the ratio of the IC50 values of DAS and other toxins. Calculated as follows:
  • CR% (IC50DAS/IC50 other toxins) ⁇ 100.
  • the affinity of DAS5G11E7 was determined by indirect non-competitive ELISA. Coat the ELISA plate with DAS-OVA at the concentration of 1.0, 0.5, 0.25, 0.125 ⁇ g/mL, 100 ⁇ L/well, 37°C, 2h; after blocking with blocking solution for 1h, the antibody diluted with PBS (dilution factor 1:2) ) Add the ELISA plate, and the other steps are the same as the indirect non-competitive ELISA method. Taking the measured OD450 value as the ordinate and the logarithmic value of the antibody concentration (mol/L) as the abscissa, 4 S-shaped curves of 4 concentrations were made.
  • the enzyme-linked immunosorbent assay (ELISA) affinity of the anti-diacetyl-fusarium enol mouse ascites antibody can reach 5.4 ⁇ 10 8 L/moL ( Figure 1).
  • mice aged 6-7 weeks were immunized with DAS-BSA, a complete antigen of fusarium diacetylenol prepared in the laboratory. After the first immunization, the complete antigen of diacetyl fusarium enol and an equal volume of complete Freund's adjuvant were emulsified, and then injected into multiple points under the skin of the back of the neck of the mouse. The second immunization was carried out 4 weeks later, with Freund's incomplete adjuvant and an equal volume of fusarium diacetylenol complete antigen emulsification, and injection into the abdominal cavity of the mouse.
  • the third immunization and the second immunization are separated by 4 weeks, and the immunization method is the same.
  • the fourth immunization is performed 3 weeks after the third immunization.
  • the immunization method is the same as the second immunization, and the same is intraperitoneal injection.
  • the same dose for the 4 immunizations was 70 ⁇ g per mouse.
  • Eight to 10 days after each immunization for the first 3 times blood was collected from the tail vein and the serum was separated, and the serum titer of the mice was detected by indirect ELISA.
  • Eight days after the third immunization blood was collected by tail-cutting, and mice corresponding to serum with relatively high titer and sensitivity were selected for the last booster immunization, and the immunization dose was twice the previous one.
  • the complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basic culture medium, 1% (weight percentage) L-glutamine, 1% (Volume percentage) HEPES, 1% (volume percentage) double antibody (10000 units per milliliter of penicillin and 10000 micrograms per milliliter of streptomycin), 2% (volume percentage) growth factor (HFCS) and 1% (weight percentage) hypoxia Purine-aminopterine-thymidine, HAT and methyl cellulose were purchased from sigma-Aldrich company.
  • the first step is to use the indirect ELISA method to screen out the positive wells that are resistant to fusarium diacetate but not the carrier protein BSA; the second step is to use the indirect competitive ELISA method to screen out the first step.
  • Extract total RNA Use Tiangen's total RNA extraction kit and follow the instructions to extract total RNA that can produce hybridoma cell line DAS5G11E7;
  • PCR method to clone variable region genes design primers according to the conserved sites of the mouse antibody gene sequence in GENBANK, and use cDNA as a template to amplify antibody heavy and light chain variable region genes.
  • the PCR program is: 94°C for 30s, 58°C for 45s, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min extension.
  • the DNA fragments were purified and recovered with the kit, and ligated into the vector pMD18-T, transformed into E. coli DH5 ⁇ competent cells, picked positive clones, and sent to Shanghai Sonny Biotech Co., Ltd. conducts sequencing.
  • the length of the gene encoding the heavy chain variable region is 351bp, and the sequence is shown in SEQ ID NO:1.
  • the sequence is shown in SEQ ID NO: 3.
  • the light chain variable region encoding gene sequence is 324 bp long, and the sequence is shown in SEQ ID NO: 2.
  • the sequence is shown in SEQ ID NO: shown in 4.
  • the anti-fumonisin B 1 monoclonal antibody is secreted and produced by the hybridoma cell line Fm7A11 with the deposit number CCTCC NO.C201636, and is specifically prepared in advance according to the method reported in the patent application number 2017101311660.
  • the preparation method is: Strain Fm7A11 was injected with BALB/c mice pre-treated with Freund’s incomplete adjuvant, the ascites of the mice were collected, and the antibody was purified by the caprylic acid-ammonium sulfate method. The specific operation was: filter the mouse ascites with double-layer filter paper.
  • Centrifuge at 12000r/min for more than 15 minutes at 12000r/min draw the supernatant, mix the resulting ascites serum with 4 times volume of acetate buffer, slowly add n-octanoic acid under stirring, the volume of n-octanoic acid required per ml of ascites is 30-35 ⁇ L , Mix at room temperature for 30-60min, and let stand at 4°C for more than 2h. Centrifuge at 12000r/min at 4°C for more than 30min, discard the precipitate, filter the supernatant obtained with double-layer filter paper, and add 1/10 of the filtrate volume to a phosphate buffer with a molar concentration of 0.1 mol/L and a pH of 7.4.
  • the acetate buffer solution is 0.29g sodium acetate, 0.141mL acetic acid is added to 100mL with water;
  • the 0.01mol/L phosphate buffer solution is 0.8g sodium chloride, 0.29g dodecahydrate hydrogen phosphate Disodium, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, dilute to 100mL with water;
  • the 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate , 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, add water to make up to 100mL.
  • the zearalenone monoclonal antibody is secreted and produced by the hybridoma cell line 2D3 with the deposit number CCTCC NO.C201328, and is specifically prepared in advance according to the method reported in the patent application number 201310115825.3.
  • the preparation method is: 2D3 injection of BALB/c mice pre-treated with Freund’s incomplete adjuvant, collect the mouse’s ascites, and use the caprylic acid-ammonium sulfate method to purify the antibody.
