WO2018032359A1 - 一种氨基甲酸乙酯半抗原组合、人工抗原组合及其制备方法与应用 - Google Patents

一种氨基甲酸乙酯半抗原组合、人工抗原组合及其制备方法与应用 Download PDF

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WO2018032359A1
WO2018032359A1 PCT/CN2016/095543 CN2016095543W WO2018032359A1 WO 2018032359 A1 WO2018032359 A1 WO 2018032359A1 CN 2016095543 W CN2016095543 W CN 2016095543W WO 2018032359 A1 WO2018032359 A1 WO 2018032359A1
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hapten
formula
urethane
artificial antigen
combination
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French (fr)
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徐振林
孙远明
罗林
杨金易
雷红涛
沈玉栋
王弘
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华南农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C269/04—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups from amines with formation of carbamate groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
    • C07C271/28—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a non-condensed six-membered aromatic ring
    • 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
    • C07K16/44—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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention relates to the technical field of food safety immunoassay, in particular to a urethane hapten combination, an artificial antigen combination, a preparation method and application thereof.
  • Ethyl Carbamate (EC), or urethane, has a molecular formula of C 3 H 7 NO 2 and is a chemical contaminant widely found in fermented foods, especially alcoholic beverages.
  • EC Ethyl Carbamate
  • urethane induced cell carcinogenesis and tumor formation in mice
  • the carcinogenicity of urethane was demonstrated in many model animals (rats, mice, hamsters, monkeys, etc.).
  • the International Agency for Research on Cancer (IARC) listed ethyl carbamate as a Class 2B carcinogenic compound.
  • Cadraney et al further confirmed that urethane can directly induce liver cancer in humans.
  • the common methods for detecting urethane at home and abroad mainly include gas chromatography, high performance liquid chromatography, gas chromatography and mass spectrometry, but these methods mainly rely on large instruments, high pretreatment requirements, complicated steps, and professional personnel.
  • the operation and detection cost are high, and it is difficult to adapt to the needs of rapid detection in many occasions, and it cannot be widely promoted in application.
  • the immunological detection method based on antigen-antibody specific binding has the advantages of simplicity, rapidity, sensitivity, and high specificity, and has been paid more and more attention. There are currently no reports of immunodetection of urethane haptens, antigens and antibodies.
  • the present invention addresses the above-discussed deficiencies of the prior art and provides a urethane hapten combination.
  • Another object of the invention is to provide a urethane artificial antigen combination.
  • a urethane hapten combination comprising an immunological hapten having a molecular structure represented by formula (I) and a coated hapten having a molecular structure represented by formula (II);
  • the immuno hapten is prepared by dropwise adding diethylpyrocarbonate to a NaHCO 3 solution having the compound of the formula (V) and stirring at room temperature. After the reaction is completed, the reaction solution is acidified, filtered, and the precipitate is washed with distilled water, and dried;
  • the coated hapten is prepared by adding diethylpyrocarbonate to an aqueous solution of the compound of the formula (VI), and stirring the reaction at room temperature overnight. After the reaction is completed, the aqueous solution is removed by rotary evaporation;
  • the molar ratio of the diethylpyrocarbonate to the compound of the formula (V) is from 1 to 2:1, and the concentration of the NaHCO 3 solution is from 0.5 to 1 mol/L.
  • the molar ratio of the diethyl pyrocarbonate to the compound of the formula (VI) is from 1 to 2:1.
  • the preparation method of the combination of the urethane artificial antigen of the formula (III) and the formula (IV), the immunological hapten represented by the formula (I) is coupled with a carrier protein to prepare an immunological artificial antigen, and the formula (II) is The coated hapten is coupled to a carrier protein to prepare a coated artificial antigen.
  • the urethane artificial antigen combination of the formula (III) and the formula (IV) is prepared by combining the immuno hapten represented by the formula (I) and the coating hapten represented by the formula (II), respectively. Reaction with N,N'-dicyclohexylcarbodiimide and N-hydroxythiosuccinimide to form an active ester; the active ester is added dropwise to the carrier protein solution for overnight reaction; the carrier protein with the hapten coupled After 3 days of dialysis purification, the artificial antigen and the artificial antigen were coated.
