CN108997161A - 一种甲霜灵半抗原与抗原的制备方法及应用 - Google Patents
一种甲霜灵半抗原与抗原的制备方法及应用 Download PDFInfo
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Abstract
一种甲霜灵半抗原与抗原的制备方法及应用,其特征在于:所述甲霜灵半抗原是由N‑(2,6‑二甲基苯基)丙氨酸甲酯与(4‑硝基苯氧基)乙酰氯反应生成硝苯基甲霜灵,再通过锌粉还原得到;所述甲霜灵抗原是由甲霜灵半抗原与载体蛋白偶联得到。本发明制备的抗原呈现出特异性的甲霜灵抗原决定簇,使得筛选出高特异性的甲霜灵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甲霜灵的快速检测。
Description
技术领域
本发明涉及一种甲霜灵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
甲霜灵(Metalaxyl)是一种强内吸性取代苯酰胺类杀菌剂,化学名N-(2-甲氧基乙酰基)-N-(二甲基苯基)-消旋-氨基苯酸甲酸,属酰胺类杀菌剂,高效低毒,主要用于卵菌纲、藻菌纲、真菌引起的各种霜霉病、晚疫病、早疫病、猝倒病以及枯腐病、果腐病等。主要抑制病菌菌丝体内蛋白质的合成,使其营养缺乏,不能正常生长而死亡。其内吸和渗透力很强,施药后30 min即可在植物体内上下双向传导,对病害植株有保护和治疗作用,对防治瓜果、蔬菜、烟草的霜毒病、疫病有较好疗效。但甲霜灵是环境中重要污染物之一,会对人类造成潜在的健康威胁,并污染生态环境,所以其在水果、蔬菜、烟草生产中的残留问题受到越来越多的关注。我国针对不同作物,制定了甲霜灵最大残留限量标准,其中黄瓜、辣椒、番茄的最大残留限量为0.5 mg/kg,糙米的最大残留限量为0.1 mg/kg,其他谷物最大残留限量为0.05 mg/kg。国际烟草科学研究合作中心(CORESTA)规定烟草中甲霜灵的指导性残留限量为2 mg/kg,在实际生产中,以2 mg/kg作为烟草最大残留量判定标准。
目前,国内外对甲霜灵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。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。免疫分析为甲霜灵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甲霜灵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:
一种甲霜灵半抗原的制备方法,是由N-(2,6-二甲基苯基)丙氨酸甲酯与(4-硝基苯氧基)乙酰氯反应生成硝苯基甲霜灵,再通过锌粉还原得到,其分子结构式为:
。
具体步骤如下:
1)取N-(2,6-二甲基苯基)丙氨酸甲酯2.00 g,加吡啶充分溶解,加2.30 g (4-硝基苯氧基)乙酰氯,搅拌,油浴加热,60℃反应2 h,TLC检测,原料基本反应完全,停止反应,恢复至室温,旋蒸,除去吡啶,加60 mL水,用1 mol/L盐酸调节pH值到5,加80 mL乙酸乙酯萃取,有机相无水硫酸钠干燥,上硅胶柱,使用体积比为1:10的乙酸乙酯/正己烷洗脱分离,得到中间产物硝苯基甲霜灵3.68 g;
2)取中间产物硝苯基甲霜灵3.60 g,加乙醇溶解,得到A液;取锌粉1.80 g,加10 mL蒸馏水,加稀盐酸0.5 mL,60℃活化20 min,加入A液,继续搅拌3 h,TLC检测,原料全部反应完成,停止反应,抽滤,除去锌粉,滤液蒸干,加50 mL水,用碳酸钠调节pH值到7,加50 mL乙酸乙酯萃取,有机相水洗,无水硫酸钠干燥蒸干,使用体积比为1:2的二氯甲烷/正己烷重结晶,得到甲霜灵半抗原产物3.22 g。
所述甲霜灵半抗原可用于制作动物免疫的抗原体系原料。
一种甲霜灵抗原的制备方法,是由甲霜灵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8 mg甲霜灵半抗原加1 mol/L的稀盐酸0.1 mL,加蒸馏水0.8 mL,0~5℃低温搅拌20 min,加含有1.7 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取50 mg牛血清白蛋白(BSA),加0.1 mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20 min,得到B液,将A液滴加到B液中,继续反应2 h。停止反应,0.02 mol/L 磷酸盐缓冲液(PBS)透析3 d,每天换液三次,分装,得到免疫原。
包被抗原的制备:取6 mg甲霜灵半抗原加1 mol/L的稀盐酸0.7 mL,加蒸馏水0.8mL,0~5℃低温搅拌20 min,加含有1.3 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取60 mg卵清蛋白(OVA),加0.1 mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20min,得到B液,将A液滴加到B液中,继续反应2 h。停止反应,0.02 mol/L PBS透析3 d,每天换液三次,分装,得到包被原。