  • the specific operation is: filter the mouse ascites with double-layer filter paper, 4°C Centrifuge at 12000r/min for 15min, aspirate the supernatant, mix the obtained ascites serum with 3 times the volume of acetate buffer, slowly add n-octanoic acid under stirring, the volume of n-octanoic acid required per ml of ascites is 33 ⁇ L, mix at room temperature for 30min Let stand for 2h at 4°C, then centrifuge at 12000r/min for 30min at 4°C, discard the precipitate, filter the supernatant obtained with double-layer filter paper, add 1/10 of the filtrate volume to a molar concentration of 0.1mol/L and pH value 7.4 phosphate buffer solution, adjust the pH value of the mixed solution to 7.4 with 2mol/L sodium hydroxide solution, pre-cool at 4°C, slowly add ammonium sulfate until the final concentration of ammonium sulfate is 0.277g/mL, statically at
  • the fully dialyzed protein solution is placed in the refrigerator at -70°C, then lyophilized with a freeze dryer, and the lyophilized powder is collected to obtain the purified anti-zearalenone monoclonal antibody. Place the antibody in the refrigerator at -20°C for use. ;
  • the acetate buffer solution is 0.29g sodium acetate, 0.141mL acetic acid is added with water and the volume is adjusted to 100mL; the 0.1mol/L phosphate buffer solution is 0.8g sodium chloride, 0.29g dodecahydrate hydrogen phosphate Disodium, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate and dilute to 100mL with water.
  • the anti-deoxynivalenol monoclonal antibody is preferably an anti-deoxynivalenol monoclonal antibody having an IC50 of less than or equal to 15 ppb.
  • IC50 of less than or equal to 15 ppb.
  • Shandong Lvdu Biotechnology Co., Ltd., etc. the specific anti-deoxynivalenol monoclonal antibody of Shandong Lvdu Biotechnology Co., Ltd. used in this embodiment has a sensitivity IC50 of 12 ppb.
  • the anti-T-2 toxin monoclonal antibody is preferably an anti-T-2 toxin monoclonal antibody with an IC50 of less than or equal to 2 ng/mL, such as Shandong Ludu Biotechnology Co., Ltd.
  • the specific use in this example is the anti-T-2 toxin monoclonal antibody of Shandong Ludu Biotechnology Co., Ltd
  • the T-2 toxin monoclonal antibody has an IC50 value of 0.8 ng/mL.
  • d is to remove excess ligands that are not coupled after coupling, and use buffers with pH values of 4 and 8, namely 0.1 mol/L acetic acid/sodium acetate buffer and 0.1 mol/L Tris-HCl buffer to treat the substrate. Carry out washing, washing at least 3 cycles, the amount of each buffer used at least 5 times the volume of the substrate. Each washing cycle step: first wash with 0.1mol/L acetic acid/sodium acetate buffer, and then wash with 0.1mol/L Tris-HCl buffer.
  • Example 3 Detection of fumonisin B1, snake toxin, T-2 toxin, zearalenone, and vomitin in rice
  • the first experiment added 500 ⁇ g/kg fumonisin B1, 10 ⁇ g/kg snake toxin, 10 ⁇ g/kg T-2 toxin, 10 ⁇ g/kg zearalenone, 10 ⁇ g/kg vomitin.
  • the second experiment addition amount 1000 ⁇ g/kg fumonisin B 1 , 20 ⁇ g/kg snake toxin, 20 ⁇ g/kg T-2 toxin, 20 ⁇ g/kg zearalenone, 20 ⁇ g/kg vomitin.
  • the addition amount of the third experiment 2000 ⁇ g / kg fumonisin B 1, 50 ⁇ g / kg snake toxin, 50 ⁇ g / kg T-2,50 ⁇ g / kg zearalenone, 50 ⁇ g / kg deoxynivalenol.
  • A 0.05% formic acid/water solution
  • B 0.05% formic acid/acetonitrile solution
  • fumonisin B1 5000, 2500, 1000, 200, 50, 5 , 1 ⁇ g/kg
  • snake toxin 100, 50, 25, 10, 5, 1, 0.1 ⁇ g/kg
  • T-2 toxin 100, 50, 25, 10, 5, 1, 0.1 ⁇ g/kg
  • Zearalenone 100, 50, 25, 10, 5, 1, 0.1 ⁇ g/kg
  • Vomiting toxin 100, 50, 25, 10, 5, 1, 0.1 ⁇ g/kg
  • measured the peak area of the standard solution under the above conditions draw the standard curve of various toxins, and then use the external standard method to calculate the content of each toxin.
  • the recovery rates of rice spiked were all between 82.5-109.1%, and the RSD was all less than 10%.
  • the results show that the method fully meets the analytical requirements for the detection of fumonisin B1, snake toxin, T-2 toxin, zearalenone and vomitin in rice. The results are shown in Table 1 to Table 5 respectively.
  • Example 4 Detection of fumonisin B1, snake toxin, T-2 toxin, zearalenone and vomitin in edible oil
  • Edible oil spiked recovery experiment adding three concentration gradients of fumonisin B 1 of 500 ⁇ g/kg, 1000 ⁇ g/kg, 2000 ⁇ g/kg and snake toxins of three concentration gradients of 10 ⁇ g/kg, 20 ⁇ g/kg and 50 ⁇ g/kg respectively , T-2 toxin, zearalenone and vomiting toxin. For each experiment, five sets of parallel experiments are done.
  • Vegetable oil liquid sample extraction accurately weigh 5.0g vegetable oil sample into a 50mL centrifuge tube, add 15.0mL 70% methanol aqueous solution, vortex mixer, shake and mix for 2min, 5,000r/min centrifugation for 2min, pipette 10.0mL methanol solution layer , Diluted with 20.0 mL of water, mixed with a mixer, filtered through glass fiber filter paper, until the filtrate was clear. Connect the composite immunoaffinity column to a 10.0 mL glass syringe.
  • A 0.05% formic acid/water solution
  • B 0.05% formic acid/acetonitrile solution
  • the results of vegetable oil recovery rates are between 88.5-109.2%, and the RSD is less than 10%.
  • the results show that the method fully meets the analytical requirements for the detection of rice fumonisins B1, snake toxin, T-2 toxin, zearalenone and vomitin.
  • the results are shown in Table 6 to Table 10 respectively.

