CN109265401B - 一种异菌脲半抗原与抗原的制备方法及应用 - Google Patents
一种异菌脲半抗原与抗原的制备方法及应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种异菌脲半抗原与抗原的制备方法及应用。属于农药免疫化学技术领域。
背景技术
异菌脲(Iprodione)属于二甲酰亚胺类,是一种广谱性触杀型保护性杀菌剂,广泛应用于烟草、果树、蔬菜的病害防治和水果的贮藏保鲜。异菌脲可以通过根部吸收起内吸作用,可有效防治对苯并咪唑类内吸杀菌剂有抗性的真菌。其主要防治对象为由葡萄孢菌、交链孢菌、核盘菌等引起的病害,如灰霉病、早疫病、黑斑病、菌核病等。我国针对不同作物,制定了异菌脲的最大残留限量标准,其中在油菜籽、黄瓜中的最大残留限量为2mg/kg,在番茄、苹果、梨中的最大残留限量为5mg/kg,在葡萄、香蕉中的最大残留限量为10mg/kg。国际烟草科学研究合作中心(CORESTA)规定烟草中异菌脲的指导性残留限量为0.5mg/kg,在实际生产中,以0.5mg/kg作为烟草最大残留量判定标准。
目前,国内外对异菌脲残留的检测方法主要有气相色谱-质谱联用法、高效液相色谱-质谱联用法、气相色谱法、高效液相色谱法,等。仪器方法具备检测灵敏度高、特异性强等优势,但是检测样本前处理繁琐、耗时,样品还需提取和净化处理,同时仪器检测方法需要昂贵的大型仪器和设备,配备专业的检测技术人员进行操作和管理,无法进行现场大规模检测,时效性差,难以推广。基于抗原抗体特异性识别的免疫分析方法可以定性定量检测样品中的农药残留。这种分析方法对仪器设备要求不高、快速简便,一般无需对样品进行复杂的预处理,灵敏度高、特异性强,对使用人员的专业技术要求不高,容易普及和推广,可满足快速分析检测的需要,尤其适宜现场筛选和大量样品的快速分析。免疫分析为异菌脲残留研究提供了一个新的分析检测途径。免疫分析目前已成为农药残留分析研究的一个崭新领域,美国化学会将免疫分析与气相色谱、液相色谱共同列为农药残留分析的三大支柱技术。我国农药免疫分析技术研究起步相对较晚,但近年来发展迅速,有关于对硫磷、甲基对硫磷、甲基对氧磷、多菌灵、毒死蜱、三唑磷、氟虫腈、二氯喹啉酸、克百威、三唑酮、甲胺磷、阿特拉津、2甲4氯等农药的人工抗原和高亲和力的特异性抗体的制备及用酶联免疫法进行样品中痕量农药分析的报道。
本发明属于农药小分子化合物免疫化学和残留分析技术领域,涉及有机合成、免疫化学及生物化学等,依靠免疫学、免疫化学基本原理和生物技术手段,设计、合成小分子目标分析物半抗原,并与载体蛋白质偶联,制备有效人工抗原。制备的抗原可以通过免疫动物制备对小分子分析物特异性识别的抗体,利用抗原抗体的特异性免疫学反应和易被检测识别的标记物的放大作用,定量的检测样品中超微量小分子目标物。半抗原的分子设计与合成是产生特异性抗体和建立农药残留免疫分析方法的关键步骤。人工抗原的制备,包括结合位点、结合方式、载体种类及半抗原与目标分析物质任何结构上的差异,诸如分子大小、形状、成分、构型、构象、极性、电子云密度等在内的拓扑性征,都可能极大的影响着相应抗体的性质。能否设计合成出性能、效果更好的半抗原与抗原,正是本发明所关注的重点。
发明内容
本发明的目的正是基于上述现有技术状况而提供一种异菌脲半抗原与抗原的制备方法及应用。
本发明的目的是通过以下技术方案来实现的:
一种异菌脲半抗原的制备方法,是由3,5-二氯苯异氰酸酯与甘氨酸乙酯盐酸盐反应生成水解得到再经过成环反应,得到3-(3,5-二氯苯基)-2,4-咪唑烷基二酮,再与6-氨基己酸甲酯盐酸盐与三光气反应得到的6-异氰酸基己酸甲酯反应生成6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯,最后在酸性条件下水解得到,其分子结构式为:
具体步骤如下:
1)取8.37g(60mmol)甘氨酸乙酯盐酸盐及16.5mL(120mmol)三乙胺加入100mL二氯甲烷中,0℃下滴加10g(53mmol)3,5-二氯苯异氰酸酯的二氯甲烷溶液,室温搅拌过夜,过滤去不溶固体,滤液分别用2N盐酸,饱和碳酸氢钠溶液,饱和食盐水洗涤,有机层用无水硫酸镁干燥,蒸去溶剂后得到白色固体
3)将上述白色固体加入100mL 20%盐酸中,搅拌加热回流(105-115℃)4h,冷却至室温,过滤得到白色固体,水洗干燥得到9.50g白色固体3-(3,5-二氯苯基)-2,4-咪唑烷基二酮。
4)反应瓶中,将2.20g(7.36mmol)三光气溶于30mL二氯甲烷中。0℃下,将3.34g(18.4mmol)6-氨基己酸甲酯盐酸盐及7.13g(55.2mmol)二异丙基乙基胺(DIEPA)的二氯甲烷溶液缓慢滴入上述反应液中,室温搅拌反应1h,蒸去二氯甲烷,残余固体中加入50mL无水乙醚搅拌,滤去不溶的盐,滤液浓缩得到6-异氰酸基己酸甲酯2.30g。
5)将3.00g(12.2mmol)3-(3,5-二氯苯基)-2,4-咪唑烷基二酮及2.67g(18.4mmol)DBU(1,8-二氮杂双环[5.4.0]-7-十一碳烯)加入50mL二氯甲烷中,0℃下,缓慢滴加2.30g(13.5mmol)6-异氰酸基己酸甲酯的二氯甲烷溶液,室温搅拌反应4h,反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为1:5的乙酸乙酯/石油醚洗脱,得到6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯1.83g。
6)将1.83g(4.4mmol)6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯溶于50mL四氢呋喃中,加入1mL 20%盐酸,50℃下反应4h,室温反应过夜。反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为30:1的二氯甲烷/甲醇洗脱,得到异菌脲半抗原6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸1.12g。
所述异菌脲半抗原可用于制作动物免疫的抗原体系原料。
一种异菌脲抗原的制备方法,是由异菌脲半抗原与载体蛋白偶联得到。所述载体蛋白为甲状腺蛋白、牛血清白蛋白、兔血清蛋白、人血清蛋白、卵清蛋白或血蓝蛋白。
具体步骤如下:
免疫抗原的制备:取9.0mg异菌脲半抗原,溶解于1.0mL二甲基甲酰胺(DMF)中,加氯甲酸异丁酯0.18mL,加吡啶0.3mL,室温搅拌5h,得到半抗原活化液A液;取50mg牛血清白蛋白(BSA),充分溶解在3.8mL磷酸盐缓冲液PBS中,得到B液,将A液滴加到B液中,室温搅拌5h,用0.01mol/L PBS在4℃透析3天,每天换液3次,以除去未反应的小分子物质,分装,得到免疫原,于-20℃保存备用。
包被抗原的制备:取7.0mg异菌脲半抗原,溶解于1.0mL DMF中,加氯化亚砜0.27mL,60℃反应6h,停止反应冷却到室温,得到半抗原活化液A液;取50mg卵清白蛋白(OVA),充分溶解在3.8mL PBS中,得到B液,将A液滴加到B液中,室温反应8h,停止反应,用0.01mol/L的PBS缓冲液透析纯化3天,每天换液3次,分装,得到包被原,于-20℃保存备用。
采用异菌脲抗原免疫动物得到的单克隆抗体,可用于建立酶联免疫吸附测定方法和胶体金试纸快速测定法,从而实现烟草及食品中异菌脲的快速检测。