采用甲霜灵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甲霜灵的快速检测。
本发明中合成的甲霜灵半抗原及抗原与申请号为201510529378.5和201510530296.2的专利中的半抗原及抗原结构不同。本发明中合成的甲霜灵半抗原既最大程度的保留了甲霜灵的化学结构,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甲霜灵抗原决定簇,使得筛选出高特异性的甲霜灵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甲霜灵的快速检测。
附图说明
图1:甲霜灵半抗原合成路线图。
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 甲霜灵半抗原的制备
1、甲霜灵半抗原的合成(合成路线见图1)
1)取N-(2,6-二甲基苯基)丙氨酸甲酯2.00 g,加吡啶充分溶解,加2.30 g (4-硝基苯氧基)乙酰氯,搅拌,油浴加热,60℃反应2 h,TLC检测,原料基本反应完全,停止反应,恢复至室温,旋蒸,除去吡啶,加60 mL水,用1 mol/L盐酸调节pH值到5,加80 mL乙酸乙酯萃取,有机相无水硫酸钠干燥,上硅胶柱,使用体积比为1:10的乙酸乙酯/正己烷洗脱分离,得到中间产物硝苯基甲霜灵3.68 g,收率98.92%;
2)取中间产物硝苯基甲霜灵3.60 g,加乙醇溶解,得到A液;取锌粉1.80 g,加10 mL蒸馏水,加稀盐酸0.5 mL,60℃活化20 min,加入A液,继续搅拌3 h,TLC检测,原料全部反应完成,停止反应,抽滤,除去锌粉,滤液蒸干,加50 mL水,用碳酸钠调节pH值到7,加50 mL乙酸乙酯萃取,有机相水洗,无水硫酸钠干燥蒸干,使用体积比为1:2的二氯甲烷/正己烷重结晶,得到甲霜灵半抗原产物3.22 g,收率96.99%。
2、甲霜灵半抗原的鉴定
核磁鉴定1H NMR(CDCl3, 300MHz)δ:4.778 (2H, q, J=7.047),3.646(3H),1.104(3H,d, J=7.047),4.52(t, 1H),7.313(1H, dd, J=7.888),2.260(t, 6H),7.41(1H, dd, J=7.888),6.865(1H, t, J=7.888),6.858(1H, ddd, J=8.804, J=0.545, J=0.000),6.272(t, 2H)。
图谱中,化学位移δ=6.272的为间隔臂上苯环氨基氢的共振吸收峰,δ=6.858、6.856的为间隔臂上苯环氢的共振吸收峰,这些峰的存在证明间隔臂偶联成功,甲霜灵半抗原结构正确。
实施例2 甲霜灵抗原的制备
1、甲霜灵免疫原的合成
甲霜灵半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取8 mg甲霜灵半抗原加1 mol/L的稀盐酸0.1 mL,加蒸馏水0.8 mL,0~5℃低温搅拌20 min,加含有1.7 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取50 mg BSA,加0.1 mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20 min,得到B液,将A液滴加到B液中,继续反应2 h。停止反应,0.02 mol/L PBS透析3 d,每天换液三次,分装,得到免疫原,-20℃保存。
2、甲霜灵包被原的合成
甲霜灵半抗原与卵清蛋白(OVA)偶联得到包被原。
取6 mg甲霜灵半抗原加1 mol/L的稀盐酸0.7 mL,加蒸馏水0.8 mL,0~5℃低温搅拌20 min,加含有1.3 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取60 mg OVA,加0.1 mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20 min,得到B液,将A液滴加到B液中,继续反应2 h。停止反应,0.02 mol/L PBS透析3 d,每天换液三次,分装,得到包被原,-20℃保存。
3、甲霜灵抗原的鉴定
按合成甲霜灵偶联抗原反应所用半抗原、载体蛋白与偶联产物的比例,进行紫外(200~ 400 nm)扫描测定,通过比较三者分别在260 nm和280 nm的吸光度值计算其结合比。偶联物甲霜灵半抗原-载体蛋白的最大吸收峰与甲霜灵半抗原、载体蛋白的最大吸收峰相比发生了明显的变化,表明甲霜灵半抗原-载体蛋白的合成是成功的。经计算,半抗原与BSA的结合比为13:1,与OVA的结合比为10:1。
实施例3 甲霜灵单克隆抗体的制备
1、杂交瘤细胞的获得
1)首次免疫:将甲霜灵半抗原-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,每只0.2 mL;
