WO2022198757A1 - 一种同时检测减肥类保健食品中三种非法添加物的广谱性抗体的制备和应用 - Google Patents

一种同时检测减肥类保健食品中三种非法添加物的广谱性抗体的制备和应用 Download PDF

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WO2022198757A1
WO2022198757A1 PCT/CN2021/091623 CN2021091623W WO2022198757A1 WO 2022198757 A1 WO2022198757 A1 WO 2022198757A1 CN 2021091623 W CN2021091623 W CN 2021091623W WO 2022198757 A1 WO2022198757 A1 WO 2022198757A1
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hapten
health food
kinds
illegal additives
detecting
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French (fr)
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雷红涛
陈沙
王锦
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华南农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Definitions

  • the invention belongs to the technical field of food safety detection. More specifically, it relates to the preparation and application of a broad-spectrum antibody for simultaneously detecting three kinds of illegal additives in diet health food.
  • Bisacodyl, deacetylbisacodyl, and sodium picosulfate are the most commonly used laxatives in clinical practice.
  • Bisacodyl mainly enhances reflex peristalsis and promotes defecation by directly contacting the mucosa and stimulating its sensory nerve endings, but excessive consumption can cause irritation or damage to the colon.
  • Deacetylbisacodyl is an impurity produced during the synthesis of bisacodyl and has the same efficacy as bisacodyl.
  • Sodium picosulfate can directly stimulate the intestinal mucosa, promote intestinal peristalsis and inhibit the absorption of water in the intestinal tract, resulting in diarrhea. Long-term use can cause abdominal pain, nausea, vomiting and other adverse symptoms.
  • the reported detection methods of this type of drugs in weight loss health food are mainly ultra-high performance liquid chromatography-tandem mass spectrometry (Song Xiaowan et al., HPLC-MS/MS method for the detection of 4 kinds of illegal additives in weight loss health care products, Food Science and Technology, Vol. 45, No. 5, 2020; Xiao Zhimin et al., Determination of 3 kinds of illegally added laxative ingredients in diet health food by ultra-high performance liquid chromatography-tandem mass spectrometry, Journal of Food Safety and Quality Inspection, No.
  • the icELISA method can detect a single object of bisacodyl and sodium picosulfate respectively. From the cross-reaction rates of other drugs in the two patents, it can be seen that the prepared antibody only meets the detection of the required object, and cannot achieve simultaneous detection. For the detection requirements of the other two illegal additives, there is currently no method for simultaneous rapid detection of bisacodyl, deacetylbisacodyl and sodium picosulfate. Therefore, it is very necessary to establish a rapid immune system for the three weight loss drugs. The key to this method is to obtain antibodies with high sensitivity to all three illegal additives. To sum up, it is very useful to prepare an antibody with strong stability and high sensitivity against the three illegal additives in weight loss health food, and to establish an efficient and rapid detection method for the simultaneous detection of three illegal additives in weight loss health food. necessary.
  • the technical problem to be solved by the present invention is to overcome the lack of detection of deacetylbisacodyl and the inability to simultaneously detect three kinds of illegally added drugs in the existing method, and to provide a wide-ranging method for simultaneously detecting three kinds of illegally added drugs in diet health food. Preparation and Application of Spectral Antibodies.
  • the purpose of the present invention is to provide a hapten 1 for simultaneously detecting three kinds of illegal additives in diet health food.
  • Another object of the present invention is to provide the application of the hapten 1 and the hapten 2 in the preparation of artificial antigens for simultaneous detection of three illegal additives in diet health food.
  • Another object of the present invention is to provide an artificial antigen for simultaneously detecting three kinds of illegal additives in slimming health food.
  • Another object of the present invention is to provide the application of the hapten 1 or the artificial antigen 1 in preparing an antibody for simultaneously detecting three kinds of illegal additives in diet health food.
  • Another object of the present invention is to provide an antibody for simultaneously detecting three kinds of illegal additives in diet health food.
  • Another object of the present invention is to provide a recombinant cell.
  • Another object of the present invention is to provide the application of the antibody or the recombinant cell in the simultaneous detection of three illegal additives in diet health food.
  • Another object of the present invention is to provide a method for simultaneously detecting three kinds of illegal additives in slimming health food.
  • the present invention provides a kind of hapten 1 for simultaneously detecting three kinds of illegal additives in diet health food, and its structural formula is shown in formula (I):
  • the hapten 1 adopts the systematic nomenclature: 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid, namely 2-(4-((4-hydroxyphenyl) (pyridin-2-yl)methyl)phenoxy)acetic acid.
  • the preparation method of the hapten 1 of the present invention is:
  • step S2 After adding the deacetylbisacodyl obtained in step S1 into a solvent to dissolve, adding ethyl bromoacetate, condensing and refluxing at 45°C to 55°C for 9 to 11 hours, purifying and eluting, and hydrolyzing to obtain a hydrolyzate;
  • step S3 Adjust acid, extract and dry the hydrolyzate obtained in step S2 to obtain the hapten 1.
  • the preparation method of the selected hapten 1 is:
  • step S2 After adding the deacetylbisacodyl obtained in step S1 into acetonitrile to dissolve, adding ethyl bromoacetate, condensing and refluxing at 45°C ⁇ 55°C for 9 ⁇ 11 hours, purifying and eluting, and hydrolyzing to obtain a hydrolyzate;
  • step S3 Adjust the pH of the hydrolyzate obtained in step S2 to 6-7, stir at room temperature for 2 hours, extract and dry to obtain the hapten 1.
  • the temperature of the condensation reflux in step S1 is 50°C, and the time is 5h.
  • the temperature of the condensation reflux in step S2 is 50°C, and the time is 10h.
  • the extraction in steps S1 and S2 is performed with ethyl acetate and water.
  • the drying in step S2 is drying with anhydrous sodium sulfate.