本发明中合成的异菌脲半抗原及抗原与申请号为201510973027.3和201510973150.5的专利中的半抗原及抗原结构不同。本发明中合成的异菌脲半抗原既最大程度的保留了异菌脲的化学结构,又有合适长度的连接臂,用该半抗原制备的免疫原去免疫动物,得到的抗体的效价、特异性、亲和力都比较好,与其他农药的交叉反应率低,特异性更好,灵敏度更高。
本发明制备的抗原呈现出特异性的异菌脲抗原决定簇,使得筛选出高特异性的异菌脲单克隆抗体成为可能。产生的抗体特异性高、灵敏度高,可用于建立酶联免疫吸附测定方法和胶体金试纸快速测定法,从而实现烟草及食品中异菌脲的快速检测。
附图说明
图1为异菌脲半抗原合成路线图;
图2为试纸剖面结构示意图,图中:1、样品吸收垫;2、反应膜;3、吸水垫;4、检测线;5、质控线;6、底板;7、保护膜;
图3为试纸俯视图;
图4为微孔试剂图,图中:8、微孔;9、微孔塞。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解,这些实施例仅用于说明本发明,而不用来限制本发明的范围。
实施例1异菌脲半抗原的制备
1、异菌脲半抗原的合成(合成路线见图1)
1)取8.37g(60mmol)甘氨酸乙酯盐酸盐及16.5mL(120mmol)三乙胺加入100mL二氯甲烷中,0℃下滴加10g(53mmol)3,5-二氯苯异氰酸酯的二氯甲烷溶液,室温搅拌过夜,过滤去不溶固体,滤液分别用2N盐酸,饱和碳酸氢钠溶液,饱和食盐水洗涤,有机层用无水硫酸镁干燥,蒸去溶剂后得到白色固体
3)将上述白色固体加入100mL 20%盐酸中,搅拌加热回流4h,冷却至室温,过滤得到白色固体,水洗干燥得到9.50g白色固体3-(3,5-二氯苯基)-2,4-咪唑烷基二酮,三步总收率73.2%。
4)反应瓶中,将2.20g(7.36mmol)三光气溶于30mL二氯甲烷中。0℃下,将3.34g(18.4mmol)6-氨基己酸甲酯盐酸盐及7.13g(55.2mmol)二异丙基乙基胺(DIEPA)的二氯甲烷溶液缓慢滴入上述反应液中,室温搅拌反应1h,蒸去二氯甲烷,残余固体中加入50mL无水乙醚搅拌,滤去不溶的盐,滤液浓缩得到6-异氰酸基己酸甲酯2.30g。
5)将3.00g(12.2mmol)3-(3,5-二氯苯基)-2,4-咪唑烷基二酮及2.67g(18.4mmol)DBU(1,8-二氮杂双环[5.4.0]-7-十一碳烯)加入50mL二氯甲烷中,0℃下,缓慢滴加2.30g(13.5mmol)6-异氰酸基己酸甲酯的二氯甲烷溶液,室温搅拌反应4h,反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为1:5的乙酸乙酯/石油醚洗脱,得到6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯1.83g,收率36.1%。
6)将1.83g(4.4mmol)6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯溶于50mL四氢呋喃中,加入1mL 20%盐酸,50℃下反应4h,室温反应过夜。反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为30:1的二氯甲烷/甲醇洗脱,得到异菌脲半抗原6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸1.12g,收率63.3%。
2、异菌脲半抗原的鉴定
核磁鉴定1H NMR(300MHz,DMSO-d6):δ12.04(1H,s),10.07(1H,s,NH),7.73(2H,d,J=1.80Hz),7.34(1H,t,J=1.80Hz),4.32(2H,s),3.44(2H,t,J=6.81Hz),2.20(2H,t,J=6.25Hz),1.62-1.44(4H,m),1.36-1.22(2H,m)。