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0.1 mL,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甲霜灵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:取出甲霜灵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0.5 mL/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,得到甲霜灵单克隆抗体溶液(-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:用甲霜灵半抗原-OVA偶联物包被酶标板,加入甲霜灵标准品溶液、甲霜灵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15 min后,加入终止液终止反应;设定酶标仪于波长450 nm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。
本实验将甲霜灵及与其结构类似的化合物(毒草胺、甲草胺、乙草胺、异丙甲草胺、丙草胺、丁草胺)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:
结果显示甲霜灵及其结构类似物的交叉反应率为:甲霜灵100%、毒草胺<1%、甲草胺<1%、乙草胺<1%、异丙甲草胺<1%、丙草胺<1%、丁草胺<1%。本发明抗体对毒草胺、甲草胺、乙草胺、异丙甲草胺、丙草胺、丁草胺等与甲霜灵结构类似的化合物无交叉反应,只针对甲霜灵有特异性结合。
Claims (8)
1.一种甲霜灵半抗原的制备方法,其特征在于:是由N-(2,6-二甲基苯基)丙氨酸甲酯与(4-硝基苯氧基)乙酰氯反应生成硝苯基甲霜灵,再通过锌粉还原得到,其分子结构式为:
。
2.如权利要求1所述的甲霜灵半抗原的制备方法,其特征在于:该制备方法的具体步骤如下:
1)取N-(2,6-二甲基苯基)丙氨酸甲酯2.00 g,加吡啶充分溶解,加2.30 g (4-硝基苯氧基)乙酰氯,搅拌,油浴加热,60℃反应2 h,TLC检测,原料基本反应完全,停止反应,恢复至室温,旋蒸,除去吡啶,加60 mL水,用1 mol/L盐酸调节pH值到5,加80 mL乙酸乙酯萃取,有机相无水硫酸钠干燥,上硅胶柱,使用体积比为1:10的乙酸乙酯/正己烷洗脱分离,得到中间产物硝苯基甲霜灵3.68 g;
2)取中间产物硝苯基甲霜灵3.60 g,加乙醇溶解,得到A液;取锌粉1.80 g,加10 mL蒸馏水,加稀盐酸0.5 mL,60℃活化20 min,加入A液,继续搅拌3 h,TLC检测,原料全部反应完成,停止反应,抽滤,除去锌粉,滤液蒸干,加50 mL水,用碳酸钠调节pH值到7,加50 mL乙酸乙酯萃取,有机相水洗,无水硫酸钠干燥蒸干,使用体积比为1:2的二氯甲烷/正己烷重结晶,得到甲霜灵半抗原产物3.22 g。
3.如权利要求1所述方法制备的甲霜灵半抗原的应用,其特征在于:所述甲霜灵半抗原可用于制作动物免疫的抗原体系原料。
4.一种甲霜灵抗原的制备方法,其特征在于:是由权利要求1制备得到的甲霜灵半抗原与载体蛋白偶联得到。
5.如权利要求4所述的甲霜灵抗原的制备方法,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所述的甲霜灵抗原的制备方法,其特征在于:具体步骤如下:取8 mg甲霜灵半抗原加1 mol/L的稀盐酸0.1 mL,加蒸馏水0.8 mL,0~5℃低温搅拌20 min,加含有1.7 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取50 mg牛血清白蛋白,加0.1mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20 min,得到B液,将A液滴加到B液中,继续反应2 h;停止反应,0.02 mol/L 磷酸盐缓冲液透析3 d,每天换液三次,得甲霜灵抗原;分装, -20℃保存。
7.如权利要求4或5所述的甲霜灵抗原的制备方法,其特征在于:具体步骤如下:取6 mg甲霜灵半抗原加1 mol/L的稀盐酸0.7 mL,加蒸馏水0.8 mL,0~5℃低温搅拌20 min,加含有1.3 mg亚硝酸钠的水溶液0.1 mL,继续搅拌1 h,得到A液;取60 mg卵清蛋白,加0.1 mol/L碳酸钠溶液6 mL溶解,0~5℃搅拌平衡温度20 min,得到B液,将A液滴加到B液中,继续反应2h;停止反应,0.02 mol/L 磷酸盐缓冲液透析3 d,每天换液三次,得甲霜灵抗原;分装,-20℃保存。
8.如权利要求4所述方法制备的甲霜灵抗原的应用,其特征在于:采用甲霜灵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甲霜灵的快速检测。
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