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Abstract

一种净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素免疫吸附剂及复合亲和柱。所述的免疫吸附剂包括固相载体和与该固相载体上偶联的伏马毒素B1单克隆抗体、蛇形毒素单克隆抗体、T-2毒素单克隆抗体、玉米赤霉烯酮单克隆抗体、呕吐毒素单克隆抗体，所述的蛇形毒素单克隆抗体为由保藏编号为CCTCC NO:C201881的杂交瘤细胞珠DAS5G11E7分泌产生的单克隆抗体。所述亲和柱可以用于伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素的高效液相色谱-质谱检测，其性能稳定。进一步地，基于此亲和柱建立了一种经济，快捷，精确，安全的检测方法，可同时用于这几种毒素样品的净化检测，五种之间无相互干涉影响。

Description

净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱 技术领域
本发明涉及一种净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素免疫吸附剂及复合亲和柱。
背景技术
伏马毒素B1是由镰刀菌属在一定的温度和湿度下产生的水溶性代谢产物，是一类由不同的多氢醇和丙三羧酸组成结构类似的双酯化合物。伏马毒素B1能在较低的浓度范围内干扰植物正常生理功能，是对植物代谢有毒害作用的非酶类化合物，属于真菌毒素和非寄主专化型毒素。主要分布在以玉米、高粱、小麦为主的农作物上，可造成秧苗枯萎，根、茎、种子腐烂等农业经济损失。伏马毒素B1对畜禽和实验动物可引起各种具有特异性的毒理作用，如马、兔的脑白质软化症，其表现为:神经性中毒，意识障碍、失明和运动失调等症状，严重者甚至造成死亡。还可造成猪的肺水肿和水胸，肝脏和食道损伤"伏马毒素B1还可引起灵长类动物的动脉粥样硬化，鼠、羔羊、小牛的肝细胞凋亡和肾毒性，还有肝毒性和致癌效应，给畜牧业带来严重经济损失。
蛇形毒素(DAS)又称二乙酸镳草镰刀菌烯醇，是镰刀菌的一些菌种的代谢产物,属于单端孢霉烯族化合物中较重要的一种单端孢霉烯族化合物是镰刀菌毒素中污染水平较高,危害较大的一类单端孢霉烯族化合物急性毒性较强。蛇形毒素为无色结晶,难溶于水,溶于极性溶剂(如甲醇等),该物质非常稳定,在烹调过程中不会被破坏蛇形毒素对大鼠的LDS为0.75mg/kg，热稳定性也较强。蛇形毒素在谷物和饲料中的污染水平仅有美国、德国、意大利、印度做了少量的调查,其含量在0.05～31.5mg/kg。蛇形毒素毒性较强,属脂溶性毒素,它引起的中毒症状与T-2毒素相似,但较其严重，中毒后临床表现为严重的皮炎、恶心、呕吐、血性腹泻、骨髓造血系统损害、神经系统紊乱厌食和死亡。
T-2毒素是镰刀菌所产生的单端孢霉烯族毒素中毒性最强的一种,它是Bamburg等在1968年发现并报告的。产生T-2毒素的真菌主要寄生在田间的谷物上,大多属于镰孢菌属，如:拟分枝孢镰刀菌,梨孢镰刀菌和三线镰刀菌等。枝孢镶刀菌的最适产毒环境为湿度40％-50％,温度为3-7℃；在玉米和黑麦中的产毒能力最强，其次为大麦、大米和小麦。可引起鸡、猪、兔、猫、大鼠、小鼠、猴 和销子等动物的中毒。其毒性主要表现在:细胞毒性、皮肤毒性、植物毒性、免疫抑制和催吐等。迄今发现，T-2毒素可能与四种已知的人类疾病有关联:一种是食物中毒性白细胞减少症(ATA)；另一种是骨病--大骨节病(KBD)、软骨损伤等；三是生殖发育系统受到损害；四是外周血淋己细胞的DNA损伤。
玉米赤霉烯酮又称F-2毒素,它首先从有赤霉病的玉米中分离得到。玉米赤霉烯酮其产毒菌主要是镰刀菌属的菌侏,如禾谷镰刀菌和三线镰刀菌。玉米赤霉烯酮主要污染玉米,小麦,大米,大麦,小米和燕麦等谷物,其中玉米的阳性检出率为45％,最高含毒量可达到2909mg/kg；小麦的检出率为20％,含毒量为0.364～11.05mg/kg。玉米赤霉烯酮的耐热性较强,110℃下处理1h才被完全破坏。玉米赤霉烯酮具有雌激素作用,主要作用于生殖系统,可使家畜,家禽和实验小鼠产生雌性激素亢进症.妊娠期的动物(包括人)食用含玉米赤霉烯酮的食物可引起流产,死胎和畸胎。食用含赤霉病麦面粉制作的各种面食也可引起中枢神经系统的中毒症状,如恶心,发冷,头痛,神智抑郁和共济失调等。
脱氧雪腐镰刀菌烯醇是由常污染小麦的雪腐镰刀菌和燕麦镰刀菌在寄生谷物的同时产生的代谢物。脱氧雪腐镰刀菌烯醇又名呕吐毒素(DON),是镰刀菌霉的次级代谢产物。DON大多在低温、潮湿和收获季节在谷物庄稼种慢慢产生,主要污染玉米、麦类等农作物,一般在小麦、大麦、燕麦、玉米种含有较高的浓度,在黑麦、高粱、大米中的浓度较低，也污染粮食制品,如面包、饼干、麦制点瓜等,另外,在动物的奶、蛋中均发现有DON残留。DON毒性较低,但它最易出现,因此在农产品中发病率最高。DON的毒性作用主要影响动物的免疫系统及胃肠道。
目前，这些真菌毒素检测方法主要有薄层层析法，酶联免疫法(ELISA)，免疫亲和层析-液相色谱法，免疫亲和层析-荧光光度法等。薄层层析法要接触大量的标准品，不利于实验者的健康，而且灵敏度很低。酶联免疫法只适用于定性检测，很容易出现假阳性和假阴性的现象。
免疫亲和层析-液相色谱法是将免疫反应与色谱分析方法相结合，利用抗原抗体结合的高度特异性和亲和力，用化学偶联键合方法将特异性抗体结合到层析吸附剂上，基于免疫学可逆结合来实现对复杂样品中目标物质的效的分离和富集净化。由此可特异性的将样品中的真菌毒素分离出来，而避免使用有毒的溶剂如氯仿和二氯甲烷。因此，制备出性能稳定的净化免疫亲和柱是建立经济， 快捷，精确，安全的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素的多毒素混合污染液相色谱检测方法的前提。
发明内容
针对现有技术的不足，本发明提供了一种净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素免疫吸附剂及复合亲和柱及其制备方法与应用。
为了实现上述目的本发明采用的技术方案是：
提供净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素免疫吸附剂，所述的免疫吸附剂包括固相载体和与该固相载体上偶联的抗伏马毒素B1单克隆抗体、抗蛇形毒素单克隆抗体、抗T-2毒素单克隆抗体、抗玉米赤霉烯酮单克隆抗体、抗呕吐毒素单克隆抗体，所述的抗蛇形毒素单克隆抗体(抗二乙酸镳草镰刀菌烯醇单克隆抗体)为由保藏编号为CCTCC NO:C201881的杂交瘤细胞株DAS5G11E7分泌产生的单克隆抗体。该杂交瘤细胞株DAS5G11E7已于2018年4月3日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO.C201881。
按上述方案，所述固相载体为琼脂糖凝胶。
提供装载有净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素抗体免疫吸附剂复合亲和柱。
净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱的制备，包括：
a)基质处理
将CNBr活化的琼脂糖凝胶基质粉末在pH2-3的条件下用HCl洗涤除杂；
CNBr活化的琼脂糖凝胶是以冻干形式提供。
b)配体偶联
使用偶联缓冲液溶解待偶联的伏马毒素B1单克隆抗体、蛇形毒素单克隆抗体、T-2毒素单克隆抗体、玉米赤霉烯酮单克隆抗体、呕吐毒素单克隆抗体，获得抗体溶液，迅速将步骤a)活化的琼脂糖凝胶基质转移到上述抗体溶液中，进行偶联；