  • the application of the hapten 1 in the preparation of artificial antigens for simultaneously detecting three kinds of illegal additives in diet health food should also fall within the protection scope of the present invention.
  • the present invention also provides an artificial antigen 1 for simultaneously detecting three kinds of illegal additives in diet health food, and its structural formula is shown in formula (II):
  • the artificial antigen 1 is obtained by coupling the hapten 1 and the carrier protein KLH by an active ester method; the carrier is keyhole limpet hemocyanin.
  • the application of the hapten 1 or the artificial antigen 1 in preparing an antibody for simultaneously detecting three kinds of illegal additives in diet health food should also fall within the protection scope of the present invention.
  • the present invention also provides a hapten 2 for coating when simultaneously detecting three kinds of illegal additives in diet health food, and its structural formula is shown in (III):
  • the hapten 2 is named 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoic acid using systematic nomenclature, namely 6-(4-((4-hydroxyphenyl) (pyridin-2-yl)methyl)phenoxy)hexanoic acid.
  • the preparation method of the hapten 2 of the present invention is:
  • step S2 after adding the deacetylbisacodyl obtained in step S1 into a solvent to dissolve, adding ethyl 6-bromohexanoate, condensing and refluxing for 9-11 hours at 45°C to 55°C, purifying and eluting, and hydrolyzing to obtain a hydrolyzate;
  • step S3 Adjust acid, extract and dry the hydrolyzate obtained in step S2 to obtain the hapten 2.
  • the preparation method of the hapten 2 is:
  • step S2 After adding the deacetylbisacodyl obtained in step S1 into acetonitrile to dissolve, adding ethyl 6-bromohexanoate, condensing and refluxing at 45°C to 55°C for 9 to 11 hours, purifying and eluting, and hydrolyzing to obtain a hydrolyzate;
  • step S3 Adjust the pH of the hydrolyzate obtained in step S2 to 6-7, stir at 50° C. for 2 hours, extract and dry to obtain the hapten 2.
  • the temperature of the condensation reflux in step S1 is 55°C, and the time is 4h.
  • the temperature of the condensation reflux in step S2 is 50°C, and the time is 10h.
  • the extraction in steps S1 and S2 is performed with ethyl acetate and water.
  • the drying in step S2 is drying with anhydrous sodium sulfate.
  • the application of the hapten 2 in the preparation of artificial antigens/coatings for simultaneously detecting three kinds of illegal additives in diet health food should also fall within the protection scope of the present invention.
  • the present invention also provides a kind of artificial antigen 2 used for coating when detecting three kinds of illegal additives in weight loss health food at the same time, and its structural formula is shown in formula (IV):
  • the artificial antigen 2 is obtained by coupling the hapten 2 and ovalbumin by an active ester method; the carrier is ovalbumin.
  • the present invention also provides an antibody for simultaneously detecting three kinds of illegal additives in diet health food, which is prepared by immunizing New Zealand white rabbits with the complete artificial antigen 1 obtained by coupling the hapten 1 with keyhole limpet hemocyanin .
  • the present invention also provides a recombinant cell, which is a cell capable of expressing the antibody.
  • the present invention also provides an immunoassay method for simultaneously detecting three kinds of illegal additives in diet health food, using the complete artificial antigen 2 obtained by coupling the hapten 2 and ovalbumin as a coating source, and using the The complete artificial antigen 1 obtained by coupling hapten 1 with keyhole limpet hemocyanin 1 was used to immunize New Zealand white rabbits for detection. Bisacodyl and sodium picosulfate.
  • the immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, and the like.
  • the present invention has the following beneficial effects:
  • the present invention prepares two kinds of haptens: hapten 1 and hapten 2.
  • the hapten 1 is used to prepare antibodies for detecting three kinds of illegal additives in weight loss health food
  • the hapten 2 is used to prepare antibodies for Coated artificial antigen 2
  • the antibody has high sensitivity recognition ability for three kinds of illegal additives in diet health food
  • the linear detection range of sacodyl is 0.36 ⁇ 7.33 ⁇ g/kg
  • the half-inhibitory concentration IC 50 is 1.63 ⁇ g/kg
  • the minimum detection limit is 0.15 ⁇ g/kg
  • the linear detection range of deacetylbisacodyl is 0.31-12.98 ⁇ g/kg
  • the half inhibitory concentration IC50 is 1.99 ⁇ g/kg
  • the minimum detection limit is 0.10 ⁇ g/kg
  • the linear detection range for sodium picosulfate was 11.61-183.70 ⁇ g/kg
  • the IC 50 at half inhibitory concentration was 46.18 ⁇ g/kg
  • the minimum detection limit was 5.18 ⁇ g/
  • the antibodies prepared by the invention for simultaneously detecting three kinds of illegal additives in weight loss health food can meet the detection requirements.
  • the detection requirement of bisacodyl in market supervision is 5 ⁇ g/kg
  • the detection requirement of sodium picosulfate is 200 ⁇ g/kg. kg
  • the preparation method of hapten 1, hapten 2, artificial antigen and antibody in this method is simple and low in cost, which is a method for the establishment of simultaneous detection of three kinds of illegal additives in weight loss health food.
  • the immunological detection method of the compound provides the core material and has broad application prospects.
  • Fig. 1 is the synthetic route diagram of the hapten 1 of the present invention.
  • Fig. 2 is a synthetic route diagram of hapten 2 of the present invention.
  • FIG. 3 is a diagram showing the identification results of artificial antigen 1 by full-wavelength UV scanning.
  • FIG. 4 is a diagram showing the identification results of artificial antigen 2 by full-wavelength UV scanning.
  • Figure 5 is a graph of the standard curve of an antibody indirect competition ELISA for the detection of bisacodyl.
  • Figure 6 is a graph of the standard curve of an antibody indirect competition ELISA for the detection of deacetylbisacodyl.
  • Figure 7 is a graph of the standard curve of the antibody indirect competition ELISA for the detection of sodium picosulfate.