  • the carrier protein is keyhole limpet hemocyanin or chicken egg albumin.
  • the urethane monoclonal antibody, polyclonal antibody or genetically engineered antibody prepared by combining the urethane artificial antigen having the molecular structure represented by the formula (III) and the formula (IV).
  • the urethane monoclonal antibody is specifically prepared by immunizing an experimental animal with a urethane artificial antigen having a molecular structure represented by the formula (III) to produce an antibody specific for urethane.
  • the antibody is an immunoglobulin G which is capable of specifically immunoreacting with urethane.
  • the specific detection method may be ELISA or other immunoassay.
  • An ELISA method for detecting urethane using an immunological artificial antigen represented by formula (III) and a coated artificial antigen combination represented by formula (IV) to establish an ELISA detection method.
  • the present invention has the following beneficial effects:
  • the invention provides several urethane haptens, antigens and preparation methods and applications of the antibodies. The idea is to retain the urethane structure, introduce an arm with an aromatic structure, increase the molecular weight and aromaticity of the hapten molecule, and it is more beneficial for the hapten molecule to induce the specificity of the animal to produce urethane. antibody.
  • the artificial antigen obtained by coupling a hapten-coupled carrier protein containing a saturated carbon chain to the arm urethane as a coating can eliminate the interference of the arm antibody induced by the aromatic structure in the immuno-hapten arm.
  • the beneficial effects of the idea are as follows: the specific antibody against urethane induced by the immunization artificial antigen represented by formula (III), and the artificial antigen represented by formula (IV) is used as a coating to establish the detection of carbamate.
  • Ethyl acetate immunoassay method enables high-throughput screening of urethane in food to improve detection efficiency and reduce detection cost.
  • the urethane artificial antigen is divided into a coating original and an immunogen according to the type of the coupled carrier protein, the carrier protein of the immunogen adopts keyhole limpet hemocyanin (KLH), and the original carrier protein is coated with chicken.
  • KLH keyhole limpet hemocyanin
  • Ovalbumin (OVA) Ovalbumin
  • the imide (NHS) was stirred under magnetic stirring at 4 ° C overnight, and the supernatant was centrifuged to be a solution A.
  • 20 mg of KLH was weighed and dissolved in 2 mL of PBS (pH 8.0) having a concentration of 0.1 mol/L, and dissolved and dissolved to prepare a solution B. Under magnetic stirring, the A solution was gradually dropped into the B solution and reacted at 4 ° C for 12 h.
  • the immunogen prepared in Example 4 was diluted to 2 mg/mL with physiological saline, and an equal volume of the immunoadjuvant (the first immunization with Freund's complete adjuvant, and later boosted with Freund's incomplete adjuvant) with a stirrer emulsification. After complete emulsification, 7-week-old female Baba/c mice were immunized with various subcutaneous injections, subcutaneous, leg muscles and ear vein veins, and each mouse was immunized with 100 ⁇ L. The first immunization was boosted every three weeks after a month. After the fourth booster immunization, blood was collected at the end of the 1st stage, and antiserum was extracted.
  • Serum titers and inhibition were determined by indirect ELISA, and the results are shown in Table 1.
  • Inhibition rate (ODmax - ODx) / ODmax ⁇ 100%; ODx is the absorbance value of the 1 mg/mL urethane hole added; ODmax is the absorbance value without adding the urethane hole.
  • monoclonal antibodies were prepared by selecting the mouse with the best serum suppression (antigen II immunized mice).
  • Splenocytes of Babl/c mice were mixed with myeloma cells SP2/0 in a volume ratio of 5:1.
  • PEG polyethylene glycol
  • the reaction was stopped by adding the basal medium, and the supernatant was removed by centrifugation at 1000 rpm/min for 10 min.
  • the cells were resuspended in 50 mL of HAT.