c)配体封闭
封闭所有残留的活性基团；
d)除去偶联后未偶联上的多余的配体；
e)装柱。
按上述方案，所述步骤a)中洗涤用HCl浓度为1mmol/L，洗涤时间为15min。
按上述方案，步骤b)中的偶联缓冲液为0.2mol/L Na 2HCO 3，pH8.3。
按上述方案，步骤b)中各抗体溶液浓度为10-15mg/mL。
按上述方案，所述步骤b)的偶联条件为：室温条件(20-25℃)下充分混匀上述混和物2-4h。按上述方案，步骤c)的配体封闭过程为：转移经步骤b)处理的琼脂糖凝胶基质至0.1mol/LTris-HCl缓冲液中，室温条件下静置2-4h。
按上述方案，步骤d)为：依次用pH值为4和pH值为8的缓冲液对经步骤c)处理后的琼脂糖凝胶基质进行洗涤，至少洗涤3个循环；
pH值为4和pH值为8的缓冲液分别可选0.1mol/L醋酸/醋酸钠缓冲液和0.1mol/L Tris-HCl缓冲液。
按上述方案，步骤e)为用5倍所述琼脂糖凝胶体积的0.01％NaN 3-PBS洗涤，并使用0.01％NaN 3-PBS保存，然后装柱。
按上述方案，优选地，抗脱氧雪腐镰刀菌烯醇(呕吐毒素)单克隆抗体的IC50小于等于15ppb；抗T-2毒素单克隆抗体的IC50小于等于2ppb；伏马毒素B1抗体可选由保藏编号为CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生的单克隆抗体；玉米赤霉烯酮抗体可选由保藏编号为CCTCC NO.C201328的杂交瘤细胞株2D3分泌产生的单克隆抗体。
在此基础上本发明建立了免疫亲和柱净化-液质联用法检测伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素含量的方法，当待检测样品通过免疫亲和柱时，免疫吸附剂会特异性的吸附伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素，其他的杂质则流出免疫亲和柱，然后用色谱级甲醇洗脱亲和柱，洗脱流速1mL/min～2mL/min，将伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素从柱子中洗脱下来，样品即得到了很好的净化，由此收集的洗脱液供高效液相色谱-质谱联用仪检测用；
基于上述复合亲和柱检测伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素含量的方法，将待检测样品通过免疫亲和柱时，免疫吸附剂会特异性的吸附伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素，其他的杂质则流出免疫亲和柱，然后用色谱级甲醇洗脱亲和柱，收集洗脱液即净化浓缩后的样品供高效液相色谱-质谱联用仪检测，得多各毒素含量；
高效液相色谱-质谱联用仪条件：
a流动相：A，0.05％甲酸/水溶液；B，0.05％甲酸/乙腈溶液；
b梯度洗脱：0-3min,15％-50％B；4-5min,50％-70％B；6.5-8min,70％-100％B；8-10min,100％-50％B；10-11min,50％-15％B；11-15min,15％B.
c色谱柱：C-18柱；
d流速：150-200μL/min；
e各种毒素检测的质谱扫描参数如表1所示
表1 各种毒素的扫描参数
Figure PCTCN2020129075-appb-000001
具体定量方法可采用如下方式：用进样器吸取不同浓度的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素标准工作液注入高效液相色谱-质谱联用仪，在上述条件下测定标准溶液的峰面积，绘制各种毒素的标准曲线，然后利用外标法标算出各毒素的含量。
按上述方案，所述的洗脱流速1mL/min～2mL/min。
本发明的有益效果：本发明制备的亲和柱可以用于伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素的高效液相色谱-质谱检测，其性能稳定。 进而本发明基于此亲和柱建立了一种经济，快捷，精确，安全的检测方法，可同时用于这几种毒素样品的净化检测，五种之间无相互干涉影响。
附图说明
图1为本发明提供的二乙酸镳草镰刀菌烯醇单克隆抗体亲和力测定数据；
图2(a)为本发明提供的二乙酸镳草镰刀菌烯醇单克隆抗体与其他真菌毒素交叉反应结果；(b)本发明提供的二乙酸镳草镰刀菌烯醇单克隆抗体建立的二乙酸镳草镰刀菌烯醇酶联免疫方法标准曲线。
具体实施方式
抗二乙酰镳草镰刀菌烯醇单克隆抗体的获得
抗二乙酰镳草镰刀菌烯醇单克隆抗体由保藏编号为CCTCC NO.C201881的杂交瘤细胞株DAS5G11E7分泌产生，制备方法为：
将杂交瘤细胞株DAS5G11E7注射预先用弗氏不完全佐剂处理过的BALB/c小鼠，收集该小鼠的腹水，采用辛酸-硫酸铵法纯化抗体，具体操作为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上。12000r/min，4℃离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH至7.4，冰浴中缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后12000r/min，4℃离心30min以上，弃上清，将所得沉淀用原腹水体积1/10体积的摩尔浓度为0.01mol/L、pH为7.4的磷酸盐缓冲液重悬，装入透析袋，用0.01mol/LPBS透析两天，再改用PB透析两天，将透析袋中蛋白溶液取出，离心，收集上清，弃沉淀，放入-70℃预冻后放入冻干机中冻干。收集冻干粉，即为纯化好的抗二乙酸镳草镰刀菌烯醇单克隆抗体；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.01mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，0.02g磷酸二氢钾，加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为8g氯化钠，2.9g十二水磷酸氢二钠，0.2g氯化钾，0.2g磷酸二氢钾，加水定容到100mL所得。
用市售亚型鉴定试剂盒鉴定杂交瘤细胞株DAS5G11E7分泌的抗二乙酸镳草镰刀菌烯醇单克隆抗体的亚型为IgG2b。
用常规非竞争酶联免疫吸附法(ELISA)测得小鼠腹水纯化得到的抗体效价可达到3.2×10 5，即抗体稀释3.2×10 5倍时溶液测定结果为阳性。用常规间接竞争ELISA测定其对二乙酸镳草镰刀菌烯醇灵敏度为3.08ng/mL。与其他真菌毒素，T2毒素、HT2毒素、呕吐毒素、3-乙酰脱氧瓜萎镰菌醇、赭曲霉毒素、伏马毒素的交叉反应均小于0.01％(表1；图2)。抗体的特异性高低可用交叉反应率来评价。采用间接竞争ELISA方法测定DAS5G11E7单克隆抗体，将DAS、T2毒素、HT2毒素、DON、3-ACDON、OTA、FB 1配制系列浓度的标准溶液，分别与等体积抗体共同加入酶标板中，孵育1h，其他步骤同间接竞争ELISA方法。以上述毒素标准品浓度为横坐标，以酶标仪测定的450nm下OD值B/B0为纵坐标，绘制竞争抑制曲线，通过计算DAS与其他毒素的IC50值比值来判定交叉反应率。计算公式如下：
CR％＝(IC50DAS/IC50其他毒素)×100。
表1.DAS5G11E7与其他毒素的交叉反应.