  • the present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the synthetic route of hapten 1 of the present invention is shown in FIG. 1
  • the synthetic route of hapten 2 is shown in FIG. 2 .
  • a preparation method for simultaneously detecting the hapten 1 of three kinds of illegal additives in diet health food comprising the following steps:
  • step S2 Take the deacetylbisacodyl obtained in step S1 and dissolve it in 10 mL of acetonitrile, add 40 ⁇ L of ethyl bromoacetate, and condense and reflux at 50 ° C for 10 h to obtain a brown substance, which is purified by silica gel column, and mixed solvent (ethyl acetate: volume of petroleum ether) The ratio is 1:2) elution separation, the obtained eluted product was dissolved in methanol, 1 mL of 3M sodium hydroxide aqueous solution was added to make the pH alkaline, stirred at room temperature for 2 h, extracted with ethyl acetate and water for 3 times, and then The pH was adjusted to 6-7 with 1 mL of 3M hydrochloric acid, and dried over anhydrous sodium sulfate to obtain hapten 1.
  • the hapten 1 prepared in Example 1 was used for 1H NMR identification and mass spectrometry.
  • Hapten 1 adopts the systematic nomenclature as: 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid, namely 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid -2-yl)methyl)phenoxy)acetic acid.
  • hapten 1 prepared in Example 1, add 1 mL of N,N-dimethylformamide (DMF) to dissolve, add 0.5 mg of N-hydroxysuccinimide (NHS), 1.5 mg of carbodiimide (EDC) 10 mg of keyhole limpet hemocyanin (KLH), dissolved in 1 mL of PBS buffer, to obtain solution B; add solution A to solution B dropwise, and avoid at 4°C.
  • the reaction was stirred with light for 12 h, purified by dialysis with PBS buffer for two days, and the medium was changed 4 times a day to obtain the artificial antigen conjugated with keyhole limpet hemocyanin, namely artificial antigen 1, which was packaged and stored at -20°C.
  • hapten 1, KLH and artificial antigen 1 were taken and identified by ultraviolet full wavelength scanning method (200-400 nm).
  • a preparation method for simultaneously detecting the hapten 2 of three kinds of illegal additives in diet health food comprising the following steps:
  • step S2 Take the deacetylbisacodyl obtained in step S1 and dissolve it in 10 mL of acetonitrile, add 64 ⁇ L of ethyl 6-bromohexanoate, and condense and reflux at 50 ° C for 10 h to obtain a dark purple substance, which is purified by silica gel column, mixed solvent (ethyl acetate: petroleum The volume ratio of ether is 1:2) elution separation, the obtained eluted product was dissolved in methanol, 1 mL of 3M aqueous sodium hydroxide solution was added to make the pH alkaline, stirred at 50 °C for 2 h, and extracted with ethyl acetate and water for 3 Next, adjust the pH to 6-7, and then dry with anhydrous sodium sulfate to obtain hapten 2.
  • the hapten 2 is named 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoic acid using systematic nomenclature, namely 6-(4-((4-hydroxyphenyl) (pyridin-2-yl)methyl)phenoxy)hexanoic acid.
  • hapten 2 prepared in Example 4, add 1 mL of N,N-dimethylformamide (DMF) to dissolve, add 2.4 mg of N-hydroxysuccinimide (NHS), carbodiimide (EDC) 8.0 mg, stirred at room temperature for 3 hours to obtain hapten solution A; take 10 mg of ovalbumin (OVA), add 1 mL of PBS buffer to dissolve to obtain B; add A dropwise to B, 4°C away from light The reaction was stirred for 12 h, dialyzed and purified with PBS buffer for two days, and the medium was changed 4 times a day to obtain the artificial antigen coupled with ovalbumin, namely artificial antigen 2, which was packaged and stored at -20°C.
  • DMF N,N-dimethylformamide
  • NHS N-hydroxysuccinimide
  • EDC carbodiimide
  • hapten 2 OVA and artificial antigen 2 were taken and identified by ultraviolet full wavelength scanning method (200-400 nm).
  • An antibody preparation method for simultaneously detecting three kinds of illegal additives in diet health food comprising the following steps:
  • Embodiment 7 Indirect competitive ELISA detection method of bisacodyl, deacetylbisacodyl and sodium picosulfate
  • An indirect competitive ELISA method for simultaneously detecting three illegal additives in diet health food comprising the following steps:
  • the artificial antigen 2 was used as the coating source, diluted to 1 ⁇ g/mL with the coating solution, coated with a 96-well ELISA plate, and 100 ⁇ L was added to each well, and incubated at 37°C overnight (12h);
  • the standard curve of antibody indirect competition ELISA used to detect three kinds of illegal additives in diet health food is shown in Figures 5-7.
  • the IC 50 is 1.63 ⁇ g/kg, the minimum detection limit is 0.15 ⁇ g/kg; the linear detection range for deacetylbisacodyl is 0.31-12.98 ⁇ g/kg, the half-inhibitory concentration IC 50 is 1.99 ⁇ g/kg, and the minimum detection limit is 0.10 ⁇ g/kg; the linear detection range for sodium picosulfate is 11.61-183.70 ⁇ g/kg, the IC50 of the half inhibitory concentration is 46.18 ⁇ g/kg, and the minimum detection limit is 5.18 ⁇ g/kg. It is indicated that the antibody prepared by the present invention for simultaneously detecting three kinds of illegal additives in weight loss health food can meet the detection requirements.
  • the specificity of the antibody was determined by comparing the cross-reaction experiments of sacodyl, deacetylbisacodyl, sodium picosulfate and their analogs. , the stronger the specificity.
  • Bisacodyl, deacetylbisacodyl, sodium picosulfate and its analogs are respectively diluted in multiple ratios, and the indirect competitive ELISA method is used to measure, and the steps are the same as the embodiment.