  • the cells were plated in 96-well plates containing feeder cells, 100 ⁇ L per well, and cultured in a carbon dioxide incubator at 37 °C. When the cells grew to 1/3 of the area of the bottom hole, the cell supernatant was taken for indirect competitive ELISA and positive wells were screened. The positive wells were subjected to limiting dilution of the subcloned positive cells until a cell line stably secreting specific for the urethane monoclonal antibody was obtained. The cell strain was expanded and passaged.
  • mice were sensitized with liquid paraffin, and the collected hybridoma cell lines were washed with basal medium and injected into the peritoneal cavity of the mice. One week later, the mice were inactive and the abdominal cavity was enlarged.
  • the ascites collected was crudely purified by caprylic acid-saturated ammonium sulfate method, and the purified ascites was stored in an environment of -20 °C. Ascites was dialyzed against 0.01 mol/L PBS at 4 °C before use.
  • a and D represent the absorbance (OD) of the drug concentration minimum and maximum, respectively, C is the midpoint concentration, and the OD value when the standard concentration is equal to C is (A+D)/2, which is at the inflection point of the curve.
  • the half-inhibition concentration is IC 50
  • B indicates the steepness of the curve, and is called the slope factor
  • IC 10 is the detection limit
  • IC 20 to IC 80 are the detection ranges.
  • the standard curve of ELISA was established with urethane standard. The results are shown in Figure 2. The relevant standard curve parameters are shown in Table 2;

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Abstract

一种氨基甲酸乙酯人工抗原组合、人工抗原组合及其制备方法与应用。所述氨基甲酸乙酯人工抗原组合具有式(Ⅲ)和式(Ⅳ)所示分子结构，该人工抗原得到的抗血清的效价可达到1.28×10 5，最低检测限为0.16mg/L，半抑制浓度为1.19mg/L，制备的抗体特异性高，灵敏度高，准确度高，可建立检测氨基甲酸乙酯的免疫分析方法，实现对食品中氨基甲酸乙酯进行高通量筛查，提高检测效率，降低检测成本。