图谱中,化学位移δ=12.04为间隔臂上羧基氢的共振吸收峰,δ=3.44、2.20、1.62-1.44、1.36-1.22为间隔臂上亚甲基氢的共振吸收峰,这些峰的存在配合其他异菌脲氢固有吸收峰的存在证明半抗原合成成功。
实施例2异菌脲抗原的制备
1、异菌脲免疫原的合成
异菌脲半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取9.0mg异菌脲半抗原,溶解于1.0mL二甲基甲酰胺(DMF)中,加氯甲酸异丁酯0.18mL,加吡啶0.3mL,室温搅拌5h,得到半抗原活化液A液;取50mg牛血清白蛋白(BSA),充分溶解在3.8mL磷酸盐缓冲液PBS中,得到B液,将A液滴加到B液中,室温搅拌5h,用0.01mol/L PBS在4℃透析3天,以除去未反应的小分子物质,得到异菌脲-BSA免疫原;于-20℃保存备用。
2、异菌脲包被原的合成
异菌脲半抗原与卵清蛋白(OVA)偶联得到包被原。
取7.0mg异菌脲半抗原,溶解于1.0mL DMF中,加氯化亚砜0.27mL,60℃反应6h,停止反应冷却到室温,得到半抗原活化液A液;取50mg卵清白蛋白(OVA),充分溶解在3.8mLPBS中,得到B液,将A液滴加到B液中,室温反应8h,停止反应,用0.01mol/L的PBS缓冲液透析纯化3天,每天换液3次,分装,得到异菌脲-OVA包被原,-20℃保存备用。
3、异菌脲抗原的鉴定
确定偶联是否成功:一般通过紫外扫描法鉴定半抗原与载体蛋白的偶联是否为有效偶联,因为半抗原与蛋白在紫外下有不同的特征吸收,当偶联成功时,偶联物的紫外吸收会有二者的叠加效应出现,因此比着单独的蛋白特征吸收会发生一定的偏移,可用于检测偶联是否成功。
偶联比的测定:用纯水稀释异菌脲半抗原、牛血清白蛋白、卵清蛋白和两种蛋白与异菌脲半抗原的结合物,配制成一定浓度的溶液,然后用紫外分光光度计进行全波长扫描,得到它们的紫外吸收光谱图。
根据公式K=A/CL分别计算出异菌脲半抗原、牛血清白蛋白、卵清蛋白的摩尔消光系数。在载体蛋白和异菌脲半抗原的最大波长处检测偶联物的光吸收值,按公式计算两种物质在偶联物中的摩尔浓度比,即偶联比:
Ca/Cb=(A偶260×KBSA280-A偶280×KBSA260)/(A偶280×K异菌脲260-A偶260×K异菌脲280)
蛋白含量的测定:将偶联物稀释到适当倍数后,测定280nm和260nm的分光光度值,按公式计算蛋白的浓度即偶联物的浓度:
蛋白质(mg/mL)=1.45×OD280-0.74×OD260
免疫原和包被原的鉴定结果:通过紫外扫描法鉴定半抗原与载体蛋白的偶联是有效偶联,根据异菌脲半抗原、载体蛋白、偶联物在特定波长的摩尔吸光系数估算半抗原与载体蛋白的结合比分别为15:1和11:1,偶联效果较好,免疫原的蛋白含量为15.1mg/mL,包被原的蛋白含量为6.9mg/mL。
实施例3异菌脲单克隆抗体的制备
1、杂交瘤细胞的获得
1)首次免疫:将异菌脲半抗原-BSA偶联物(免疫原)与等量的弗氏完全佐剂充分乳化,皮下注射6周龄的Balb/c小鼠,免疫剂量为150μg/只;
2)加强免疫两次:从首次免疫开始,每两周加强免疫一次,用弗式不完全佐剂代替弗氏完全佐剂,方法和剂量同首次免疫;
3)最后一次加强免疫一周后眼底静脉采血测效价和抑制,有抑制且效价达到1:10000以上时进行如下末次免疫:腹腔注射不加任何佐剂的免疫原溶液0.1mL,三天后处死小鼠,取其脾脏与骨髓瘤细胞融合;