  • the sensitivity verification method of 7 was used to obtain the IC 50 value of each analog, and the cross-reaction rate (CR) was calculated according to the following formula:
  • CR(%) IC50 (desacetylbisacodyl) /IC50 (analog) ⁇ 100%.
  • the cross-reaction results of the three illegal additives in diet health food and their analogs are shown in Table 1. It can be seen that the cross-reaction rate of the antibodies used to detect the three illegal additives in diet health food to deacetylbisacodyl 100%, IC 50 of 1.99 ⁇ g/kg, 122% cross-reactivity to sacodyl, 1.63 ⁇ g/kg IC 50 , 4% cross-reactivity to sodium picosulfate, IC 50 of 46.18 ⁇ g /kg, the cross-reaction rate with diacetin, phenolphthalein, and sibutramine were all less than 1%; it can be seen that the antibody has strong specificity to the three illegal additives in the weight-loss health food in this study, and does not interact with Cross-reaction occurs with other weight loss drugs with the same effect.
  • the green plum, slimming tea and slimming capsule samples that were determined to not contain the three illegal additives in the diet health food in this study were added with standard solutions of bisacodyl, deacetylbisacodyl and sodium picosulfate to make the total concentration Addition recovery was measured at 0 ⁇ g/kg, 5 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg, and 20 ⁇ g/kg, respectively.

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Abstract

一种用于同时检测的广谱性抗体的制备方法和应用。首先提供了两种半抗原：半抗原1和半抗原2，其中半抗原1结构式如式(Ⅰ)所示；半抗原2结构式如式(Ⅲ)所示。应用半抗原1制备得到了用于同时检测减肥类保健食品中三种非法添加物的抗体，应用半抗原2制备得到了用于包被的人工抗原2，该抗体对减肥类保健食品中三种非法添加物均具有较高的灵敏度和特异性，满足市场监管所需的检出限，同时建立了一种低成本、高灵敏、稳定的同时检测减肥类保健食品中三种非法添加物的免疫分析方法，具有良好的应用前景。

Description

一种同时检测减肥类保健食品中三种非法添加物的广谱性抗体的制备和应用 技术领域
本发明属于食品安全检测技术领域。更具体地，涉及一种同时检测减肥类保健食品中三种非法添加物的广谱性抗体的制备和应用。
背景技术
比沙可啶、脱乙酰比沙可啶、匹可硫酸钠是临床上使用率高的缓泻药物。比沙可啶主要通过直接接触黏膜并刺激其感觉神经末梢而增强反射性蠕动，促进排便，但食用过量会对结肠造成刺激或损伤作用。脱乙酰比沙可啶是比沙可啶合成过程中产生的一种杂质，具有与比沙可啶同样的功效。匹可硫酸钠可直接刺激肠道黏膜，促进肠道蠕动并抑制肠道中水分的吸收，从而导致腹泻，长期服用会导致腹痛、恶心、呕吐等不良症状。随着人们生活水平的提高，人们对饮食健康和体重方面也逐渐重视，对减肥类保健食品的需求日益增加，不法商家利用三种减肥类药物致泻的功效将其添加在青梅、玫瑰茄等果脯、蜜饯类保健食品等减肥类保健品中，使达到快速减肥的目的，但对人体危害巨大。
已报道的该类药物在减肥类保健食品中的检测方法主要是超高效液相色谱-串联质谱法(宋晓婉等，HPLC-MS/MS法检测减肥类保健品中4种减肥类非法添加物，食品科技，2020年第45卷第5期；肖之敏等，超高效液相色谱-串联质谱法测定减肥类保健食品中3种违法添加的缓泻药成分的含量，食品安全质量检测学报，2020年第11卷第5期)和icELISA方法(专利CN112142651A，一种快速检测比沙可啶的半抗原BIS、人工抗原及其抗体和检测方法；专利CN112250617A，一种匹可硫酸钠半抗原、人工抗原、抗体及其制备方法和应用)，超高效液相色谱-串联质谱方法对不同的食品基质净化效果好，灵敏度、准确度和精密度高，回收率稳定，可用于实验室的检测，但该方法需要大型仪器，且仪器昂贵，需专业人员操作，无法满足市场监管局的现场快速检测的要求。icELISA方法分别有针对比沙可啶和匹可硫酸钠单一对象的检测，从两者专利中对其他药物的交叉反应率可以看出，制备得到的抗体仅满足所需对象的检测，无法达到同时检测另外两种非法添加物的检测需求，目前未有对比沙可啶、脱乙酰比沙可啶和匹可硫酸钠同时进行快速检测的方法，因此十分有必要建立一种针对三种减肥 药的快速免疫学检测方法，该方法的关键在于获得对三种非法添加物均具有高灵敏度的抗体。综上，制备一种稳定性强、灵敏度高的针对减肥类保健食品中三种非法添加物的抗体，建立一种高效快速同时检测减肥类保健食品中三种非法添加物的检测方法是十分有必要的。