Figure PCTCN2020129075-appb-000002
Figure PCTCN2020129075-appb-000003
利用间接非竞争ELISA测定DAS5G11E7的亲和力。用DAS-OVA按1.0、0.5、0.25、0.125μg/mL浓度包被酶标板，100μL/孔，37℃，2h；封闭液封闭1h后，将用PBS稀释好的抗体(稀释因子1:2)加入酶标板，其余步骤同间接非竞争ELISA方法。以测定的OD450值为纵坐标，抗体浓度(mol/L)的对数值为横坐标，做出4个浓度的4条S形曲线。找出每条S曲线最顶部的最大OD值即ODmax，找出每条曲线50％ODmax值对应的抗体浓度。将4个浓度任意两两一组，根据公式Ka＝(n-1)/2(n[Ab’]t-[Ab]t)计算抗体的亲和力常数，其中[Ab’]t、[Ab]t为每组中两个50％最大OD值对应的抗体浓度，n为每组中包被抗原浓度的倍数(包括1：2，1：4，1：8三个比值)，共得到6个Ka值。将得到的六个Ka值取平均，得抗二乙酰镳草镰刀菌烯醇小鼠腹水抗体酶联免疫吸附分析(ELISA)法亲和力可达5.4×10 8L/moL(图1)。
杂交瘤细胞株DAS5G11E7的筛选
1.动物免疫
采用实验室制备的二乙酰镳草镰刀菌烯醇完全抗原DAS-BSA对6-7周龄BALB/c小鼠进行免疫。第一次免疫将二乙酰镳草镰刀菌烯醇完全抗原与等体积的弗氏完全佐剂乳化后，于小鼠颈背部皮下多点注射。第二次免疫于4周后进行，采用福氏不完全佐剂与等体积的二乙酰镳草镰刀菌烯醇完全抗原乳化，于小鼠腹腔注射。第三次免疫与第二次免疫间隔4周，免疫方式与其相同，第四次免疫于第三次免疫3周后进行，免疫方式与第二次免疫相同，同样为腹腔注射。4次免疫剂量相同，均为每鼠70μg。前3次每次免疫后8～10天，尾静脉采血，分离血清，采用间接ELISA对小鼠的血清效价进行检测。第3次免疫后8天，断尾采血，选择效价、灵敏度均相对较高的血清对应的小鼠进行最后一次加强免疫，免疫剂量为前面的2倍。
2.细胞融合
加强免疫3天后，采用重量百分数为50％的聚乙二醇分子量为1450的PEG作融合剂，按常规方法进行细胞融合，具体步骤：无菌条件下脱颈处死小鼠，取出脾脏，用均质器碾碎脾脏，采用过滤网分离脾细胞，与鼠源骨髓瘤细胞SP2/0以5︰1的个数比混合，离心，用RPMI-1640基础培养液重悬混合细胞，离心，弃上清。加入50％PEG 1-2mL，共用时1分钟，贴壁加入RPMI-1640基础培养液10-20mL，离心，弃上清，管底的融合细胞用20mL含1％HAT的细胞完全培养基重悬，将悬起的细胞加入到80mL半固体培养基中，混匀后加到6孔细胞培养板上，1.5mL/孔，置于37℃二氧化碳培养箱培养。所述的含1％HAT的细胞完全培养基含有20％(体积百分数)胎牛血清，75％(体积百分数)RPMI-1640基础培养液，1％(重量百分数)L-谷氨酰胺，1％(体积百分数)HEPES，1％(体积百分数)双抗(10000单位每毫升青霉素和10000微克每毫升链霉素)，2％(体积百分数)生长因子(HFCS)和1％(重量百分数)次黄嘌呤-氨基蝶岭-胸腺嘧啶核苷即HAT和甲基纤维素购于sigma-Aldrich公司。
细胞株的筛选及克隆
待细胞融合后2-3周，细胞集落长至肉眼可见时，用微量移液器将克隆从培养基中挑出，转移至96孔细胞培养板采用HAT液体培养，待细胞长至2/3孔底时，吸取培养上清进行检测。采用两步筛选法，第一步采用间接ELISA方法，筛选出抗二乙酸镳草镰刀菌烯醇而不抗载体蛋白BSA的阳性孔；第二步采用间接竞争ELISA法对第一步筛选出的阳性孔进行检测，用二乙酸镳草镰刀菌烯醇作为竞争原，选择吸光值和灵敏度均较高的孔(吸光值较高指竞争原为0的孔即阳性对照孔的最终测定值较高，灵敏度较高指抑制率为50％时的竞争原浓度亦IC 50值较小)，采用有限稀释法进行亚克隆，亚克隆后采用同样的两步法进行检测，如此重复亚克隆4-5次后，获得杂交瘤细胞株DAS5G11E7。该杂交瘤细胞株已于2018年4月3日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO:C201881。
抗二乙酸镳草镰刀菌烯醇单克隆抗体杂交瘤细胞株DAS5G11E7抗体可变区序列测定
(1)提取总RNA：采用天根公司的总RNA提取试剂盒并按照说明书提取可产生杂交瘤细胞株DAS5G11E7的总RNA；
(2)合成cDNA：以步骤1获得的总RNA为模板，oligo(dT)15为引物，按照SuperScript TM-2II反转录酶说明书进行反转录，合成cDNA第一链；引物oligo(dT)15由Invitrogen购得；
(3)PCR法克隆可变区基因：根据GENBANK中小鼠抗体基因序列的保守位点设计引物，以CDNA为模版扩增抗体重链、轻链可变区基因。PCR程序为：94℃30s、58℃45s、72℃1min，扩增30个循环，最后72℃延伸10min。PCR产物经过1％(重量百分数)的琼脂糖凝胶电泳分离后，用试剂盒纯化回收DNA片段，连接在载体pMD18-T中，转化大肠杆菌DH5α感受态细胞，挑取阳性克隆，送至上海桑尼生物科技有限公司进行测序。其中引物的序列分别为：重链可变区引物为5’-CAG GTS MAR CTG MAG GAG TCW G-3’(22mer)和5’-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3’(28mer),其中S、M、R和W为兼并碱基，M＝A/C，R＝A/G，S＝G/C，W＝A/T，轻链可变区引物为5’-GAC ATC AAG ATG ACC CAG TCT CCA-3’(24mer)和5’-CCG TTT TAT TTC CAG CTT GGT CCC-3’(24mer)。
得到的基因序列结果：重链可变区编码基因序列长351bp，序列如SEQ ID NO:1所示，根据所获得的基因序列推导出该基因序列所编码的重链可变区由117个氨基酸组成，序列如SEQ ID NO:3所示。轻链可变区编码基因序列长324bp,序列如SEQ ID NO:2所示，根据所获得的基因序列推导出该基因序列所编码的轻链可变区由108个氨基酸组成，序列如SEQ ID NO:4所示。
抗伏马毒素B 1单克隆抗体的获得：