Description

一种氨基甲酸乙酯半抗原组合、人工抗原组合及其制备方法与应用 技术领域
本发明涉及食品安全免疫检测技术领域，具体地，涉及一种氨基甲酸乙酯半抗原组合、人工抗原组合及其制备方法与应用。
背景技术
氨基甲酸乙酯(Ethyl Carbamate，简称EC)或称为尿烷(Urethane)，分子式为C3H7NO2，是一种广泛存在于发酵食品尤其是酒精饮料中的化学污染物。1943年Nettleship等发现氨基甲酸乙酯可以导致小鼠细胞癌变和肿瘤的形成后，在许多模式动物(大鼠、小鼠、仓鼠和猴子等)试验中都证明了氨基甲酸乙酯的致癌性。1974年国际癌症研究机构(IARC)将氨基甲酸乙酯列为2B类的致癌化合物。1993年Cadraney等进一步证实氨基甲酸乙酯可以直接诱发人患肝癌，因此，2007年国际癌症研究机构(IARC)进一步将氨基甲酸乙酯的致癌等级由2B提升到了2A。随着检测技术的发展，越来越多发酵食品尤其是酒精饮品中陆续检测出氨基甲酸乙酯。由于氨基甲酸乙酯的致癌性，很多国家对酒精饮品中的氨基甲酸乙酯残留都做出严格规定。例如，加拿大早在1985就限定佐餐葡萄酒中的氨基甲酸乙酯含量不得超过30ng/mL，加烈葡萄酒100ng/mL、蒸馏酒150ng/mL、清酒200ng/mL、白兰地400ng/mL。随后，美国、法国、德国等也都设立了相关标准。目前，我国暂未正式制定公布氨基甲酸乙酯限量标准，但食品安全部门已经在认真的考虑中，相信不久将来就会出台相关的氨基甲酸乙酯残留限量标准条例。
目前，国内外检测氨基甲酸乙酯的常用方法主要有气相色谱、高效液相色谱、气相色谱与-质谱等方法，但这些方法样品主要依赖大型仪器、预处理要求高、步骤复杂、需专业人员操作、检测费用高、很难适应许多场合快速检测的需要，在应用上不能广泛推广。基于抗原抗体特异性结合的的免疫学检测方法具有简便、快速、灵敏、高特异性等优点，越来越被关注。目前尚无免疫检测的氨基甲酸乙酯半抗原、抗原和抗体的相关报道。
发明内容
本发明针对现有技术的上述不足，提供一种氨基甲酸乙酯半抗原组合。
本发明的另一个目的是提供一种氨基甲酸乙酯人工抗原组合。
本发明的再一个目的是提供一种氨基甲酸乙酯半抗原组合的制备方法。
本发明的再一个目的是提供一种氨基甲酸乙酯人工抗原组合的制备方法。
本发明的再一个目的是提供一种氨基甲酸乙酯抗体。
本发明的上述目的是通过以下技术方案予以实现的。
一种氨基甲酸乙酯半抗原组合，包括免疫半抗原和包被半抗原，免疫半抗原具有式(I)所示分子结构，包被半抗原具有式(Ⅱ)所示分子结构；
Figure PCTCN2016095543-appb-000001
式(I)；其中m＝0～2；
Figure PCTCN2016095543-appb-000002
式(Ⅱ)；其中：n＝1～5。
如上所述氨基甲酸乙酯半抗原组合对应的人工抗原组合，包括免疫人工抗原和包被人工抗原，免疫人工抗原具有式(Ⅲ)所示分子结构，包被人工抗原具有式(Ⅳ)所示分子结构；
Figure PCTCN2016095543-appb-000003
式(Ⅲ)，其中m＝0～2；
Figure PCTCN2016095543-appb-000004
式(Ⅳ)，其中n＝1～5。
优选地，如上所述氨基甲酸乙酯半抗原组合的制备方法，免疫半抗原的制备 方法为：将焦碳酸二乙酯滴加到具有式(Ⅴ)所示化合物的NaHCO3溶液中，室温搅拌反应过夜，反应结束后将反应液调酸，过滤，用蒸馏水洗涤沉淀，烘干即得；
Figure PCTCN2016095543-appb-000005
式(Ⅴ)；其中m＝0～2；
包被半抗原的制备方法为：将焦碳酸二乙酯滴加到式(Ⅵ)所示化合物的水溶液中，室温搅拌反应过夜，反应完后，旋蒸除去水溶液即得；
Figure PCTCN2016095543-appb-000006
式(Ⅵ)；其中n＝1～5。
更优选地，所述焦碳酸二乙酯与式(Ⅴ)所示化合物的摩尔比为1～2:1，NaHCO3溶液的浓度为0.5～1mol/L。
