CN109232286A - 一种二甲戊灵半抗原与抗原的制备方法及应用 - Google Patents
一种二甲戊灵半抗原与抗原的制备方法及应用 Download PDFInfo
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- CN109232286A CN109232286A CN201811118385.6A CN201811118385A CN109232286A CN 109232286 A CN109232286 A CN 109232286A CN 201811118385 A CN201811118385 A CN 201811118385A CN 109232286 A CN109232286 A CN 109232286A
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- dimethylaniline
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/16—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions not involving the amino or carboxyl groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/04—Formation of amino groups in compounds containing carboxyl groups
- C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
- C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
- C07C227/20—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters by hydrolysis of N-acylated amino-acids or derivatives thereof, e.g. hydrolysis of carbamates
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- 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
- C07K14/765—Serum albumin, e.g. HSA
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- 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
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Abstract
一种二甲戊灵半抗原与抗原的制备方法及应用,其特征在于:所述二甲戊灵半抗原是由N‑(1‑乙基丙基)‑3,4‑二甲基苯胺与4‑溴丁酸乙酯反应生成烃基化N‑(1‑乙基丙基)‑3,4‑二甲基苯胺,再与浓硝酸反应生成硝基烃化N‑(1‑乙基丙基)‑3,4‑二甲基苯胺,然后与氢氧化钾反应得到;所述二甲戊灵抗原是由二甲戊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二甲戊灵抗原决定簇,使得筛选出高特异性的二甲戊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二甲戊灵的快速检测。
Description
技术领域
本发明涉及一种二甲戊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
二甲戊灵(Pendimethalin)属于二硝基苯胺类除草剂,其作用机制为抑制分生组织细胞分裂。双子叶植物吸收二甲戊灵的部位为下胚轴,单子叶植物吸收部位为幼芽。二甲戊灵适用于玉米、大豆、小麦、花生、棉花等多种旱地作物防除马唐、狗尾草、马齿苋等一年生禾本科和阔叶杂草。烟草上主要用于化学抑芽和烟草杂草防除。依据中国农药急性毒性分级标准,二甲戊灵被划分为一种低毒的化合物。依据美国环保局EPA的毒物分类标准,二甲戊灵属于III类,低毒化合物,安全摄入量为每天0.13 mg/kg。根据我国2016年发布的国家标准《GB2763-2016食品中农药最大残留限量》的规定,稻谷、韭菜、结球甘蓝、普通白菜、菠菜、芹菜、大白菜中二甲戊灵的最大残留限量为0.2 mg/kg,糙米、玉米、棉籽、大蒜、莴苣中二甲戊灵的最大残留限量为0.1 mg/kg。国际烟草科学研究合作中心(CORESTA)规定烟草中二甲戊灵的指导性残留限量为5 mg/kg。