抗伏马毒素B 1单克隆抗体由保藏编号为CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生，具体根据申请号为2017101311660的专利中报道的方法预先制得，制备方法为：将杂交瘤细胞株Fm7A11注射预先用弗氏不完全佐剂处理过的BALB/c小鼠，收集该小鼠的腹水，采用辛酸-硫酸铵法纯化抗体，具体操作为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上。12000r/min，4℃离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH至7.4，冰浴中缓慢 加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后12000r/min，4℃离心30min以上，弃上清，将所得沉淀用原腹水体积1/10体积的摩尔浓度为0.01mol/L、pH为7.4的磷酸盐缓冲液重悬，装入透析袋，用0.01mol/LPBS透析两天，再改用PB透析两天，将透析袋中蛋白溶液取出，离心，收集上清，弃沉淀，放入-70℃预冻后放入冻干机中冻干。收集冻干粉，即为纯化好的抗伏马毒素B 1单克隆抗体；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.01mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，0.02g磷酸二氢钾，加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为8g氯化钠，2.9g十二水磷酸氢二钠，0.2g氯化钾，0.2g磷酸二氢钾，加水定容到100mL所得。
抗玉米赤霉烯酮单克隆抗体的获得
玉米赤霉烯酮单克隆抗体由保藏编号为CCTCC NO.C201328的杂交瘤细胞株2D3分泌产生，具体根据申请号为201310115825.3的专利中报道的方法预先制得，制备方法为：将杂交瘤细胞株2D3注射预先用福氏不完全佐剂处理过的BALB/c小鼠，收集该小鼠的腹水，采用辛酸-硫酸铵法纯化抗体，具体操作为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min，吸取上清，将所得腹水上清与3倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为33μL，室温混合30min，4℃静置2h，然后4℃，12000r/min离心30min，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH值为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH值至7.4，4℃预冷，缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h，然后4℃，12000r/min离心30min，弃上清，将所得沉淀用原腹水体积1/10的0.01mol/L磷酸盐缓冲液重悬，装入透析袋，对纯水透析，将充分透析好的蛋白溶液置-70℃冰箱冷冻，之后用冷冻干燥机冻干，收集冻干粉，即得纯化好的抗玉米赤霉烯酮单克隆抗体，将抗体置-20℃冰箱中备用；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容至100mL所得；所述的0.1mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，磷酸二氢钾0.02g加水定容至100mL所得。
抗脱氧雪腐镰刀菌烯醇单克隆抗体优选IC50小于等于15ppb的抗脱氧雪腐镰刀菌烯醇单克隆抗体。如山东绿都生物科技有限公司等，本实施例具体使用的为山东绿都生物科技有限公司的抗脱氧雪腐镰刀菌烯醇单克隆抗体，灵敏度IC50为12ppb。
抗T-2毒素单克隆抗体优选IC50小于等于2ng/mL的抗T-2毒素单克隆抗体，如山东绿都生物科技有限公司，本实施例具体使用的为山东绿都生物科技有限公司的抗T-2毒素单克隆抗体，经检测为IC50值为0.8ng/mL。
实例二：
伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素复合免疫亲和柱的制备
1.基质制备
称取所需1gCNBr活化的琼脂糖凝胶(Sepharose)冻干基质粉末(每克冻干基质粉末可形成3.5mL终体积的溶胀基质)，溶于1mmol/L HCl中。基质将会立即溶胀，然后置于烧结玻璃过滤器中使用1mmol/L HCl洗涤15min。
2.配体(抗体)偶联
a使用偶联缓冲液0.2mol/L NaHCO 3pH8.3溶解待偶联的上述伏马毒素B1抗体、蛇形毒素、T-2毒素抗体、玉米赤霉烯酮抗体和呕吐毒素抗体，各抗体浓度为12.5mg/mL，溶解的抗体置于冰浴中暂存。在一个带盖的可完全密封的容器中加入上述含有抗体的偶联缓冲液。迅速将CNBr活化的Sepharose转移到抗体溶液中。室温条件(20-25℃)下充分混匀上述的混和物2-4h。
b偶联率的计算：2,000rpm离心，将sepharose离心至管底，将上清液转移至新的离心管中，测定上清液的蛋白质含量值。计算偶联率为98.5％(说明偶联很成功)。取离心至管底的sepharose，使用偶联缓冲液进行洗涤，除去多余的配体。
c封闭：转移基质至0.1mol/L Tris-HCl缓冲液中。室温条件下静置2-4h，封闭所有残留的活性基团。
d为除去偶联后未偶联上的多余的配体，依次用pH值为4和8的缓冲液即0.1mol/L醋酸/醋酸钠缓冲液和0.1mol/L Tris-HCl缓冲液对基质进行洗涤，至少洗涤3个循环，每种缓冲液的使用量至少5倍基质体积。每个洗涤循环步骤：先用0.1mol/L醋酸/醋酸钠缓冲液洗涤，接着再用0.1mol/L Tris-HCl缓冲液进行洗涤。
e用5倍胶体积的0.01％NaN 3-PBS洗涤，并使用0.01％NaN 3-PBS保存。
3.装柱使用结合缓冲液制备浆液，以75％沉降基质和25％磷酸盐缓冲液(pH7.0)的比例进行混合。以连续性的操作向柱内倾入浆液。使用一个斜靠在柱内壁上的玻璃棒进行填柱操作，将有助于减少气泡的产生。填柱后，关闭亲和柱下端的开口，并取下亲和柱的顶端部件。仔细操作，使用pH7.0的PBS缓冲液加入充填亲和柱的余下部分，以在亲和柱的顶端形成一个向上的弯液面。将顶端筛板以一定的角度插入到亲和柱中，确保在筛板的下方没有空气。将筛板锁定在基质表面适当的位置上，打开亲和柱下方的开口，用5倍柱床体积的无菌过滤的0.01％NaN 3-PBS过柱，并使用0.01％NaN 3-PBS保存，至此伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素亲和柱已装填并平衡完毕，可直接供使用。
