CN106674138B - 一种戊唑醇新型半抗原及其合成方法和应用 - Google Patents
一种戊唑醇新型半抗原及其合成方法和应用 Download PDFInfo
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- CN106674138B CN106674138B CN201611019736.9A CN201611019736A CN106674138B CN 106674138 B CN106674138 B CN 106674138B CN 201611019736 A CN201611019736 A CN 201611019736A CN 106674138 B CN106674138 B CN 106674138B
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- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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- 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
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
本发明涉及一种戊唑醇新型半抗原及其合成方法和应用,戊唑醇新型半抗原的结构式为:。所述戊唑醇新型半抗原以戊唑醇为原料,其合成路线如下:先将戊唑醇溶于无水二氯甲烷中,溶解后,再加入三乙胺,冰浴下搅拌,再加入琥珀酸单乙酯单酰氯,室温下搅拌过夜,洗涤,干燥,过滤,浓缩至干得中间产物;将中间产物溶于无水甲醇和四氢呋喃混合溶液中,然后加一水合氢氧化锂的水溶液,室温搅拌过夜,在冰浴下,调pH值,采用反相柱纯化,浓缩冻干,回收得到无色油状固体,即为戊唑醇新型半抗原。本发明方法戊唑醇新型半抗原合成方法简单,采用该半抗原制备戊唑醇抗体,所得抗体的灵敏度高,可用于建立戊唑醇残留检测的酶联免疫分析法。
Description
技术领域
本发明涉及一种半抗原,具体的说是一种戊唑醇新型半抗原及其合成方法和应用。
背景技术
戊唑醇,英文名为Tebuconazole,是一种羟乙基***衍生物,属低毒、高效、广谱的***类内吸杀菌剂,是甾醇脱甲基化抑制剂,通过破坏和阻止病菌的细胞膜中麦角甾醇的生物合成,使病原菌因不能形成细胞膜而致死,能有效防治禾谷类作物的锈病和白粉病;苹果和香蕉的叶斑病、轮纹病和黑星病等病害。然而,长期大量地将戊唑醇用于田间病害防治时,由于其持效期长,造成了严重的“3R”(抗性Resistance、再增猖獗Resurgence和残留Residue)问题;另外,研究还发现戊唑醇对斑马鱼胚胎发育具有一定的致死和致畸效应,对老鼠具有毒性蓄积和遗传效应,美国环境保护署(Enviroment Protection Agency, EPA)把戊唑醇列为“人类可能致癌物质”,因此,亟需建立快速、高灵敏地检测戊唑醇杀菌剂的方法。
目前,检测戊唑醇残留的方法主要有高效液相色谱法、气相色谱法和气相色谱联用质谱法。虽然仪器分析方法灵敏、准确和重现性好,但样品的预处理步骤繁琐,仪器设备昂贵,需要专业人员操作,检测费用高,需要消耗大量有毒的有机溶剂,尤其不适合大量样品的快速筛选。近年来,研究者还采用荧光光谱法和分子印迹电化学传感器对农产品中戊唑醇残留进行检测,具有快速灵敏的优点,但这两种方法的检测成本较高,稳定性差。而酶联免疫分析法具有高通量、检测成本低和快速简便的优点,正好可以弥补上述方法的不足。到目前为止,国内尚没有关于戊唑醇残留检测的酶联免疫分析法报道。而建立戊唑醇酶联免疫分析方法的关键在于其半抗原的设计和合成。
发明内容
