CN109053477A - 一种仲丁灵半抗原与抗原的制备方法及应用 - Google Patents
一种仲丁灵半抗原与抗原的制备方法及应用 Download PDFInfo
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Abstract
一种仲丁灵半抗原与抗原的制备方法及应用,其特征在于:所述仲丁灵半抗原是由4‑氯‑3,5‑二硝基苯乙酸与丁胺反应得到;所述仲丁灵抗原是由仲丁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仲丁灵抗原决定簇,使得筛选出高特异性的仲丁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仲丁灵的快速检测。
Description
技术领域
本发明涉及一种仲丁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
仲丁灵(Butralin)又名地乐胺、双丁乐灵、硝苯胺灵、比达宁、止芽素,化学名称为N-仲丁基-4-特丁基-2,6-二硝基苯胺,分子式为C14H21N3O4。仲丁灵为二硝基苯胺类除草剂,纯品为橙黄色结晶体,易溶于有机溶剂,而难溶于水,有挥发性,受光后易分解,对人畜等动物类毒性较低。该药为选择性萌前除草剂,其作用与氟乐灵相似。药剂进入植物体后,主要抑制分生组织的细胞***,从而抑制杂草的幼芽及幼根生长,导致杂草死亡。仲丁灵可应用于大田作物抑制杂草生长,也可用于水产养殖清除水体中的杂藻,仲丁灵在烟草生产中应用较多,主要用来抑制烟草叶芽生长。国际烟草科学研究合作中心(CORESTA)规定烟草中仲丁灵的指导性残留限量为5 mg/kg。
目前,有关仲丁灵残留检测的研究多集中在气相色谱法、液相色谱法、气相色谱串联质谱法、液相色谱串联质谱等色谱质谱方法。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。免疫分析为仲丁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、***磷、氟虫腈、二氯喹啉酸、克百威、***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。目前关于仲丁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仲丁灵半抗原与抗原的制备方法。
本发明的目的是通过以下技术方案来实现的:
一种仲丁灵半抗原的制备方法,是由4-氯-3,5-二硝基苯乙酸与丁胺反应得到,其分子结构式为:
。
具体步骤如下:
取4-氯-3,5-二硝基苯乙酸1.0 g,加50 mL乙醇溶解,加碳酸钠0.45 g,搅拌,加丁胺0.31 g,80℃油浴加热,搅拌3 h,检测,原料基本反应完全;停止反应,冷却到室温,旋蒸,除去乙醇,加80 mL水,用1 mol/L盐酸调节pH值到4,有大量浑浊,加80 mL乙酸乙酯萃取,水洗,上硅胶柱,用体积比为5:1的二氯甲烷/甲醇洗脱分离,得到仲丁灵半抗原产物1.05 g。
所述仲丁灵半抗原可用于制作动物免疫的抗原体系原料。
一种仲丁灵抗原的制备方法,是由所述仲丁灵半抗原与载体蛋白偶联得到。所述仲丁灵抗原为仲丁灵半抗原与载体蛋白的偶联物,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:取仲丁灵半抗原11 mg,加二甲基亚砜(DMSO)0.2 mL溶解,加三乙胺20 μL,搅拌,混匀,加氯甲酸异丁酯6 mg,搅拌2 h,得到半抗原活化液A液;取50 mg牛血清白蛋白(BSA)加0.1 mol/L pH值为7.2的PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,继续搅拌5 h,停止反应,0.02 mol/L PBS透析3 d,每天换液3次,得到仲丁灵-BSA偶联物,即为免疫原。
包被抗原的制备:取仲丁灵半抗原6 mg,加N,N-二甲基甲酰胺(DMF)0.2 mL溶解,加碳二亚胺(EDC)4.5 mg,搅拌,澄清,加N-琥珀酰亚胺(NHS)2.24 mg,室温搅拌活化2 h,得到A液;取卵清蛋白(OVA)50 mg,加6 mL 0.05 mol/L pH值为7.2的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 mol/L PBS透析3 d,每天换液3次,得到仲丁灵-OVA偶联物,即为包被原。