4)采用间接竞争酶联免疫分析方法测定细胞上清液,筛选阳性孔。利用有限稀释法对阳性孔进行克隆化,得到并建立稳定分泌异菌脲单克隆抗体的杂交瘤细胞株,取处于对数生长期的杂交瘤细胞用冻存液制成细胞悬液,分装于冻存管,在液氮中长期保存。
2、单克隆抗体的制备
1)细胞复苏:取出异菌脲单克隆抗体杂交瘤细胞株冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养;
2)制备腹水与抗体纯化:采用体内诱生法,将Balb/c小鼠(8周龄)腹腔注入灭菌石蜡油0.5mL/只,7天后腹腔注射杂交瘤细胞5×105个/只,7天后采集腹水。用辛酸-饱和硫酸铵法进行纯化,得到异菌脲单克隆抗体溶液(-20℃保存)。
3、单克隆抗体效价的测定
用间接竞争ELISA法测定抗体的效价为1:(100000~300000)。
间接竞争ELISA方法:用异菌脲半抗原-OVA偶联物包被酶标板,加入异菌脲标准品溶液、异菌脲单克隆抗体溶液和辣根过氧化物酶标记的羊抗鼠抗抗体溶液,25℃反应30min,倒出孔内液体,用洗涤液洗涤3~5次,用吸水纸拍干;加入底物显色液,25℃反应15min后,加入终止液终止反应;设定酶标仪于波长450nm处测定每孔吸光度值。
4、单克隆抗体特异性的测定
抗体特异性是指它同特异性抗原结合的能力与同该类抗原类似物结合能力的比较,常用交叉反应率作为评价标准。交叉反应越小,抗体的特异性则越高。
本实验将异菌脲及与其他二甲酰亚胺类杀菌剂(腐霉利、菌核净、乙烯菌核利)做系列稀释,分别与单克隆抗体进行间接竞争ELISA,制作标准曲线,分析得到IC50,然后按下式计算交叉反应率:
结果显示异菌脲及其结构类似物的交叉反应率为:异菌脲100%、腐霉利<1%、菌核净<1%、乙烯菌核利<1%。本发明抗体对腐霉利、菌核净、乙烯菌核利等其他二甲酰亚胺类杀菌剂无交叉反应,只针对异菌脲有特异性结合。
实施例4异菌脲胶体金试纸条的制备
1、异菌脲单克隆抗体-胶体金标记物的制备
(1)胶体金的制备
用双蒸去离子水将质量分数为1%的氯金酸溶液稀释成0.01%,取100mL置于锥形瓶中,用恒温电磁搅拌器加热至沸腾,在持续高温、持续搅拌下加入1.5mL质量分数为1%的柠檬酸三钠溶液,继续匀速搅拌加热至溶液呈透亮的酒红色时停止,冷却至室温后用去离子水恢复到原体积,4℃保存。制备良好的胶体金用肉眼观察是清亮透明的,没有混浊,液体表面无漂浮物,在日光下观察胶体金的颜色为酒红色。
(2)异菌脲单克隆抗体-胶体金标记物的制备
在磁力搅拌下,用0.2mol/L碳酸钾溶液调胶体金的pH值至7.2(不同抗体的pH值标记范围在7~8之间,可以变化),按每毫升胶体金溶液中加入20~50μg抗体的标准向胶体金溶液中加入上述异菌脲单克隆抗体,搅拌混匀,室温静置10min,加入10%BSA使其在胶体金溶液中的终质量分数为1%,静置10min。12000r/min,4℃离心40min,弃上清液,沉淀用复溶缓冲液洗涤两次,用体积为初始胶体金体积1/10的复溶缓冲液将沉淀重悬,置4℃备用。
复溶缓冲液:含BSA的质量分数为0.1%~0.3%、吐温-80的质量分数为0.05%~0.2%、pH值为7.2的0.02mol/L磷酸盐缓冲液。
2、微孔试剂的制备向微孔试剂微孔中加入100μL异菌脲单克隆抗体-胶体金标记物,放入冷冻干燥机中,在冷阱温度为-50℃条件下,预冻3h后,再真空干燥15h,即可取出,得到冻干有异菌脲单克隆抗体-胶体金标记物的微孔试剂,密封保存。
3、样品吸收垫的制备
将样品吸收垫置于体积分数为0.5%牛血清白蛋白、pH值为7.2的0.1mol/L磷酸盐缓冲液中浸泡2h,37℃烘2h备用。