发明内容
本发明要解决的技术问题是克服现有方法对脱乙酰比沙可啶检测的缺失和无法同时检测三种非法添加药物的不足，提供一种同时检测减肥类保健食品中三种非法添加物的广谱性抗体的制备和应用。
本发明的目的是提供一种同时检测减肥类保健食品中三种非法添加物的半抗原1。
本发明另一目的是提供所述半抗原1、半抗原2在制备用于同时检测减肥类保健食品中三种非法添加物的人工抗原中的应用。
本发明另一目的是提供一种用于同时检测减肥类保健食品中三种非法添加物的人工抗原。
本发明另一目的是提供所述半抗原1或所述人工抗原1在制备用于同时检测减肥类保健食品中三种非法添加物的抗体中的应用。
本发明另一目的是提供一种用于同时检测减肥类保健食品中三种非法添加物的抗体。
本发明另一目的是提供一种重组细胞。
本发明另一目的是提供所述抗体或所述重组细胞在同时检测减肥类保健食品中三种非法添加物的应用。
本发明另一目的是提供一种同时检测减肥类保健食品中三种非法添加物的方法。
本发明上述目的通过以下技术方案实现：
本发明提供了一种用于同时检测减肥类保健食品中三种非法添加物的半抗原1，其结构式如式(I)所示：
Figure PCTCN2021091623-appb-000001
所述半抗原1采用系统命名法为：2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid，即2-(4-((4-羟苯基)(吡啶-2-基)甲基)苯氧基)乙酸。
本发明所述半抗原1的制备方法为：
S1.将比沙可啶溶解于溶剂中，加入碱性水溶液，45℃～55℃冷凝回流4～6h，调酸，萃取，得到脱乙酰比沙可啶；
S2.将步骤S1所得脱乙酰比沙可啶加入溶剂中溶解后，加入溴乙酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
S3.将步骤S2所得水解产物调酸、萃取、干燥，得所述半抗原1。
优选地，所选半抗原1的制备方法为：
S1.将比沙可啶充分溶解于乙腈中，加入3mol/L的氢氧化钠水溶液，45℃～55℃冷凝回流4～6h，冷却，用1mol/L盐酸调节pH至6～7，萃取，取酯层，旋蒸后得到脱乙酰比沙可啶；
S2.将步骤S1所得脱乙酰比沙可啶加入乙腈中溶解后，加入溴乙酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
S3.将步骤S2所得水解产物调节pH至6～7，室温搅拌2h，萃取、干燥，得所述半抗原1。
优选地，步骤S1所述冷凝回流的温度为50℃，时间为5h。
优选地，步骤S2所述冷凝回流的温度为50℃，时间为10h。
优选地，步骤S1、S2所述萃取是用乙酸乙酯和水进行萃取。
优选地，步骤S2所述干燥是无水硫酸钠进行干燥。
所述半抗原1在制备用于同时检测减肥类保健食品中三种非法添加物的人 工抗原中的应用，也应在本发明的保护范围之内。
本发明还提供了一种用于同时检测减肥类保健食品中三种非法添加物的人工抗原1，其结构式如式(II)所示：
Figure PCTCN2021091623-appb-000002
优选地，所述人工抗原1由所述半抗原1与载体蛋白KLH用活泼酯法偶联得到；所述载体为钥孔血蓝蛋白。
所述半抗原1或所述人工抗原1在制备用于同时检测减肥类保健食品中三种非法添加物的抗体中的应用，也应在本发明的保护范围之内。
本发明还提供了一种用于同时检测减肥类保健食品中三种非法添加物时用于包被的半抗原2，其结构式如(Ⅲ)所示：
Figure PCTCN2021091623-appb-000003
所述半抗原2采用系统命名法命名为6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoic acid，即6-(4-((4-羟基苯基)(吡啶-2-基)甲基)苯氧基)己酸。
本发明所述半抗原2的制备方法为：
S1.将比沙可啶充分溶解于溶剂中，加入碱性水溶液，45℃～55℃冷凝回流 4～6h，调酸，萃取，得到脱乙酰比沙可啶；
S2.将步骤S1所得脱乙酰比沙可啶加入溶剂中溶解后，加入6-溴己酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
S3.将步骤S2所得水解产物调酸、萃取、干燥，得所述半抗原2。
优选地，所述半抗原2的制备方法为：
S1.将比沙可啶充分溶解于乙腈中，加入3mol/L氢氧化钠水溶液，45℃～55℃冷凝回流4～6h，冷却，用盐酸调节pH至6～7，萃取，取酯层，旋蒸后得到脱乙酰比沙可啶；
S2.将步骤S1所得脱乙酰比沙可啶加入乙腈中溶解后，加入6-溴己酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
S3.将步骤S2所得水解产物调节pH至6～7，50℃下搅拌2h，萃取、干燥，得所述半抗原2。
优选地，步骤S1所述冷凝回流的温度为55℃，时间为4h。
优选地，步骤S2所述冷凝回流的温度为50℃，时间为10h。
优选地，步骤S1、S2所述萃取是用乙酸乙酯和水进行萃取。
优选地，步骤S2所述干燥是无水硫酸钠进行干燥。
所述半抗原2在制备用于同时检测减肥类保健食品中三种非法添加物的人工抗原/包被原中的应用，也应在本发明的保护范围之内。