目前二甲戊灵的检测方法主要是仪器检测方法,如气相色谱法、气相色谱-质谱法、气相色谱串联质谱法、高效液相色谱法、液相色谱串联质谱法,等。但是由于这些分析方法需要昂贵的大型仪器设备和专业的检测人员、前处理过程复杂、操作繁琐、检测成本高、分析速度慢,难以满足现场监测和大量样本中农药残留量快速筛查的需要。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。免疫分析为二甲戊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ELISA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。目前关于二甲戊灵半抗原及抗原的制备方法尚未见报道。
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二甲戊灵半抗原与抗原的制备方法及其应用。
本发明的目的是通过以下技术方案来实现的:
一种二甲戊灵半抗原的制备方法,是由N-(1-乙基丙基)-3,4-二甲基苯胺与4-溴丁酸乙酯反应生成烃基化N-(1-乙基丙基)-3,4-二甲基苯胺,再与浓硝酸反应生成硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺,然后与氢氧化钾反应得到的。其分子结构式为:
。
具体步骤如下:
1)取1.00 g N-(1-乙基丙基)-3,4-二甲基苯胺,加80 mL丙酮溶解,加0.35 g 氢氧化钾,加1.21 g 4-溴丁酸乙酯,加热回流反应3 h,TLC检测,原料全部反应完成,停止反应,冷却到室温,旋蒸,除去丙酮,加60 mL水,加50 mL二氯甲烷萃取,有机相无水硫酸钠干燥,蒸干浓缩,上硅胶柱,使用体积比为10:1的石油醚/乙酸乙酯洗脱分离,得到中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺1.51 g;
2)取1.50 g中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺,加5 mL浓硫酸溶解澄清,在冰浴下缓慢滴加浓硝酸3 mL,恢复至室温,继续搅拌4 h,停止反应,冰浴下加蒸馏水30mL,加氢氧化钠溶液调节pH值到7,加50 mL乙酸乙酯萃取,无水硫酸钠干燥蒸干,得到红色油状物,使用体积比为1:5的二氯甲烷/正己烷重结晶,得到硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺1.81 g;
3)取1.80 g硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺加2 mol/L氢氧化钾水溶液,60℃搅拌3 h,TLC检测,原料无剩余,停止反应,冷却到室温,加稀盐酸,调节pH值到6,加80 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1:1的乙醚/正己烷重结晶,得到二甲戊灵半抗原产物1.60 g。
所制备的二甲戊灵半抗原可用于制作动物免疫的抗原体系原料。
一种二甲戊灵抗原的制备方法,是由所述二甲戊灵半抗原与载体蛋白偶联得到。所述载体蛋白为牛血清白蛋白、鼠血清蛋白、兔血清蛋白、甲状腺蛋白、卵清蛋白、血蓝蛋白或人血清白蛋白。
具体步骤如下:
免疫抗原的制备:取二甲戊灵半抗原10 mg,加二甲基亚砜(DMSO)溶解,加碳二亚胺(EDC)5.7 mg,搅拌,澄清,加N-羟基琥珀酰亚胺(NHS)3.8 mg,室温搅拌活化3 h,得到A液;取人血清白蛋白(HSA)50 mg,加8 mL 0.1 mol/L PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h。停止反应,0.02 M PBS缓冲液透析3天,每天换液三次,得到二甲戊灵-HSA免疫原。
包被抗原的制备:取二甲戊灵半抗原6 mg,加0.2 mL 二甲基甲酰胺(DMF)溶解,澄清,加三乙胺20 μL,搅拌5 min,加氯甲酸异丁酯6 μL,搅拌2 h,得到半抗原活化液A液;取卵清蛋白(OVA)50 mg,加8 mL 0.05 mol/L pH 7.2 PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h。停止反应,0.02 M PBS缓冲液透析3天,每天换液三次,得到二甲戊灵-OVA包被原。
采用二甲戊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二甲戊灵的快速检测。
本发明中合成的二甲戊灵半抗原既最大程度的保留了二甲戊灵的化学结构,又有合适长度的连接臂,本发明制备的抗原呈现出特异性的二甲戊灵抗原决定簇,使得筛选出高特异性的二甲戊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二甲戊灵的快速检测。
附图说明
图1:二甲戊灵半抗原合成路线图,
图2:二甲戊灵半抗原核磁共振氢谱图。