4、反应膜的制备包被过程:用磷酸缓冲液将异菌脲半抗原-卵清蛋白偶联物稀释到1mg/mL,用Isoflow点膜仪将其包被于硝酸纤维素膜上的检测线(T),包被量为1.0μL/cm;用0.01mol/L、pH值为7.4的磷酸盐缓冲液将羊抗鼠抗抗体稀释到200μg/mL,用Isoflow点膜仪将其包被于硝酸纤维素膜上的质控线(C),包被量为1.0μL/cm。将包被好的反应膜置于37℃条件下干燥2h,备用。
5、各部件的组装
(1)试纸的组装
将所述样品吸收垫、反应膜、吸水垫依次按顺序粘贴在所述底板上;样品吸收垫的末端与反应膜的始端相连,反应膜的末端与吸水垫的始端相连,样品吸收垫的始端与底板的始端对齐,吸水垫的末端与底板的末端对齐;在组装好的试纸的样品吸收垫上粘贴保护膜,保护膜上印有MAX标记线。
(2)试纸条的组装
将上述步骤1得到的试纸与微孔试剂组装成试纸条,在2~8℃的环境中贮存,有效期12个月。
实施例5检测异菌脲的试纸条的应用
1、样品的前处理
称取1.0±0.05g粉碎后的待测样品至50mL离心管中,加入10mL 50%甲醇水溶液,涡旋1min,3000rpm以上离心5min;取100μL上清液,加入400μL样本复溶液,混匀待测。
2、用试纸条进行检测
用微量移液器吸取100μL待测样本溶液于微孔试剂中,缓慢抽吸且充分与微孔中试剂混匀,室温(20~25℃)孵育3min后,将试纸标有MAX标记线端向下插入孵育后的微孔试剂中,液体流动时开始计时,反应10min,根据示意图判定结果。
3、分析检测结果
阴性(-):T线显色比C线显色深或与C线显色一致,表示样品中异菌脲浓度低于检测限。
阳性(+):T线显色比C线显色浅或T线不显色,表示样品中异菌脲浓度等于或高于检测限。
无效:未出现C线,表明不正确的操作过程或试纸条已变质失效,在此情况下,应再次仔细阅读说明书,并用新的试纸条重新测试。
实施例6检测异菌脲的试纸条技术参数的确定
1、检测限试验
取空白农产品及烟草样品,在其中分别添加异菌脲至终浓度为0.25、0.5、1mg/kg,取试纸条进行检测,每个样品重复测定三次。
用试纸条检测农产品及烟草样品时,当其中异菌脲添加浓度为0.25mg/kg时,试纸条上显示出T线显色比C线显色深或与C线显色一致,呈阴性;当其中异菌脲添加浓度为0.5、1mg/kg时,试纸条上显示出T线显色比C线显色浅或T线不显色,呈阳性,表明本试纸条对农产品及烟草中异菌脲的检测限为0.5mg/kg。
2、假阳性率、假阴性率试验
取已知异菌脲含量大于0.5mg/kg的阳性样品20份,阴性样品20份,用三批试纸条进行检测,计算其阴阳性率。
结果表明:用3个批次生产的试纸条检测阳性样品时,结果全为阳性,可知阳性样品符合率为100%,假阴性率为0;检测阴性样品时,结果全为阴性,可知阴性样品符合率为100%,假阳性率为0。说明本发明的检测异菌脲的试纸条可以对农产品及烟草中的异菌脲进行快速检测。
3、特异性试验
将腐霉利、菌核净、乙烯菌核利等其他二甲酰亚胺类杀菌剂用pH值为7.2、0.2mol/L的磷酸盐缓冲液稀释至1mg/L,用异菌脲试纸条进行检测。结果显示,用该试纸条检测1mg/L的腐霉利、菌核净、乙烯菌核利时,试纸条T线显色比C线显色深或与C线显色一致,呈阴性。说明本试纸条对腐霉利、菌核净、乙烯菌核利等与异菌脲结构类似的化合物无交叉反应,特异性良好。
Claims (8)
1.一种异菌脲半抗原的制备方法,其特征在于:是由3,5-二氯苯异氰酸酯与甘氨酸乙酯盐酸盐反应生成水解得到再经过成环反应,得到3-(3,5-二氯苯基)-2,4-咪唑烷基二酮,再与6-氨基己酸甲酯盐酸盐与三光气反应得到的6-异氰酸基己酸甲酯反应生成6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯,最后在酸性条件下水解得到,其分子结构式为:
所述异菌脲半抗原的制备方法的具体步骤如下:
1)取8.37g甘氨酸乙酯盐酸盐及16.5mL三乙胺加入100mL二氯甲烷中,0℃下滴加10g3,5-二氯苯异氰酸酯的二氯甲烷溶液,室温搅拌过夜,过滤去不溶固体,滤液分别用2N盐酸,饱和碳酸氢钠溶液,饱和食盐水洗涤,有机层用无水硫酸镁干燥,蒸去溶剂后得到白色固体
3)将上述白色固体加入100mL 20%盐酸中,搅拌加热回流4h,冷却至室温,过滤得到白色固体,水洗干燥得到9.50g白色固体3-(3,5-二氯苯基)-2,4-咪唑烷基二酮;
4)反应瓶中,将2.20g三光气溶于30mL二氯甲烷中,0℃下,将3.34g 6-氨基己酸甲酯盐酸盐及7.13g二异丙基乙基胺的二氯甲烷溶液缓慢滴入上述反应液中,室温搅拌反应1h,蒸去二氯甲烷,残余固体中加入50mL无水乙醚搅拌,滤去不溶的盐,滤液浓缩得到6-异氰酸基己酸甲酯2.30g;
5)将3.00g 3-(3,5-二氯苯基)-2,4-咪唑烷基二酮及2.67g 1,8-二氮杂双环[5.4.0]-7-十一碳烯加入50mL二氯甲烷中,0℃下,缓慢滴加2.30g 6-异氰酸基己酸甲酯的二氯甲烷溶液,室温搅拌反应4h,反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为1:5的乙酸乙酯/石油醚洗脱,得到6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯1.83g;
6)将1.83g 6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸甲酯溶于50mL四氢呋喃中,加入1mL 20%盐酸,50℃下反应4h,室温反应过夜;反应液蒸去溶剂,残余物经硅胶柱层析纯化,用体积比为30:1的二氯甲烷/甲醇洗脱,得到异菌脲半抗原6-(3-(3,5-二氯苯基)-2,4-二氧咪唑烷基-1-甲酰胺基)己酸1.12g。
2.如权利要求1所述的异菌脲半抗原的制备方法,其特征在于:步骤3)中的加热回流时的温度为105-115℃。
3.如权利要求1所述方法制备的异菌脲半抗原的应用,其特征在于:所述异菌脲半抗原用于制作动物免疫的抗原体系原料。
4.一种异菌脲抗原的制备方法,其特征在于:是由权利要求1制备得到的异菌脲半抗原与载体蛋白偶联得到。
5.如权利要求4所述的异菌脲抗原的制备方法,其特征在于:所述载体蛋白为甲状腺蛋白、牛血清白蛋白、兔血清蛋白、人血清蛋白、卵清蛋白或血蓝蛋白。
6.如权利要求4或5所述的异菌脲抗原的制备方法,其特征在于:具体方法如下:取9.0mg异菌脲半抗原,溶解于1.0mL二甲基甲酰胺中,加氯甲酸异丁酯0.18mL,吡啶0.3mL,室温搅拌5h,得到半抗原活化液A液;取50mg牛血清白蛋白,充分溶解在3.8mL磷酸盐缓冲液PBS中,得到B液,将A液滴加到B液中,室温搅拌5h,用0.01mol/L PBS在4℃透析3天,每天换液3次,以除去未反应的小分子物质,得异菌脲抗原;分装,于-20℃保存备用。
7.如权利要求4或5所述的异菌脲抗原的制备方法,其特征在于:具体方法如下:取7.0mg异菌脲半抗原,溶解于1.0mL二甲基甲酰胺中,加氯化亚砜0.27mL,60℃反应6h,停止反应冷却到室温,得到半抗原活化液A液;取50mg卵清白蛋白,充分溶解在3.8mL磷酸盐缓冲液PBS中,得到B液,将A液滴加到B液中,室温反应8h,停止反应,用0.01mol/L的PBS缓冲液透析纯化3天,每天换液3次,得异菌脲抗原;分装,于-20℃保存备用。
8.如权利要求4所述方法制备的异菌脲抗原的应用,其特征在于:采用异菌脲抗原免疫动物得到的单克隆抗体,用于建立酶联免疫吸附测定方法和胶体金试纸快速测定法,从而实现烟草及食品中异菌脲的快速检测。
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