实例三：大米中的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素的检测
1.0大米中的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素的检测
大米添加回收实验，分别添加500μg/kg，1000μg/kg，2000μg/kg三个浓度梯度的伏马毒素B1和10μg/kg，20μg/kg，50μg/kg三个浓度梯度的蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素。每个实验做五组平行试验。
三个梯度：
第1个实验添加量：500μg/kg伏马毒素B1、10μg/kg蛇形毒素、10μg/kg T-2毒素、10μg/kg玉米赤霉烯酮、10μg/kg呕吐毒素。
第2个实验添加量：1000μg/kg伏马毒素B 1、20μg/kg蛇形毒素、20μg/kg T-2毒素、20μg/kg玉米赤霉烯酮、20μg/kg呕吐毒素。
第3个实验添加量：2000μg/kg伏马毒素B 1、50μg/kg蛇形毒素、50μg/kg T-2、50μg/kg玉米赤霉烯酮、50μg/kg呕吐毒素。
大米中伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素的提取：
准确称取经过磨细(粒度小于2mm)的试样20.0g于均质机中，加入100mL乙腈/水/甲酸(80+18+2)，均质高速搅拌提取2min。定量滤纸过滤，准确移取5.0mL滤液并加入15.0mL PH7.0PBS溶液稀释，用玻璃纤维滤纸过滤1～2次，至滤液澄清。将复合免疫亲和柱连接于10.0mL玻璃注射器下。准确移取10.0mL样品提 取液注入玻璃注射器中，将空气压力泵与玻璃注射器连接，调节压力使溶液以约6mL/min流速缓慢通过复合免疫亲和柱，直至2～3mL空气通过柱体。以10.0mL水淋洗柱子2次，弃去全部流出液，并使2mL～3mL空气通过柱体。准确加入1.0mL色谱级甲醇洗脱，流速为1mL/min～2mL/min，收集全部洗脱液于玻璃试管中，供检测用。
2.0高效液相色谱-质谱条件
a流动相：A，0.05％甲酸/水溶液；B，0.05％甲酸/乙腈溶液
b梯度洗脱：0-3min,15％-50％B；4-5min,50％-70％B；6.5-8min,70％-100％B；8-10min,100％-50％B；10-11min,50％-15％B；11-15min,15％B.
c色谱柱：C-18柱(柱长50mm，内径2.1mm，填料直径1.7μm)
d流速：200μL/min
e各种毒素检测的质谱扫描参数如表1所示
3.0定量
用进样器吸取不同浓度的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素标准工作液，伏马毒素B1(5000、2500、1000、200、50、5、1μg/kg)；蛇形毒素(100、50、25、10、5、1、0.1μg/kg)；T-2毒素(100、50、25、10、5、1、0.1μg/kg)；玉米赤霉烯酮(100、50、25、10、5、1、0.1μg/kg)；呕吐毒素(100、50、25、10、5、1、0.1μg/kg)注入高效液相色谱-质谱联用仪，在上述条件下测定标准溶液的峰面积，绘制各种毒素的标准曲线，然后利用外标法标算出各毒素的含量。
4.0结果
大米加标回收率结果都在82.5-109.1％之间，RSD均小于10％。结果表明该方法完全满足大米中伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素检测的分析要求。结果分别见表1-表5。
表1 大米中伏马毒素B 1添加回收率结果
Figure PCTCN2020129075-appb-000004
Figure PCTCN2020129075-appb-000005
表2 大米中蛇形毒素添加回收率结果
Figure PCTCN2020129075-appb-000006
表3 大米中T-2毒素添加回收率结果
Figure PCTCN2020129075-appb-000007
表4 大米中玉米赤霉烯酮添加回收率结果
Figure PCTCN2020129075-appb-000008
表5 大米中呕吐毒素添加回收率结果
Figure PCTCN2020129075-appb-000009
实例四：食用油中的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素的检测
1.0食用油中的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素的检测
食用油加标回收实验，分别添加500μg/kg，1000μg/kg，2000μg/kg三个浓度 梯度的伏马毒素B 1和10μg/kg，20μg/kg，50μg/kg三个浓度梯度的蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素。每个实验做五组平行试验。
食用油中伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素的提取：
植物油液态样品提取：准确称取5.0g植物油试样于50mL离心管中，加入15.0mL 70％甲醇水溶液，漩涡混合器，振荡混匀2min，5 000r/min离心2min，移取10.0mL甲醇溶液层，用20.0mL水稀释，混合器混匀，经玻璃纤维滤纸过滤，至滤液澄清。将复合免疫亲和柱连接于10.0mL玻璃注射器下。准确移取10.0mL样品提取液注入玻璃注射器中，将空气压力泵与玻璃注射器连接，调节压力使溶液以约6mL/min流速缓慢通过复合免疫亲和柱，直至2～3mL空气通过柱体。以10.0mL水淋洗柱子2次，弃去全部流出液，并使2mL～3mL空气通过柱体。准确加入1.0mL色谱级甲醇洗脱，流速为1mL/min～2mL/min，收集全部洗脱液于玻璃试管中，供检测用。
2.0高效液相色谱-质谱条件
a流动相：A，0.05％甲酸/水溶液；B，0.05％甲酸/乙腈溶液
b梯度洗脱：0-3min,15％-50％B；4-5min,50％-70％B；6.5-8min,70％-100％B；8-10min,100％-50％B；10-11min,50％-15％B；11-15min,15％B.