本发明目的是为解决上述技术问题的不足,提供一种戊唑醇新型半抗原及其合成方法和应用,该合成方法简单,采用所合成的戊唑醇新型半抗原制备戊唑醇抗体,所得抗体灵敏度高,半数抑制浓度(IC50)为1.46±0.32 ng/mL;特异性强,与其类似物的交叉反应率均低于10%,
一种戊唑醇新型半抗原,其结构式为:。
所述戊唑醇新型半抗原,通过液质联用技术和核磁共振的氢谱和碳谱进行表征,结果为:
所述戊唑醇新型半抗原以戊唑醇为原料,经过2步化学反应获得,其合成路线如下:
其中DCM代表二氯甲烷;Et3N代表三乙胺;LiOH-H2O代表一水氢氧化锂;MeOH代表甲醇;THF代表四氢呋喃。
所述戊唑醇新型半抗原的合成方法,包括以下步骤:
(1)中间产物的合成
先将戊唑醇 (184 mg, 0.6 mmol) 溶于无水二氯甲烷 (CH2Cl2,15 mL) 中,溶解后,再加入三乙胺(C6H15N,730 mg, 7.2 mmol),冰浴下搅拌5分钟后,再加入琥珀酸单乙酯单酰氯(C6H9ClO3,988 mg, 6 mmol),室温下搅拌过夜,然后依次用水、饱和盐水洗涤,用无水硫酸钠干燥二氯甲烷过滤,减压蒸发浓缩至干,得中间产物约400 mg,直接用于下一步水解。该产物的结构通过液质联用技术得到证实。
所述饱和盐水为饱和NaCl水溶液。所述依次用水、饱和盐水洗涤,即采用15 mL水洗涤3次,再采用饱和盐水洗涤3次。
(2)戊唑醇新型半抗原的合成
将粗品 400 mg(约0.6 mmol)溶于无水甲醇-四氢呋喃 (9 mL-9 mL) 混合液中,加入一水合氢氧化锂的水溶液 (LiOH·H2O,252 mg, 6 mmol, 3 mL H2O),室温搅拌过夜,除去有机溶剂,冰浴下,采用1 mol/L盐酸将溶液的pH调到2.0-3.0,采用反相柱纯化,流动相为体积浓度为40%乙腈水溶液,浓缩冻干,回收得到无色油状固体( 70mg ),得率29%。
所述戊唑醇新型半抗原在制备免疫原中的应用,包括以下步骤:
(1)称取戊唑醇半抗原(4.1 mg,0.01 mmoL)加到N-N-二甲基甲酰胺(DMF,1 mL)中,搅拌溶解后,加入的1-乙基-碳二亚胺盐酸盐(EDC,4.5 mg)和N-羟基琥珀酰亚胺(NHS,4.4 mg),室温反应6小时,得到A液;
(2)取血蓝蛋白(KLH,20.92 mg/mL,0.3 mL)加到的硼酸盐缓冲溶液中(0.2 mol/L,3.5 mL,pH 8.8),搅拌混匀,得到B液;在冰浴下,将A液逐滴加到B液中,在4℃下搅拌反应8-10小时;
(3)采用磷酸盐缓冲溶液(0.01mol/L, pH 7.2)对反应液透析3天,每6小时更换一次透析液,更换8次;所得的戊唑醇免疫原分装,冻存和备用。
所述戊唑醇新型半抗原可应用于制备抗戊唑醇多克隆抗体。
所述戊唑醇新型半抗原可应用于农产品中戊唑醇残留检测的免疫分析。
所述戊唑醇新型半抗原可应用于制备抗戊唑醇多克隆抗体。
所述戊唑醇新型半抗原可应用于农产品中戊唑醇残留检测的免疫分析。
有益效果是:
本发明方法戊唑醇新型半抗原合成方法简单,合成了戊唑醇杀菌剂的新型半抗原,采用该半抗原制备戊唑醇抗体,所得抗体灵敏度高,半数抑制浓度(IC50)为1.46±0.32ng/mL;特异性强,与其类似物的交叉反应率均低于10%,可用于建立戊唑醇残留检测的酶联免疫分析法,为建立农产品中戊唑醇残留检测的免疫分析方法提供原料。
附图说明
图1 是中间产物的液相色谱图;
图2是中间产物的质谱图;
图3 戊唑醇半抗原的液相色谱图;
图4戊唑醇半抗原的质谱图;
图5 戊唑醇半抗原的核磁氢谱图;
图6 戊唑醇半抗原的核磁碳谱图;
图7 不同浓度戊唑醇半抗原的紫外扫描图;
图8 戊唑醇免疫原的紫外表征图;
图9 戊唑醇包被原的紫外表征图;
图10测定戊唑醇的标准抑制曲线图。
具体实施方式