更优选地，所述焦碳酸二乙酯与式(Ⅵ)所示化合物的摩尔比为1～2:1。
式(Ⅲ)和式(Ⅳ)所述氨基甲酸乙酯人工抗原组合的制备方法，将式(I)所示的免疫半抗原与载体蛋白偶联制备得到免疫人工抗原，将式(Ⅱ)所示的包被半抗原与载体蛋白偶联制备得到包被人工抗原。
优选地，式(Ⅲ)和式(Ⅳ)所述氨基甲酸乙酯人工抗原组合的制备方法为：将式(I)所示的免疫半抗原和式(Ⅱ)所示的包被半抗原分别与N,N’-二环己基碳二亚胺和N-羟基硫代琥珀酰亚胺反应形成活泼酯；将活泼酯滴加入载体蛋白溶液中，反应过夜；偶连有半抗原的载体蛋白经过3天的透析纯化，即得免疫人工抗原和包被人工抗原。
更优选地，所述载体蛋白为钥孔血蓝蛋白或鸡卵白蛋白。
采用式(Ⅲ)和式(Ⅳ)所示分子结构的氨基甲酸乙酯人工抗原组合制备得到的氨基甲酸乙酯单克隆抗体、多克隆抗体或基因工程抗体。所述氨基甲酸乙酯单克隆抗体的具体制备方法为：用式(Ⅲ)所示分子结构的氨基甲酸乙酯人工抗原免疫实验动物，使其机体产生特异地针对氨基甲酸乙酯的抗体，所述抗体是能与氨基甲酸乙酯发生特异性免疫反应的免疫球蛋白G。
式(Ⅲ)和式(Ⅳ)所示分子结构的氨基甲酸乙酯人工抗原组合在检测氨基 甲酸乙酯中的应用，具体的检测方法可以是ELISA法或其他的免疫检测法。
一种氨基甲酸乙酯的ELISA检测方法，利用式(Ⅲ)所示免疫人工抗原和式(Ⅳ)所示包被人工抗原组合，建立ELISA检测方法。
与现有技术相比，本发明具有以下有益效果：
当前检测氨基甲酸乙酯的方法大多基于液相、气相色谱及其与质谱联用的仪器检测方法，这些方法成本高，样品前处理复杂。目前，还未发现针对氨基甲酸乙酯免疫检测技术的报道。本发明提供了几种氨基甲酸乙酯半抗原、抗原及抗体制备方法及应用。其思路是保留氨基甲酸乙酯结构的前提下，引入带有芳香性结构的手臂，增加半抗原分子的分子量及芳香性，更有益于半抗原分子诱导动物机体产生识别氨基甲酸乙酯的特异性抗体。以含饱和碳链为手臂氨基甲酸乙酯的半抗原偶联载体蛋白得到的人工抗原作为包被原，可以消除免疫半抗原手臂中芳香结构诱导产生的臂抗体的干扰。该思路的有益效果体现在：以式(Ⅲ)所示免疫人工抗原诱导产生的针对氨基甲酸乙酯的特异性抗体，以式(Ⅳ)所示人工抗原作为包被原，可建立检测氨基甲酸乙酯的免疫分析方法，实现对食品中氨基甲酸乙酯进行高通量筛查，提高检测效率，降低检测成本。
附图说明
图1为半抗原AⅠ(m＝1)及其免疫抗原、包被抗原紫外扫描图。
图2为以载体蛋白为KLH的人工抗原I(m＝1,KLH为载体蛋白)为免疫原所制备的单克隆抗体在以人工抗原Ⅱ(n＝3，OVA为载体蛋白)对氨基甲酸乙酯的ELISA抑制曲线。
具体实施方式
下面结合说明书附图和具体实施例对本发明做进一步详细说明，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。
实施例1 氨基甲酸乙酯半抗原AⅠ(m＝0)制备方法
将1.37g 4-氨基苯甲酸溶于30mL的1mol/L的碳酸氢钠溶液，向该溶液滴加焦碳酸二乙酯2mL，室温搅拌反应过夜，反应完后，将反应混合液调酸至pH为1～2，有白色沉淀析出，过滤沉淀，蒸馏水洗涤烘干得到白色固体1.78g即为半抗原AⅠ(m＝0)。ESI-MS analysis(negative)m/z 190[M-H]–；1H NMR(600MHz, MeOD)δ7.97–7.93(m,2H),7.55(d,J＝8.8Hz,2H),4.21(q,J＝7.1Hz,2H),1.32(t,J＝7.1Hz,3H).
实施例2 氨基甲酸乙酯半抗原AⅠ(m＝1)制备方法
将1.51g 4-氨甲基苯甲酸溶于30mL的1mol/L碳酸氢钠溶液，向该溶液滴加焦碳酸二乙酯2mL，室温搅拌反应过夜，反应完后，将反应混合液调酸至pH为1～2，有白色沉淀析出，过滤沉淀，蒸馏水洗涤烘干得到白色固体2.04g即为半抗原AⅠ(m＝1)。ESI-MS analysis(negative)m/z 222[M-H]–；1H NMR(600MHz,MeOD)δ7.99(d,J＝8.2Hz,2H),7.39(d,J＝8.1Hz,2H),4.35(s,2H),4.12(q,J＝7.1Hz,2H),1.26(t,J＝7.1Hz,3H).