c色谱柱：C-18柱(柱长50mm，内径2.1mm，填料直径1.7μm)
d流速：200μL/min
e各种毒素检测的质谱扫描参数如表1所示
3.0定量
用进样器吸取不同浓度的伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素标准工作液注入高效液相色谱-质谱联用仪，在上述条件下测定标准溶液的峰面积，绘制各种毒素的标准曲线，然后利用外标法标算出各毒素的含量。
4.0结果
植物油添加回收率结果都在88.5-109.2％之间，RSD均小于10％。结果表明该方法完全满足大米伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮和呕吐毒素检测的分析要求。结果分别见表6-表10。
表6 植物油中伏马毒素B 1添加回收率结果
Figure PCTCN2020129075-appb-000010
表7 植物油中蛇形毒素添加回收率结果
Figure PCTCN2020129075-appb-000011
表8 植物油中T-2毒素添加回收率结果
Figure PCTCN2020129075-appb-000012
表9 植物油中玉米赤霉烯酮添加回收率结果
Figure PCTCN2020129075-appb-000013
表10 植物油中呕吐毒素添加回收率结果
Figure PCTCN2020129075-appb-000014

Claims (10)

  1. 净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素免疫吸附剂，其特征在于：所述的免疫吸附剂包括固相载体和与该固相载体上偶联的抗伏马毒素B1单克隆抗体、抗蛇形毒素单克隆抗体、抗T-2毒素单克隆抗体、抗玉米赤霉烯酮单克隆抗体、抗呕吐毒素单克隆抗体，所述的抗蛇形毒素单克隆抗体为由保藏编号为CCTCC NO:C201881的杂交瘤细胞珠DAS5G11E7分泌产生的单克隆抗体。
  2. 根据权利要求1所述的免疫吸附剂，其特征在于：所述固相载体为琼脂糖凝胶。
  3. 根据权利要求1所述的免疫吸附剂，其特征在于：抗呕吐毒素单克隆抗体的IC50小于等于15ppb；抗T-2毒素单克隆抗体的IC50小于等于2ppb；抗伏马毒素B1单克隆抗体选自由保藏编号为CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生的单克隆抗体；抗玉米赤霉烯酮单克隆抗体选自由保藏编号为CCTCC NO.C201328的杂交瘤细胞株2D3分泌产生的单克隆抗体。
  4. 装载有净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素抗体免疫吸附剂的净化伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱。
  5. 权利要求4所述的复合亲和柱的制备方法，其特征在于：包括：
    a)基质处理
    将CNBr活化的琼脂糖凝胶基质粉末在pH2-3的条件下用HCl洗涤除杂；
    CNBr活化的琼脂糖凝胶是以冻干形式提供。
    b)配体偶联
    使用偶联缓冲液溶解待偶联的伏马毒素B1单克隆抗体、蛇形毒素单克隆抗体、T-2毒素单克隆抗体、玉米赤霉烯酮单克隆抗体、呕吐毒素单克隆抗体，获得抗体溶液，迅速将步骤a)活化的琼脂糖凝胶基质转移到上述抗体溶液中，进行偶联；
    c)配体封闭
    封闭所有残留的活性基团；
    d)除去偶联后未偶联上的多余的配体；
    e)装柱。
  6. 根据权利要求5所述的复合亲和柱的制备方法，其特征在于：所述步骤a) 中洗涤用HCl浓度为1mmol/L，洗涤时间为15min；步骤b)中的偶联缓冲液为0.2mol/L Na 2HCO 3，pH8.3。
  7. 根据权利要求5所述的复合亲和柱的制备方法，其特征在于：步骤b)中各抗体溶液浓度为10-15mg/mL；
    所述步骤b)的偶联条件为：室温条件20-25℃下充分混匀上述混和物2-4h。
  8. 根据权利要求3所述的复合亲和柱的制备方法，其特征在于：步骤c)的配体封闭过程为：转移经步骤b)处理的琼脂糖凝胶基质至0.1mol/LTris-HCl缓冲液中，室温条件下静置2-4h；
  9. 根据权利要求5所述的复合亲和柱的制备方法，其特征在于：步骤d)为：依次用pH值为4和pH值为8的缓冲液对经步骤c)处理后的琼脂糖凝胶基质进行洗涤，至少洗涤3个循环；
    pH值为4和pH值为8的缓冲液分别可选0.1mol/L醋酸/醋酸钠缓冲液和0.1mol/L Tris-HCl缓冲液；
    经步骤d)处理后，用5倍所述琼脂糖凝胶体积的0.01％NaN 3-PBS洗涤，并使用0.01％NaN 3-PBS保存，然后装柱。
  10. 基于权利要求4的复合亲和柱检测伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素含量的方法，其特征在于：将待检测样品通过权利要求4所述的免疫亲和柱时，免疫吸附剂会特异性的吸附伏马毒素B1、蛇形毒素、T-2毒素、玉米赤霉烯酮、呕吐毒素，其他的杂质则流出免疫亲和柱，然后用色谱级甲醇洗脱亲和柱，收集洗脱液即净化浓缩后的样品供高效液相色谱-质谱联用仪检测，得多各毒素含量。
PCT/CN2020/129075 2019-11-15 2020-11-16 净化伏马毒素b1、蛇形毒素、t-2毒素、玉米赤霉烯酮、呕吐毒素复合亲和柱 WO2021093887A1 (zh)

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