本发明还提供了一种同时检测减肥类保健食品中三种非法添加物时用于包被的的人工抗原2，其结构式如式(Ⅳ)所示：
Figure PCTCN2021091623-appb-000004
优选地，所述人工抗原2由所述半抗原2与卵清蛋白用活泼酯法偶联得到；所述载体为卵清蛋白。
本发明还提供了一种用于同时检测减肥类保健食品中三种非法添加物的抗体，由所述半抗原1与钥孔血蓝蛋白偶联得到的完全人工抗原1免疫新西兰大白兔制备得到。
本发明还提供了一种重组细胞，所述重组细胞为能够表达所述抗体的细胞。
所述抗体或所述重组细胞在同时检测减肥类保健食品中三种非法添加物的应用，也应在本发明的保护范围之内。
本发明还提供了一种同时检测减肥类保健食品中三种非法添加物的免疫分析方法，以所述半抗原2与卵清蛋白偶联得到的完全人工抗原2作为包被原，以所述半抗原1与钥孔血蓝蛋白偶联得到的完全人工抗原1免疫新西兰大白兔制备得到的抗体作为检测抗体进行检测；所述减肥类保健食品中三种非法添加物为比沙可啶、脱乙酰比沙可啶及匹可硫酸钠。所述免疫分析方法包括但不局限于酶免疫分析、免疫层析、免疫传感等。
与现有技术相比，本发明具有以下有益效果：
本发明制备得到了两种半抗原：半抗原1和半抗原2，应用半抗原1制备得到了用于检测减肥类保健食品中三种非法添加物的抗体，应用半抗原2制备得到了用于包被的人工抗原2，该抗体对减肥类保健食品中三种非法添加物具有高灵敏度的识别能力，对比沙可啶的线性检测范围为0.36～7.33μg/kg，半抑制浓度IC 50为1.63μg/kg，最低检测限为0.15μg/kg；对脱乙酰比沙可啶的线性检测范围为0.31～12.98μg/kg，半抑制浓度IC 50为1.99μg/kg，最低检测限为0.10μg/kg；对匹可硫酸钠的线性检测范围为11.61～183.70μg/kg，半抑制浓度IC 50为46.18μg/kg，最低检测限为5.18μg/kg。不与双醋酚丁、酚酞等减肥药物发生交叉反应。本发明制备得到的同时检测减肥类保健食品中三种非法添加物的抗体能够满足检测要求，市场监管目前对比沙可啶的检测需求为5μg/kg，对匹可硫酸钠的检测需求为200μg/kg，对脱乙酰比沙可啶无确切说明；另外，该方法中半抗原1、半抗原2、人工抗原及抗体的制备方法简单、成本低，为建立同时检测减肥类保健食品中三种非法添加物的免疫学检测方法提供了核心材料，具有广阔的应用前景。
附图说明
图1为本发明半抗原1的合成路线图。
图2为本发明半抗原2的合成路线图。
图3为人工抗原1的紫外全波长扫描鉴定结果图。
图4为人工抗原2的紫外全波长扫描鉴定结果图。
图5为用于检测比沙可啶的抗体间接竞争ELISA标准曲线图。
图6为用于检测脱乙酰比沙可啶的抗体间接竞争ELISA标准曲线图。
图7为用于检测匹可硫酸钠的抗体间接竞争ELISA标准曲线图。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明半抗原1的合成路线图如图1所示，半抗原2的合成路线如图2所示。
实施例1 半抗原1的制备
一种同时检测减肥类保健食品中三种非法添加物的半抗原1的制备方法，包括以下步骤：
S1.将1g比沙可啶溶解在10mL乙腈中，加入12mL 3M的氢氧化钠水溶液使pH呈碱性，50℃冷凝回流5h，用1M盐酸水溶液酸化，再用乙酸乙酯和水进行萃取，收集酯层，得到脱乙酰比沙可啶10.99g，转化率为99％。
S2.取步骤S1所得脱乙酰比沙可啶溶解于10mL乙腈，加入40μL溴乙酸乙酯，50℃冷凝回流10h，得到棕褐色物质，用硅胶柱纯化，混合溶剂(乙酸乙酯：石油醚的体积比为1:2)洗脱分离，将得到的洗脱产物溶于甲醇中，加入1mL 3M的氢氧化钠水溶液使pH呈碱性，室温搅拌2h，用乙酸乙酯和水萃取3次，再用1mL 3M的盐酸调节pH至6～7，无水硫酸钠进行干燥，得到半抗原1。
实施例2 半抗原1的鉴定
1、实验方法
以实施例1中的制备得到的半抗原1进行核磁共振氢谱鉴定和质谱确定。
2、实验结果
半抗原1的MS-ESI +结果如下：MS:C 20H 17NO 4:335，ESI +[M-H] +:336。
半抗原1的 1H-NMR结果如下： 1H NMR(600MHz,Methanol-d4) δ8.47(ddd,J＝5.0,1.9,0.9Hz,2H),7.80(td,J＝7.7,1.8Hz,2H),7.31(ddd,J＝7.5,5.0,1.2Hz,2H),7.19(dt,J＝7.9,1.1Hz,2H),7.06-7.01(m,4H),6.95-6.88(m,6H),6.89(d,J ＝6.7Hz,2H),6.76-6.70(m,4H),5.58(s,2H),4.85(s,39H),4.63(s,4H),4.11(q,J＝7.1Hz,1H),2.01(d,J＝13.3Hz,2H),1.35(s,1H),1.34(s,1H),1.33-1.22(m,8H),0.94-0.89(m,1H)。
根据核磁共振氢谱和质谱结果可以看出，衍生位点正确且成功，说明本发明成功制备得到了半抗原1，其结构式如式(I)所示：
Figure PCTCN2021091623-appb-000005
半抗原1采用系统命名法为：2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid，即2-(4-((4-羟苯基)(吡啶-2-基)甲基)苯氧基)乙酸。
实施例3 人工免疫抗原1的合成与鉴定
1、合成
取1mg实施例1制备得到的半抗原1，加入N,N-二甲基甲酰胺(DMF)1mL溶解，加N-羟基琥珀酰亚胺(NHS)0.5mg、碳二亚胺(EDC)1.5mg，室温搅拌反应3h，得到半抗原溶液A液；取钥孔血蓝蛋白(KLH)10mg，加1mL PBS缓冲液溶解，得到B液；将A液逐滴加到B液中，4℃避光搅拌反应12h，用PBS缓冲液透析纯化两天，每天换液4次，得到与钥孔血蓝蛋白偶联的人工抗原，即人工抗原1，分装，-20℃保存。