实施例3 氨基甲酸乙酯半抗原AⅠ(m＝2)制备方法
将1.65g 4-(2-氨乙基)苯甲酸溶于30mL的1mol/L的碳酸氢钠溶液，向该溶液滴加焦碳酸二乙酯2mL，室温搅拌反应过夜，反应完后，将反应混合液调酸至pH为1～2，有白色沉淀析出，过滤沉淀，蒸馏水洗涤烘干得到白色固体2.13g即为半抗原AⅠ(m＝2)。ESI-MS analysis(negative)m/z 236[M-H]–；1H NMR(600MHz,MeOD)δ7.96(d,J＝8.2Hz,2H),7.33(d,J＝8.1Hz,2H),4.06(q,J＝7.1Hz,2H),3.36(t,J＝7.3Hz,2H),2.86(t,J＝7.3Hz,2H),1.22(t,J＝7.1Hz,3H).
实施例4 氨基甲酸乙酯半抗原BⅡ(n＝3)制备方法
将1.03g 4-氨基丁酸溶于30mL蒸馏水，向溶液中滴加2mL焦碳酸二乙酯，室温搅拌过夜，旋转蒸发除尽水溶液，得油状透明物1.57g即为BⅡ(n＝3)。ESI-MSanalysis(negative)m/z 174[M-H]–；1H NMR(600MHz,MeOD)δ4.07(q,J＝7.1Hz,2H),3.18–3.12(m,2H),2.36–2.31(m,2H),1.81–1.75(m,2H),1.23(t,J＝7.1Hz,3H).
氨基甲酸乙酯半抗原BⅡ(n＝3)的结构式如下所示：
Figure PCTCN2016095543-appb-000007
其中，n＝3。
实施例5 氨基甲酸乙酯人工抗原的制备
氨基甲酸乙酯人工抗原按照偶联载体蛋白的类型不同分为包被原和免疫原，所述免疫原的载体蛋白采用钥孔血蓝蛋白(KLH)，所述包被原的载体蛋白采用鸡卵白蛋白(OVA)。以下所述以免疫原的制备方法为例。
活泼酯法：取半抗原AⅠ(m＝1)0.1mmoL溶于0.5mL二甲基甲酰胺(DMF)中，搅拌加入0.1mmol二环己基碳二亚胺(DCC)和0.1mmoL N-羟基琥珀酰亚胺(NHS)，4℃下磁力搅拌反应过夜，离心后上清液为A液。称取KLH 20mg溶于2mL浓度为0.1mol/L的PBS(pH8.0)中，搅拌溶解制备B液。磁力搅拌下，A液逐渐滴入B液中，4℃下反应12h。离心后，取上清液，4℃下用生理盐水透析3d，每天更换4次透析液。得到的免疫原Ⅰ(m＝1)，以1mg/mL的浓度分装于0.5mL离心管中。冻存于-20℃冰箱中。
对载体蛋白KLH，半抗原AⅠ(m＝1)、及其相应的免疫原进行紫外扫描测定(200～400nm)，发现免疫原同时具备半抗原和载体蛋白的特征吸收峰(图1)，说明免疫抗原制备成功。
参考上述免疫原的制备方法，分别制备得到包被原Ⅰ0(m＝0)、包被原Ⅰ1(m＝1)、包被原Ⅰ2(m＝2)、包被原Ⅱ3(n＝3)；免疫原Ⅰ0(m＝0)、免疫原Ⅰ1(m＝1)、免疫原Ⅰ2(m＝2)、免疫原Ⅱ3(n＝3)。
实施例6 抗体的制备及鉴定
实施例4制备的免疫原用生理盐水稀释至2mg/mL，与等体积的免疫佐剂(第1次免疫用弗氏完全佐剂，以后加强免疫均用弗氏不完全佐剂)用搅拌器乳化。完全乳化后，采用背部皮下、各部位皮下、腿部肌肉和耳缘静脉多种注射方式免疫7周龄雌性巴比赛(Babl/c)小鼠，每只小鼠免疫100μL。第一次免疫隔一个月后每三周加强免疫一次。第4次加强免疫后1段尾采血，提取抗血清。
利用间接ELISA测定血清效价与抑制，结果如表1。
表1抗体的制备及鉴定(效价的单位均为×103，抑制率的单位为％)