一种戊唑醇新型半抗原的合成方法,以戊唑醇为原料,经过2步化学反应,获得戊唑醇的新型半抗原。其合成路线如下:
说明:DCM代表二氯甲烷;Et3N代表三乙胺;LiOH-H2O代表一水氢氧化锂;MeOH代表甲醇;THF代表四氢呋喃。
其合成步骤为:
(1)半抗原的合成
①、粗品的合成
先将戊唑醇 (184 mg, 0.6 mmol) 溶于无水二氯甲烷 (CH2Cl2,15 mL) 中,溶解后,再加入三乙胺(C6H15N,730 mg, 7.2 mmol),冰浴下搅拌5分钟后,再加入琥珀酸单乙酯单酰氯(C6H9ClO3, 988 mg, 6 mmol),室温下搅拌过夜,然后依次用水、饱和盐水洗涤,用无水硫酸钠干燥二氯甲烷过滤,减压蒸发浓缩至干,得中间产物(约400 mg),直接用于下一步水解。该产物的结构通过液质联用技术得到证实。结果见图1和图2所示, LCMS (ESI) m/z= 564.8/566.8 ([M+H]+)。
所述饱和盐水为饱和NaCl水溶液。所述依次用水、饱和盐水洗涤,即采用15 mL水洗涤3次,再采用饱和盐水洗涤3次。
②戊唑醇新型半抗原的合成
粗品 (400 mg, 约0.6 mmol) 溶于无水甲醇-四氢呋喃 (9 mL-9 mL) 混合液中,加入一水合氢氧化锂的水溶液 (LiOH·H2O,252 mg, 6 mmol, 3 mL H2O),室温搅拌过夜,除去有机溶剂,冰浴下,采用1 mol/L盐酸将溶液的pH调到2.0-3.0,采用反相柱纯化,流动相为体积浓度为40%的乙腈水溶液,浓缩冻干,回收得到无色油状固体( 70mg ),得率29%。
该产物的结构通过液质联用技术和核磁共振的氢谱和碳谱进行表征。结果见图3-图6所示:
(2)免疫原的制备
称取戊唑醇半抗原(4.1 mg, 0.01 mmoL)加到N-N-二甲基甲酰胺(DMF,1 mL)中,搅拌溶解后,加入的1-乙基-碳二亚胺盐酸盐(EDC,4.5 mg)和N-羟基琥珀酰亚胺(NHS,4.4mg),室温反应6小时,得到A液。
取血蓝蛋白(KLH,20.92 mg/mL,0.3 mL)加到的硼酸盐缓冲溶液中(0.2 mol/L,3.5 mL,pH 8.8),搅拌混匀,得到B液。在冰浴下,将A液逐滴加到B液中,在4℃下搅拌反应8-10小时。
采用磷酸盐缓冲溶液(0.01mol/L, pH 7.2)对反应液透析3天,每6小时更换一次透析液,更换8次。所得的戊唑醇免疫原分装,冻存和备用。
(3)包被原的制备
称取戊唑醇半抗原(4.1 mg,0.01 mmol)溶于N-N-二甲基甲酰胺(DMF,1 mL),再加入三丁胺(8μL)和氯甲酸异丁酯(5 μL),室温反应30分钟,得到A液。
称量牛血清白蛋白(20 mg)或鸡卵清蛋白(15 mg)加到硼酸盐缓冲液(0.2 mol/L,5.0 mL,pH 8.8),搅拌混匀,得到B液。然后在搅拌状态下将A液逐滴加入到B液中,4℃下反应6小时。
采用磷酸盐缓冲溶液(0.01mol/L, pH 7.2)对反应液透析3天,每6小时更换一次透析液,更换8次。所得的戊唑醇包被原分装,冻存和备用。
(2)人工抗原的鉴定
戊唑醇半抗原最大吸收波长的确定:先配制1 mg/mL的戊唑醇半抗原甲醇溶液,经过稀释,配制一系列的戊唑醇半抗原浓度(0,25,50,75μg/mL),扫描波长200-500 nm的紫外线。紫外测定结果见图7,由结果可知,戊唑醇半抗原的最大吸收波长为247 nm,每个浓度做3个平行样。
摩尔吸光系数(ε)的计算:摩尔吸光系数的计算公式ε=最大吸收波长下吸光值/摩尔浓度。本实验计算得戊唑醇抗原ε=4029.3 L/moL。
偶联物蛋白浓度的测定:因为所得的抗原溶液为棕黄色的澄清溶液,故蛋白浓度的计算公式:
C=M/V
M为载体蛋白的质量,g;V为抗原透析后的体积,mL;C为抗原的蛋白浓度。因此,在抗原透析后,直接测定透析后抗原的总体积。通过计算可得,抗原的蛋白浓度为3.72mg•mL-1。