采用所述仲丁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仲丁灵的快速检测。
本发明中合成的仲丁灵半抗原既最大程度的保留了仲丁灵的化学结构,又有合适长度的连接臂,用该半抗原制备的抗原呈现出特异性的仲丁灵抗原决定簇,使得筛选出高特异性的仲丁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仲丁灵的快速检测。
附图说明
图1:仲丁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 仲丁灵半抗原的制备
1、仲丁灵半抗原的合成(合成路线见附图1)
取4-氯-3,5-二硝基苯乙酸1.00 g,加50 mL乙醇溶解,加碳酸钠0.45 g,搅拌,加丁胺0.31 g,80℃油浴加热,搅拌3 h,检测,原料基本反应完全;停止反应,冷却到室温,旋蒸,除去乙醇,加80 mL水,用1 mol/L盐酸调节pH值到4,有大量浑浊,加80 mL乙酸乙酯萃取,水洗,上硅胶柱,用体积比为5:1的二氯甲烷/甲醇洗脱分离,得到仲丁灵半抗原产物1.05 g,收率92.11%。
2、仲丁灵半抗原的鉴定
核磁鉴定1H NMR(CDCl3, 300MHz)δ:11.01(1H, d, J=0.000),8.281(1H, d, J=0.000),4.011(1H, tq, J=6.914, J=6.757),8.281(1H, d, J=0.000),3.822(t, 2H),2.791(1H, ddd, J=7.114, J=6.914),1.591(2H, d, J=6.757),1.251(3H, t, J=7.114),0.901(3H, t, J=7.114)。
图谱中,化学位移δ=1.591、1.251、0.901、2.791的为原料丁胺上甲基和亚甲基氢共振吸收峰,δ=4.011的为原料丁胺反应后形成亚胺氢的共振吸收峰,这些峰的存在证明间隔臂偶联成功,仲丁灵半抗原结构正确。
实施例2 仲丁灵抗原的制备
1、仲丁灵免疫抗原的合成
仲丁灵半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取仲丁灵半抗原11 mg,加二甲基亚砜(DMSO)0.2 mL溶解,加三乙胺20 μL,搅拌,混匀,加氯甲酸异丁酯6 mg,搅拌2 h,得到半抗原活化液A液;取50 mg BSA加0.1 mol/L pH值为7.2的PB缓冲液溶解,得到B液,将A液缓慢滴加到B液中,继续搅拌5 h,停止反应,0.02mol/L PBS透析3 d,每天换液3次,得到仲丁灵-BSA偶联物,即为免疫原,分装,-20℃保存。
2、仲丁灵包被抗原的合成
仲丁灵半抗原与卵清蛋白(OVA)偶联得到包被原。
取仲丁灵半抗原6 mg,加N,N-二甲基甲酰胺(DMF)0.2 mL溶解,加碳二亚胺(EDC)4.5 mg,搅拌,澄清,加N-琥珀酰亚胺(NHS)2.24 mg,室温搅拌活化2 h,得到A液;取OVA 50mg,加6 mL 0.05 mol/L pH值为7.2的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 mol/L PBS透析3 d,每天换液3次,得到仲丁灵-OVA偶联物,即为包被原,分装,-20℃保存。
3、仲丁灵抗原的鉴定
按合成仲丁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。偶联物仲丁灵半抗原-载体蛋白的最大吸收峰与仲丁灵半抗原、载体蛋白的最大吸收峰相比发生了明显的变化,表明仲丁灵半抗原-载体蛋白的合成是成功的。经计算,半抗原与BSA的结合比为12:1,与OVA的结合比为9:1。
实施例3 仲丁灵单克隆抗体的制备
1、杂交瘤细胞的获得
1)首次免疫:将仲丁灵半抗原-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仲丁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:取出仲丁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,得到仲丁灵单克隆抗体溶液(-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:(150000~400000)。