取上述半抗原1、KLH、人工抗原1，分别用紫外全波长扫描法(200～400nm)进行鉴定。
2、鉴定
紫外全波长扫描鉴定结果如图3所示，比较偶连前后的KLH、半抗原1、人工抗原1的吸收曲线在280nm处的吸光值可知：人工抗原1的吸收曲线在此处的吸光值与半抗原1的吸收曲线吸光值接近，明显区别于KLH的吸收曲线，且相较于KLH的吸收曲线有一定的偏移。说明半抗原1与KLH偶联成功，本发明 成功制备得到了人工免疫抗原1。
实施例4 半抗原2的合成与鉴定
1、合成步骤
一种同时检测减肥类保健食品中三种非法添加物的半抗原2的制备方法，包括以下步骤：
S1.将1g比沙可啶溶解在10mL乙腈中，加入12mL 3M的氢氧化钠水溶液使pH呈碱性，55℃冷凝回流4h，用1M盐酸水溶液酸化，再用乙酸乙酯和水进行萃取，收集酯层，得到脱乙酰比沙可啶10.99g，转化率为99％。
S2.取步骤S1所得脱乙酰比沙可啶溶解于10mL乙腈，加入64μL 6-溴己酸乙酯，50℃冷凝回流10h，得到深紫色物质，用硅胶柱纯化，混合溶剂(乙酸乙酯：石油醚的体积比为1:2)洗脱分离，将得到的洗脱产物溶于甲醇中，加入1mL3M的氢氧化钠水溶液使pH呈碱性，50℃搅拌2h，用乙酸乙酯和水萃取3次，调节pH至6～7，再用无水硫酸钠进行干燥，即得到半抗原2。
2、鉴定
半抗原2的MS-ESI +结果如下：MS:C 24H 25NO 4:391，ESI +[M-H] +:392。
半抗原2的 1H-NMR结果如下： 1H NMR(600MHz,DMSO-d6) δ8.51–8.46(m,2H),7.68(td,J＝7.7,1.9Hz,2H),7.21–7.14(m,4H),7.05(d,J＝2.0Hz,3H),7.04(s,1H),6.97–6.91(m,4H),6.83–6.78(m,4H),6.69–6.66(m,3H),5.44(s,2H),4.02(q,J＝7.1Hz,1H),3.87(t,J＝6.5Hz,4H),3.17(s,1H),2.09(s,1H),2.07(d,J＝7.4Hz,3H),1.97(s,2H),1.77(s,13H),1.65(p,J＝6.7Hz,4H),1.52(d,J＝7.6Hz,2H),1.49(d,J＝7.5Hz,2H),1.37(h,J＝7.1,6.3Hz,4H),1.16(t,J＝7.1Hz,2H)。
根据核磁共振氢谱和质谱结果可以看出，衍生位点正确且成功，说明本发明成功制备得到了半抗原2，其结构式如式(Ⅲ)所示：
Figure PCTCN2021091623-appb-000006
所述半抗原2采用系统命名法命名为6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoic acid，即6-(4-((4-羟基苯基)(吡啶-2-基)甲基)苯氧基)己酸。
实施例5 人工包被抗原2的合成与鉴定
1、合成
取5.5mg实施例4制备得到的半抗原2，加入N,N-二甲基甲酰胺(DMF)1mL溶解，加N-羟基琥珀酰亚胺(NHS)2.4mg、碳二亚胺(EDC)8.0mg，室温搅拌反应3h，得到半抗原溶液A液；取卵清蛋白(OVA)10mg，加1mL PBS缓冲液溶解，得到B液；将A液逐滴加到B液中，4℃避光搅拌反应12h，用PBS缓冲液透析纯化两天，每天换液4次，得到与卵清蛋白偶联的人工抗原，即人工抗原2，分装，-20℃保存。
取上述半抗原2、OVA、人工抗原2，分别用紫外全波长扫描法(200～400nm)进行鉴定。
2、鉴定
紫外全波长扫描鉴定结果如图4所示，比较偶连前后的OVA、半抗原2、人工抗原2的吸收曲线在280nm处的吸光值可得：人工抗原2吸收曲线在该处的吸收值为半抗原2和OVA吸收曲线在该处吸收值的叠加，明显不同于OVA的吸收曲线，说明半抗原2与OVA成功偶联，成功制备得到了人工免疫抗原2。
实施例6 抗体制备
用于同时检测减肥类保健食品中三种非法添加物的抗体制备方法，包括以下步骤：
(1)取健康的6周的新西兰大白兔两只(雌、雄各一只)，初次免疫用弗氏完全佐剂乳化后背部皮下多点注射，每只免疫剂量为500μg人工抗原1；之后每隔三周加强免疫，用弗氏不完全佐剂进行乳化，共加强免疫四次。
(2)第三次免疫后隔一周兔耳静脉取血，离心取上清，-20℃保存，用于ELISA检测免疫效果。
(3)第五次免疫后进行心脏取血，将所得兔血在37℃温育2h后，在4℃条件下放置过夜(12h)，第二天将水层清液取出，然后在4℃条件下，3000r/min离心10min，去除沉淀，获得上清液，分装，标记，-20℃保存，即得用于同时检测减肥类保健食品中三种非法添加物的抗体。
实施例7 比沙可啶、脱乙酰比沙可啶、匹可硫酸钠的间接竞争ELISA检测方法
1、实验方法
一种同时检测减肥类保健食品中三种非法添加物的间接竞争ELISA方法，包括以下步骤：
(1)将人工抗原2作为包被原，用包被液稀释至1μg/mL，包被96孔酶标板，每孔加入100μL，37℃孵育过夜(12h)；
(2)弃去包被液，洗涤2次，拍干；
(3)每孔加入120μL封闭液(即1％鱼胶蛋白)，37℃封闭30min；
(4)弃去封闭液，拍板，37℃烘干30min后取出，用自封袋装好备用；
(5)用PBST 1:8000倍稀释抗体，并将比沙可啶、脱乙酰比沙可啶、匹可硫酸钠标准品分别稀释至1000ng/mL，100ng/mL，10ng/mL，1ng/mL，0.1ng/mL，0.01ng/mL，0.001ng/mL；
(6)每孔加入50μL比沙可啶稀释液、50μL脱乙酰比沙可啶稀释液和50μL匹可硫酸钠稀释液(分别做三组平行)，最后一行不加药物稀释液，加50μLPBST，再加入50μL/孔的兔血清稀释液，37℃孵育40min，洗涤5次，拍干；