Figure PCTCN2016095543-appb-000008
注：血清Ⅰ0、血清Ⅰ1、血清Ⅰ2、血清Ⅱ3分别为由免疫原Ⅰ0(m＝0)、免疫 原Ⅰ1(m＝1)、免疫原Ⅰ2(m＝2)、免疫原Ⅱ3(n＝3)免疫小鼠得到；
包被原Ⅰ0、包被原Ⅰ1、包被原Ⅰ2、包被原Ⅱ3分别为载体蛋白为OVA的人工抗原I(m＝0、1、2)及载体蛋白为OVA的抗原Ⅱ(n＝3)；
抑制率＝(ODmax-ODx)/ODmax×100％；ODx为添加了1mg/mL氨基甲酸乙酯孔的吸光值；ODmax为不添加氨基甲酸乙酯孔的吸光值。
根据表1的结果，选取血清抑制最好的小鼠(抗原Ⅱ免疫的小鼠)制备单克隆抗体。将Babl/c小鼠的脾细胞与骨髓瘤细胞SP2/0按5:1体积比混合。在37℃水浴条件下，1min内往混合的细胞中滴加1mL聚乙二醇(PEG)，静止90s，加入基础培养基终止反应，1000rpm/min离心10min除去上清，细胞用50mLHAT重悬，铺于含饲养细胞的96孔板，每孔100μL，于二氧化碳培养箱中37℃培养。当细胞长至底孔面积的1/3时，取细胞上清进行间接竞争ELISA测定，筛选出阳性孔。将阳性孔进行有限稀释亚克隆阳性细胞，直到得到稳定分泌特异性针对氨基甲酸乙酯单克隆抗体的细胞株。将细胞株进行扩大培养及传代。
用液体石蜡致敏Balb/c小鼠，将收集得到的杂交瘤细胞株，用基础培养基进行清洗，并注射入小鼠的腹腔，一周后可见小鼠状态不活跃并且腹腔肿大。采集到的腹水，用辛酸-饱和硫酸铵法粗纯化，纯化后的腹水放入-20℃环境保存。腹水在使用前经过0.01mol/L PBS于4℃环境下透析。
实施例7 抗体的特异性及灵敏度
依据如上效果，使用所纯化的单克隆抗体绘制酶联免疫分析(ELISA)标准曲线；使用磷酸盐吐温缓冲液(PBST,100mmol/L，pH 7.4)作为所有样品的稀释液；以抗原Ⅱ(n＝3，载体蛋白为OVA)作为包被原；将50μL系列浓度的氨基甲酸乙酯标准品和50μL适当稀释倍数的氨基甲酸乙酯多特异性单克隆抗体加入96孔酶标板中，竞争反应后通过酶标分析仪测定吸光值(OD)。以OD值为纵坐标，相应标准品浓度对数值为横坐标，应用originPro 7.5软件四参数对数函数进行曲线拟合：
y＝(A-D)/[1+(x/C)B]+D  (1)
其中，A和D分别代表药物浓度最小和最大时的吸光值(OD)，C为中点浓度，当标准品浓度等于C时的OD值为(A+D)/2，正处于曲线的拐点处，半数抑制量浓度为IC50，B表示曲线的陡峭程度，称斜率因子；以IC10为检测限，以IC20～IC80为检测范围。以氨基甲酸乙酯标准品建立ELISA的标准曲线，结果 如图2，相关标准曲线参数见表2；
表2所制备的单克隆抗体建立的ELISA方法对氨基甲酸乙酯的检测参数
IC50(mg/L) 线性范围(mg/L) 最低检测限(mg/L) 相关系数R2
1.19 0.24～5.78 0.16 0.9988
表3所制备的单克隆抗对氨基甲酸乙酯及其结构类似的交叉反应率
Figure PCTCN2016095543-appb-000009