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 二甲戊灵半抗原的制备
1、二甲戊灵半抗原的合成(合成路线见附图1)
1)取1.00 g N-(1-乙基丙基)-3,4-二甲基苯胺,加80 mL丙酮溶解,加0.35 g 氢氧化钾,加1.21 g 4-溴丁酸乙酯,加热回流反应3 h,TLC检测,原料全部反应完成,停止反应,冷却到室温,旋蒸,除去丙酮,加60 mL水,加50 mL二氯甲烷萃取,有机相无水硫酸钠干燥,蒸干浓缩,上硅胶柱,使用体积比为10:1的石油醚/乙酸乙酯洗脱分离,得到中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺1.51g,收率94.97%;
2)取1.50 g中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺,加5 mL浓硫酸溶解澄清,在冰浴下缓慢滴加浓硝酸3 mL,恢复至室温,继续搅拌4 h,停止反应,冰浴下加蒸馏水30mL,加氢氧化钠溶液调节pH值到7,加50 mL乙酸乙酯萃取,无水硫酸钠干燥蒸干,得到红色油状物,使用体积比为1:5的二氯甲烷/正己烷重结晶,得到硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺1.81g,收率93.30%;
3)取1.80 g硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺加2 mol/L氢氧化钾水溶液,60℃搅拌3 h,TLC检测,原料无剩余,停止反应,冷却到室温,加稀盐酸,调节pH值到6,加80 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1:1的乙醚/正己烷重结晶,得到二甲戊灵半抗原产物1.60 g,收率95.81%。
2、二甲戊灵半抗原的鉴定
核磁鉴定1H NMR(CDCl3,300MHZ)δ: 11.00 (1H, s), 2.398 (6H, s), 2.61 (1H,t), 3.655 (2H, s, J=7.500), 8.213 (14, 1H), 1.419 (4H, qd, J=7.126, J=7.017),1.923 (2H, tt, J=7.500, J=7.367), 0.829 (6H, t, J=7.126)。
化学位移δ=11.0的为间隔臂羧基氢共振吸收峰,δ=3.655、1.923、2.398的为间隔臂上亚甲基氢的共振吸收峰,这些峰的存在,证明间隔臂偶联成功。
实施例2 二甲戊灵抗原的制备
1、二甲戊灵免疫原的合成
二甲戊灵半抗原与人血清白蛋白(HSA)偶联得到免疫原。
取二甲戊灵半抗原10 mg,加二甲基亚砜(DMSO)溶解,加碳二亚胺(EDC)5.7 mg,搅拌,澄清,加N-羟基琥珀酰亚胺(NHS)3.8 mg,室温搅拌活化3 h,得到A液;取HAS 50 mg,加8mL 0.1 mol/L PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h。停止反应,0.02 M PBS缓冲液透析3天,每天换液三次,得到二甲戊灵-HSA免疫原,分装,-20℃保存。
2、二甲戊灵包被原的合成
二甲戊灵半抗原与卵清蛋白(OVA)偶联得到包被原。
取二甲戊灵半抗原6 mg,加0.2 mL 二甲基甲酰胺(DMF)溶解,澄清,加三乙胺20 μL,搅拌5 min,加氯甲酸异丁酯6 μL,搅拌2 h,得到半抗原活化液A液;取OVA 50 mg,加8 mL0.05 mol/L pH 7.2 PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h。停止反应,0.02 M PBS缓冲液透析3天,每天换液三次,得到二甲戊灵-OVA包被原,分装,-20℃保存。
3、二甲戊灵抗原的鉴定
按合成二甲戊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。偶联物二甲戊灵半抗原-载体蛋白的最大吸收峰与二甲戊灵半抗原、载体蛋白的最大吸收峰相比发生了明显的变化,表明二甲戊灵半抗原-载体蛋白的合成是成功的。经计算,半抗原与HSA的结合比为14:1,与OVA的结合比为9:1。
实施例3 二甲戊灵单克隆抗体的制备
1、杂交瘤细胞的获得
1)首次免疫:将二甲戊灵半抗原-HSA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二甲戊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:取出二甲戊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,得到二甲戊灵单克隆抗体溶液(-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:(200000~500000)。