偶联比的测定:配制0.2 mg/mL载体蛋白的水溶液,抗原采用水溶液稀释到0.2mg/mL,分别测定在278 nm处的吸光值,测出的吸光值为A1,A2,则偶联比率r为:
r=((A2-A1)/ε)/(200×10-3/载体蛋白的分子质量)
其中ε为摩尔吸光系数(L/moL),本实验计算得:r≈15。由此得出,半抗原与载体蛋白的比例为15:1。
实施例1戊唑醇半抗原的合成与鉴定
步骤一、中间产物的合成
首先将戊唑醇 (184 mg, 0.6 mmol) 溶于无水二氯甲烷 (CH2Cl2,15 mL) 中,再加入三乙胺(C6H15N, 730 mg, 7.2 mmol),冰浴搅拌5分钟后,加入琥珀酸单乙酯单酰氯(C6H9ClO3, 988 mg, 6 mmol),室温下搅拌过夜,依次以水、饱和盐水洗涤(分别为15 mL×3),用无水硫酸钠干燥二氯甲烷后,过滤,减压蒸发浓缩至干,得到中间产物(约400mg),直接用于下一步水解。该产物结构通过液质联用技术进行表征。
步骤二、戊唑醇半抗原的合成
中间产物 (400 mg, 0.6 mmol) 溶于无水甲醇-四氢呋喃 (9 mL:9 mL) 混合液中,加入一水合氢氧化锂的水溶液 (LiOH·H2O,252 mg, 6 mmol, 3 mL水),室温搅拌过夜,除去有机溶剂。冰浴下,采用1 mol/L盐酸将溶液的pH调到2.0-3.0,采用反相柱纯化,流动相为体积浓度为40%乙腈水溶液,浓缩冻干,回收得到无色油状固体(约70mg ),得率29%。该产物结构采用液质联用技术和核磁共振的氢谱和碳谱进行分析。戊唑醇半抗原的结构如下,
实施例2戊唑醇抗原的制备与鉴定
1、将戊唑醇半抗原采用活性酯法(EDC法)和血蓝蛋白(KLH)偶联,用作免疫原,具体反应路线如下:
将戊唑醇半抗原采用混合酸酐法和鸡卵清蛋白(OVA)或牛血清白蛋白(BSA)偶联,用作包被原,具体反应路线如下:
说明:其中Protein是蛋白质。
2、人工抗原的纯化
采用透析法对人工抗原进行脱盐纯化。
3、人工抗原的鉴定
偶联物蛋白浓度的测定:本方法所制备的抗原为棕黄色的澄清溶液,蛋白浓度的计算公式为:
C=M/V
M为载体蛋白的质量,g;V为抗原透析后的体积,mL;C为抗原的蛋白浓度。因此,在抗原透析后,直接测定透析后抗原的总体积。通过计算可得,抗原的蛋白浓度为3.72 mg/mL。
偶联比测定:配制0.2 mg/mL载体蛋白的水溶液,抗原采用蒸馏水稀释到0.2 mg/mL,分别测定在278 nm处测吸光值,测出的吸光值为A1,A2。偶联比率r的计算公式为:
;其中ε为摩尔吸光系数(L/moL),本实验计算得出:r≈15,因此,根据上述公式估算戊唑醇免疫原的偶联比15:1。
多克隆抗体的效价为1:16000,半数抑制浓度(IC50)为1.46±0.32 ng/mL,与其类似物的的交叉反应率低于10%;按照如下步骤产生:
步骤一 抗戊唑醇多克隆抗体的制备
1、动物免疫:选择体重为2.0 kg左右的新西兰大白兔为试验动物,每2只为一组,共3组。免疫原为戊唑醇半抗原和血蓝蛋白通过活性酯法制备的偶联物。初免采用氟氏完全佐剂乳化,免疫原乳化后,采用背部多点注射的方式进行免疫,初免剂量为1 mg/kg体重(以蛋白含量计),四周后进行加强免。加强免采用不完全佐剂进行乳化,免疫剂量0.65 mg/kg体重,每隔3周加强免一次。
2、抗血清的筛选:第四次免疫后7-10天,通过耳廓外缘静脉采血,采血后,室温下静置30分钟, 离心(5000 rpm,10 min),吸出上清液,获得抗血清。采用间接竞争酶联免疫法测定抗血清的效价和抑制,选出效价高且抑制好的血清,通过心脏采血的方式对选出的兔子采血,收集于50 mL灭菌塑料离心管中,按照上述方法制备抗血清。
3、抗体的纯化