(7)加入羊抗兔二抗-HRP(4000倍稀释)，37℃温育30min，洗涤5次，拍干；
(8)加入显色液，每孔100μL，显色10min；
(9)加入50μL 10％H 2SO 4终止反应，并在450nm处读取OD值。
2、实验结果
用于检测减肥类保健食品中三种非法添加物的抗体间接竞争ELISA标准曲线如图5～7所示，可知该抗体对比沙可啶的线性检测范围为0.36～7.33μg/kg，半抑制浓度IC 50为1.63μg/kg，最低检测限为0.15μg/kg；对脱乙酰比沙可啶的线性检测范围为0.31～12.98μg/kg，半抑制浓度IC 50为1.99μg/kg，最低检测限为0.10μg/kg；对匹可硫酸钠的线性检测范围为11.61～183.70μg/kg，半抑制浓度IC 50为46.18μg/kg，最低检测限为5.18μg/kg。说明本发明制备得到的用于同时检测减肥类保健食品中三种非法添加物的抗体可以满足检测需求。
实施例8 抗体的特异性评价
1、实验方法
通过对比沙可啶、脱乙酰比沙可啶、匹可硫酸钠与其类似物进行交叉反应实 验来确定该抗体的特异性，其抗体的特异性用交叉反应率(CR)表示，交叉反应率越小，特异性越强。将比沙可啶、脱乙酰比沙可啶、匹可硫酸钠及其类似物(双醋酚丁、酚酞、西布曲明)分别作倍比稀释，采用间接竞争ELISA法进行测定，步骤同实施例7的灵敏度验证方法，得到各类似物的IC 50值，按照以下公式计算交叉反应率(CR)：
CR(％)＝IC 50(脱乙酰比沙可啶)/IC 50(类似物)×100％。
2、实验结果
减肥类保健食品中的三种非法添加物与其类似物的交叉反应结果如表1所示，可知用于检测减肥类保健食品中三种非法添加物的抗体对脱乙酰比沙可啶的交叉反应率为100％，IC 50为1.99μg/kg，对比沙可啶的交叉反应率为122％，IC 50为1.63μg/kg，对匹可硫酸钠的交叉反应率为4％，IC 50为46.18μg/kg，与双醋酚丁、酚酞、西布曲明的交叉反应率均小于1％；可以看出该抗体对本研究的减肥类保健食品中三种非法添加物的特异性较强，不与其他具有同种功效的减肥药发生交叉反应。
表1 交叉反应实验结果
Figure PCTCN2021091623-appb-000007
注：NR表示无反应。
实施例9 添加回收率
1、实验方法
采用经测定不含有本研究的减肥类保健食品中三种非法添加物的青梅、减肥茶、减肥胶囊样品，分别添加比沙可啶、脱乙酰比沙可啶、匹可硫酸钠标准溶液，使总浓度分别为0μg/kg、5μg/kg、10μg/kg、15μg/kg、20μg/kg，进行添加回收率的测定。
2、实验结果
减肥类保健食品中三种非法添加物的添加回收率结果如表2所示，可知减肥类保健食品中三种非法添加物在5种不同总浓度时的添加回收率为90％～110％。
表2 减肥类保健食品中三种非法添加物的添加回收率结果
Figure PCTCN2021091623-appb-000008
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种用于同时检测减肥类保健食品中三种非法添加物的半抗原1，其特征在于，所述半抗原1的结构式如式(Ⅰ)所示：
    Figure PCTCN2021091623-appb-100001
  2. 权利要求1所述半抗原1的制备方法，其特征在于，包括如下步骤：
    S1.将比沙可啶溶解于溶剂中，加入碱性水溶液，45℃～55℃冷凝回流4～6h，调酸，萃取，得到脱乙酰比沙可啶；
    S2.将步骤S1所得脱乙酰比沙可啶加入溶剂中溶解后，加入溴乙酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
    S3.将步骤S2所得水解产物调酸、萃取、干燥，得所述半抗原1。
  3. 权利要求1所述半抗原1在制备用于检测减肥类保健食品中三种非法添加物的人工抗原中的应用。
  4. 一种用于检测减肥类保健食品中三种非法添加物的人工抗原1，其特征在于，其结构式如式(II)所示：
    Figure PCTCN2021091623-appb-100002
    其中，KLH为钥孔血蓝蛋白。
  5. 一种用于检测减肥类保健食品中三种非法添加物的半抗原2，其特征在于，其结构式如式(Ⅲ)所示：
    Figure PCTCN2021091623-appb-100003
  6. 权利要求5所述的半抗原2的制备方法，其特征在于，包括如下步骤：
    S1.将比沙可啶充分溶解于溶剂中，加入碱性水溶液，45℃～55℃冷凝回流4～6h，调酸，萃取，得到脱乙酰比沙可啶；
    S2.将步骤S1所得脱乙酰比沙可啶加入溶剂中溶解后，加入6-溴己酸乙酯，45℃～55℃冷凝回流9～11h，纯化洗脱，水解，得到水解产物；
    S3.将步骤S2所得水解产物调酸、萃取、干燥，得所述半抗原2。
  7. 一种用于检测减肥类保健食品中三种非法添加物的包被人工抗原2，其特征在于，其结构式如式(Ⅳ)所示：
    Figure PCTCN2021091623-appb-100004
    其中，OVA为卵清蛋白。
  8. 权利要求1所述半抗原1或权利要求4所述人工抗原1在制备用于检测减肥类保健食品中三种非法添加物的抗体中的应用。
  9. 一种用于检测减肥类保健食品中三种非法添加物的抗体，其特征在于，由权利要求4所述人工抗原1免疫新西兰大白兔制备得到。
  10. 一种检测减肥类保健食品中三种非法添加物的免疫分析方法，其特征在于，以权利要求7所述包被人工抗原2为包被原，以权利要求9所述抗体作为检测抗体进行检测；所述减肥类保健食品中三种非法添加物为比沙可啶、脱乙酰比沙可啶及匹可硫酸钠。
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