间接竞争ELISA方法:用二甲戊灵半抗原-OVA偶联物包被酶标板,加入二甲戊灵标准品溶液、二甲戊灵单克隆抗体溶液,25℃反应30 min,倒出孔内液体,用洗涤液洗涤3~5次,用吸水纸拍干;加入辣根过氧化物酶标记的羊抗鼠抗抗体溶液,25℃反应30 min,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。
本实验将二甲戊灵、氟节胺、仲丁灵、氟乐灵、乙丁氟灵等二硝基苯胺类除草剂做系列稀释,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:
结果显示各类似物的交叉反应率为:二甲戊灵100%、氟节胺<1%、仲丁灵<1%、氟乐灵<1%、乙丁氟灵<1%。本发明抗体对氟节胺、仲丁灵、氟乐灵、乙丁氟灵等其他二硝基苯胺类除草剂无交叉反应,只针对二甲戊灵有特异性结合。
Claims (7)
1.一种二甲戊灵半抗原的制备方法,其特征在于:是由N-(1-乙基丙基)-3,4-二甲基苯胺与4-溴丁酸乙酯反应生成烃基化N-(1-乙基丙基)-3,4-二甲基苯胺,再与浓硝酸反应生成硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺,然后与氢氧化钾反应得到,其分子结构式为:
。
2.如权利要求1所述的二甲戊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1.00 g N-(1-乙基丙基)-3,4-二甲基苯胺,加80 mL丙酮溶解,加0.35 g 氢氧化钾,加1.21 g 4-溴丁酸乙酯,加热回流反应3 h,TLC检测,原料全部反应完成,停止反应,冷却到室温,旋蒸,除去丙酮,加60 mL水,加50 mL二氯甲烷萃取,有机相无水硫酸钠干燥,蒸干浓缩,上硅胶柱,使用体积比为10:1的石油醚/乙酸乙酯洗脱分离,得到中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺1.51 g;
2)取1.50 g中间体烃基化N-(1-乙基丙基)-3,4-二甲基苯胺,加5 mL浓硫酸溶解澄清,在冰浴下缓慢滴加浓硝酸3 mL,恢复至室温,继续搅拌4 h,停止反应,冰浴下加蒸馏水30mL,加氢氧化钠溶液调节pH值到7,加50 mL乙酸乙酯萃取,无水硫酸钠干燥蒸干,得到红色油状物,使用体积比为1:5的二氯甲烷/正己烷重结晶,得到硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺1.81 g;
3)取1.80 g硝基烃化N-(1-乙基丙基)-3,4-二甲基苯胺加2 mol/L氢氧化钾水溶液,60℃搅拌3 h,TLC检测,原料无剩余,停止反应,冷却到室温,加稀盐酸,调节pH值到6,加80 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1:1的乙醚/正己烷重结晶,得到二甲戊灵半抗原产物1.60 g。
3.如权利要求1所述方法制备的二甲戊灵半抗原的应用,其特征在于:所述二甲戊灵半抗原可用于制作动物免疫的抗原体系原料。
4.一种二甲戊灵抗原的制备方法,其特征在于:是由权利要求1制备得到的二甲戊灵半抗原与载体蛋白偶联得到,所述载体蛋白为牛血清白蛋白、鼠血清蛋白、兔血清蛋白、甲状腺蛋白、卵清蛋白、血蓝蛋白或人血清白蛋白。
5.如权利要求4所述的二甲戊灵抗原的制备方法,其特征在于:具体步骤如下:取二甲戊灵半抗原10 mg,加二甲基亚砜(DMSO)溶解,加碳二亚胺(EDC)5.7 mg,搅拌,澄清,加N-羟基琥珀酰亚胺(NHS)3.8 mg,室温搅拌活化3 h,得到A液;取人血清白蛋白50 mg,加8 mL0.1 mol/L磷酸盐缓冲液(PB)溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h;停止反应,0.02 M磷酸盐缓冲液(PBS)透析3天,每天换液3次,得二甲戊灵抗原;分装,-20℃保存。
6.如权利要求4所述的二甲戊灵抗原的制备方法,其特征在于:具体步骤如下:取二甲戊灵半抗原6 mg,加0.2 mL 二甲基甲酰胺(DMF)溶解,澄清,加三乙胺20 μL,搅拌5 min,加氯甲酸异丁酯6 μL,搅拌2 h,得到半抗原活化液A液;取卵清蛋白50 mg,加8 mL 0.05 mol/L pH 7.2 PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,室温搅拌反应5 h;停止反应,0.02 M PBS缓冲液透析3天,每天换液3次,得二甲戊灵抗原;分装,-20℃保存。
7.如权利要求4所述方法制备的二甲戊灵抗原的应用,其特征在于:采用所制备的二甲戊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二甲戊灵的快速检测。
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