由图2、表2和表3的结果可知，以氨基甲酸乙酯为标准品建立的标准曲线具备典型的S型曲线，检测限到达0.16mg/L，特异性良好，除与氨基甲酸甲酯有一定交叉反应外，与其他结构类似物均不纯在明显的交叉反应(表3)。考虑到食品中氨基甲酸甲酯的含量特别低，有文献报道其含量通常仅为氨基甲酸乙酯的1/100(Shin,H.-S.,&Yang,E.-Y.(2012).Simultaneous determination of methylcarbamate and ethylcarbamate in fermented foods and beverages by derivatization and GC-MS analysis.Chemistry Central Journal 6:157)，因此该抗体应用于实际检测时，受到的干扰很小，对可用于酒精饮品中氨基甲酸乙酯的检测，同时也为后续开发更灵敏的检测氨基甲酸乙酯的免疫分析方法奠定基础。

Claims (10)

  1. 一种氨基甲酸乙酯半抗原组合，包括免疫半抗原和包被半抗原，其特征在于，免疫半抗原具有式(I)所示分子结构，包被半抗原具有式(Ⅱ)所示分子结构；
    Figure PCTCN2016095543-appb-100001
    式(I)；其中m＝0～2；
    Figure PCTCN2016095543-appb-100002
    式(Ⅱ)；其中：n＝1～5。
  2. 权利要求1所述氨基甲酸乙酯半抗原组合对应的人工抗原组合，包括免疫人工抗原和包被人工抗原，其特征在于，免疫人工抗原具有式(Ⅲ)所示分子结构，包被人工抗原具有式(Ⅳ)所示分子结构；
    Figure PCTCN2016095543-appb-100003
    式(Ⅲ)，其中m＝0～2；
    Figure PCTCN2016095543-appb-100004
    式(Ⅳ)，其中n＝1～5。
  3. 权利要求1所述氨基甲酸乙酯半抗原组合的制备方法，其特征在于，免疫半抗原的制备方法为：将焦碳酸二乙酯滴加到具有式(Ⅴ)所示化合物的NaHCO3溶液中，室温搅拌反应过夜，反应结束后将反应液调酸，过滤，用蒸馏水洗涤沉 淀，烘干即得；
    Figure PCTCN2016095543-appb-100005
    式(Ⅴ)；其中m＝0～2；
    包被半抗原的制备方法为：将焦碳酸二乙酯滴加到式(Ⅵ)所示化合物的水溶液中，室温搅拌反应过夜，反应完后，旋蒸除去水溶液即得；
    Figure PCTCN2016095543-appb-100006
    式(Ⅵ)；其中n＝1～5。
  4. 根据权利要求3所述的制备方法，其特征在于，所述焦碳酸二乙酯与式(Ⅴ)所示化合物的摩尔比为1～2:1，NaHCO3溶液的浓度为0.5～1mol/L。
  5. 权利要求2所述氨基甲酸乙酯人工抗原组合的制备方法，其特征在于，将权利要求1的免疫半抗原与载体蛋白偶联制备得到免疫人工抗原，将权利要求1的包被半抗原与载体蛋白偶联制备得到包被人工抗原。
  6. 根据权利要求5所述氨基甲酸乙酯人工抗原组合的制备方法，其特征在于，将权利要求1的免疫半抗原和包被半抗原分别与N,N’-二环己基碳二亚胺和N-羟基硫代琥珀酰亚胺反应形成活泼酯；将活泼酯滴加入载体蛋白溶液中，反应过夜；偶连有半抗原的载体蛋白经过3天的透析纯化，即得免疫人工抗原和包被人工抗原。
  7. 根据权利要求5或6氨基甲酸乙酯人工抗原的制备方法，其特征在于，所述载体蛋白为钥孔血蓝蛋白或鸡卵白蛋白。
  8. 一种采用权利要求2所述的人工抗原制备得到的氨基甲酸乙酯单克隆抗体、多克隆抗体或基因工程抗体。
  9. 权利要求8所述的抗体在检测氨基甲酸乙酯中的应用。
  10. 权利要求2所述的氨基甲酸乙酯人工抗原组合在检测氨基甲酸乙酯中的应用。
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