间接竞争ELISA方法:用仲丁灵半抗原-OVA偶联物包被酶标板,加入仲丁灵标准品溶液、仲丁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。
本实验将仲丁灵及烟草中常见的其他二硝基苯胺类除草剂(氟节胺、异丙乐灵、二甲戊灵、乙丁氟灵、氟乐灵)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:
结果显示仲丁灵及烟草中常见的其他二硝基苯胺类除草剂的交叉反应率为:仲丁灵100%、氟节胺<1%、异丙乐灵<1%、二甲戊灵<1%、乙丁氟灵<1%、氟乐灵<1%。本发明抗体对氟节胺、异丙乐灵、二甲戊灵、乙丁氟灵、氟乐灵等常见的其他二硝基苯胺类除草剂无交叉反应,只针对仲丁灵有特异性结合。
Claims (7)
1.一种仲丁灵半抗原的制备方法,其特征在于:是由4-氯-3,5-二硝基苯乙酸与丁胺反应得到,其分子结构式为:
。
2.如权利要求1所述的仲丁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:
取4-氯-3,5-二硝基苯乙酸1.0 g,加50 mL乙醇溶解,加碳酸钠0.45 g,搅拌,加丁胺0.31 g,80℃油浴加热,搅拌3 h,检测,原料基本反应完全;停止反应,冷却到室温,旋蒸,除去乙醇,加80 mL水,用1 mol/L盐酸调节pH值到4,有大量浑浊,加80 mL乙酸乙酯萃取,水洗,上硅胶柱,用体积比为5:1的二氯甲烷/甲醇洗脱分离,得到仲丁灵半抗原产物1.05 g。
3.如权利要求1所述方法制备的仲丁灵半抗原的应用,其特征在于:所述仲丁灵半抗原可用于制作动物免疫的抗原体系原料。
4.一种仲丁灵抗原的制备方法,其特征在于:所述仲丁灵抗原为仲丁灵半抗原与载体蛋白的偶联物,是由权利要求1所述仲丁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。
5.如权利要求4所述的仲丁灵抗原的制备方法,其特征在于:具体步骤如下:取仲丁灵半抗原11 mg,加二甲基亚砜(DMSO)0.2 mL溶解,加三乙胺20 μL,搅拌,混匀,加氯甲酸异丁酯6 mg,搅拌2 h,得到半抗原活化液A液;取50 mg牛血清白蛋白(BSA)加0.1 mol/L pH值为7.2的磷酸盐缓冲液(PB)溶解,得到B液,将A液缓慢滴加到B液中,继续搅拌5 h,停止反应,0.02 mol/L PBS透析3天,每天换液3次,得到仲丁灵-BSA偶联物即仲丁灵抗原,分装,-20℃保存。
6.如权利要求4所述的仲丁灵抗原的制备方法,其特征在于:具体步骤如下:取仲丁灵半抗原6 mg,加N,N-二甲基甲酰胺(DMF)0.2 mL溶解,加碳二亚胺(EDC)4.5 mg,搅拌,澄清,加N-琥珀酰亚胺(NHS)2.24 mg,室温搅拌活化2 h,得到A液;取卵清蛋白(OVA)50 mg,加6mL 0.05 mol/L pH值为7.2的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 mol/L PBS透析3天,每天换液3次,得到仲丁灵-OVA偶联物即仲丁灵抗原,分装,-20℃保存。
7.如权利要求4所述方法制备的仲丁灵抗原的应用,其特征在于:采用仲丁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仲丁灵的快速检测。
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CN114480295A (zh) * | 2022-01-18 | 2022-05-13 | 江南大学 | 一株分泌抗仲丁灵单克隆抗体杂交瘤细胞株及其应用 |
CN114480295B (zh) * | 2022-01-18 | 2023-08-22 | 江南大学 | 一株分泌抗仲丁灵单克隆抗体杂交瘤细胞株及其应用 |
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