采用硫酸铵沉淀法进行纯化。
步骤二、戊唑醇抗体特性的测定
1、抗体效价的测定
采用间接竞争酶联免疫法(ic-ELISA)测定戊唑醇抗体的效价,得出抗体的效价为1:16000。采用戊唑醇半抗原-牛血清白蛋白的偶联物包被酶标板;封闭后,加入倍比稀释的一抗血清,37℃下反应30 分钟;洗涤拍干后,加入酶标二抗,37℃下反应30 分钟;洗涤拍干后,37℃下显色15 分钟,终止。采用酶标仪测定450 nm下的吸光值。
2、灵敏度的测定
采用间接竞争酶联免疫法(ic-ELISA)测定抗体对戊唑醇农药的抑制效果。选择包被原浓度为0.3μg/mL,抗体的蛋白浓度为0.04μg/mL,测定结果用origin 8.5软件进行四参数回归拟合,结果见图10。根据拟合的回归方程,计算戊唑醇抗体的半数抑制浓度,即IC50为1.46±0.32 ng/mL;标准曲线的线性范围在0-40 ng/mL之间。
3、特异性的测定
采用间接竞争酶联免疫法测定戊唑醇抗体对其类似物(***醇、***酮、烯唑醇、腈菌唑和联苯***醇)的抑制作用。根据图10的四参数回归方程计算戊唑醇类似物的半数抑制浓度,再利用下式计算戊唑醇类似物的交叉反应率。
交叉反应率(CR,%)= IC50(戊唑醇)/ IC50(戊唑醇类似物)。戊唑醇抗体的交叉反应的结果见表1。
表1:
Claims (7)
1.一种戊唑醇半抗原,其特征在于:其结构式为:
2.如权利要求1所述戊唑醇半抗原的合成方法,其特征在于:所述戊唑醇半抗原以戊唑醇为原料,经过2步化学反应获得,其合成路线如下:
其中DCM代表二氯甲烷;Et3N代表三乙胺;LiOH-H2O代表一水氢氧化锂;MeOH代表甲醇;THF代表四氢呋喃。
3.如权利要求2所述戊唑醇半抗原的合成方法,其特征在于:每70mg戊唑醇半抗原的合成,包括以下步骤:
(1)中间产物的合成
先将0.6mmol戊唑醇溶于15mL无水二氯甲烷中,溶解后,再加入7.2mmol三乙胺,冰浴下搅拌5分钟后,再加入6mmol琥珀酸单乙酯单酰氯,室温下搅拌过夜,然后依次用水、饱和氯化钠水溶液洗涤,用无水硫酸钠干燥,用二氯甲烷过滤,减压蒸发浓缩至干,得中间产物,直接用于下一步水解;
(2)戊唑醇半抗原的合成
将粗品溶于18mL体积比为1:1的无水甲醇和四氢呋喃混合溶液中,然后加3-4mL0.002mmol/L的一水合氢氧化锂的水溶液室温搅拌过夜,得到混合溶液C;在冰浴下,采用1mol/L盐酸将混合溶液C的pH值调到2.0-3.0,采用反相柱纯化,流动相为体积浓度为40%乙腈水溶液,浓缩冻干,回收得到无色油状固体,即为戊唑醇半抗原。
4.如权利要求1所述戊唑醇半抗原在制备免疫原中的应用。
5.如权利要求4所述戊唑醇半抗原在制备免疫原中的应用,其特征在于:每4.1mg戊唑醇半抗原制备免疫原,包括以下步骤:
(1)称取戊唑醇半抗原4.1mg加入到1mL N-N’-二甲基甲酰胺中,搅拌溶解后,加入4.5mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4.4mg N-羟基琥珀酰亚胺,室温反应6小时,得到A液;
(2)取0.3mL浓度为20.92mg/mL血蓝蛋白溶液,加到3.5mL浓度为0.2mol/L,pH值为8.8的硼酸盐缓冲溶液中,搅拌混匀,得到B液;在冰浴下,将A液逐滴加到B液中,在4℃下搅拌反应8-10小时,得到反应液;
(3)采用浓度为0.01mol/L,pH值为7.2的磷酸盐缓冲溶液对反应液透析3天,每6小时更换一次透析液,更换8次,得到戊唑醇免疫原,分装、冻存和备用。
6.如权利要求1所述戊唑醇半抗原在制备抗戊唑醇多克隆抗体中的应用。
7.如权利要求1所述戊唑醇半抗原在农产品中戊唑醇残留检测的免疫分析中应用。
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