CN109111394B - 一种啶虫脒半抗原与抗原的制备方法及应用 - Google Patents
一种啶虫脒半抗原与抗原的制备方法及应用 Download PDFInfo
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- CN109111394B CN109111394B CN201811104907.7A CN201811104907A CN109111394B CN 109111394 B CN109111394 B CN 109111394B CN 201811104907 A CN201811104907 A CN 201811104907A CN 109111394 B CN109111394 B CN 109111394B
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- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
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Abstract
一种啶虫脒半抗原与抗原的制备方法及应用,其特征在于:所述啶虫脒半抗原是由E‑N‑(4‑(6‑氯吡啶‑3‑基)2‑丁烯‑2‑基)氰胺与氨基丙酸反应得到;所述啶虫脒抗原是由啶虫脒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啶虫脒抗原决定簇,使得筛选出高特异性的啶虫脒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法和胶体金试纸快速测定法,从而实现烟草及食品中啶虫脒的快速检测。
Description
技术领域
本发明涉及一种啶虫脒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
啶虫脒(acetamiprid)属于新烟碱类杀虫剂,其主要通过对昆虫的神经突触后膜内的乙酰胆碱受体产生作用,进而有效杀灭昆虫,对蚜虫、叶蝉、烟粉虱、潜叶蛾等害虫防治效果较为理想,是一种广谱、高效、低毒的杀虫剂,在实际生产生活中得到广泛使用。
烟草作为一种吸食产品,其安全性越来越受到人们的关注,而农药残留是影响烟叶安全性的主要因素之一。啶虫脒作为一种烟碱类高效杀虫剂,虽然安全性高,但是其对人体的危害不容忽视,此外,近来欧盟由于担心烟碱类农药对蜜蜂等授粉昆虫造成的潜在风险而有可能提高该类杀虫剂的残留标准或禁止其使用。烟草作为一种出口商品,应未雨绸缪,提早应对欧盟等组织的严格禁令。因此,为避免啶虫脒等烟碱类农药残留对人体造成危害以及突破国外贸易壁垒,建立简单、快速、准确、可靠的啶虫脒残留量的检测方法很有必要。
目前,有关啶虫脒残留分析的研究主要集中于气相色谱、液相色谱及气相色谱-质谱联用技术、液相色谱-质谱联用技术。仪器分析方法虽可以实现准确定性定量,但需要复杂昂贵的仪器设备及专业操作人员,加之样品前处理繁琐费时、检测时间长、检测费用高,对于现场检测工作的开展具有局限性。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。免疫分析为啶虫脒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。目前关于啶虫脒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啶虫脒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:
一种啶虫脒半抗原的制备方法,是由E-N-(4-(6-氯吡啶-3-基)2-丁烯-2-基)氰胺与氨基丙酸反应得到,其分子结构为:
具体步骤如下:
取E-N-(4-(6-氯吡啶-3-基)2-丁烯-2-基)氰胺0.50 g,加甲醇50 mL溶解,加氨基丙酸0.30 g,搅拌,加37%甲醛水溶液0.37 mL,搅拌,混匀,80℃反应4 h;停止反应,旋蒸除去甲醇,加水50 mL,加乙酸乙酯100 mL萃取,萃取三次,合并有机相,旋蒸蒸干,上硅胶柱,二氯甲烷/甲醇(V/V,10/1)洗脱分离,得到半抗原产物羧基啶虫脒0.69 g。
所述啶虫脒半抗原可用于制作动物免疫的抗原体系原料。
一种啶虫脒抗原的制备方法,是由所述啶虫脒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、人血清白蛋白、卵清蛋白或血蓝蛋白。
具体步骤如下:
免疫抗原的制备:
取半抗原羧基啶虫脒11 mg,加二甲基亚砜1 mL溶解,加氯甲酸异丁酯0.18 mL,三乙胺0.1 mL,0~4℃反应1 h,得到半抗原活化液A液;取BSA 50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02 mol/L PB透析纯化3天,每天换液3次,得到免疫原,分装,-20℃保存。
包被抗原的制备:取半抗原羧基啶虫脒5 mg,加二甲基亚砜1 mL溶解,加二环己基碳二亚胺(DCC)9 mg,N-羟基琥珀酰亚胺(NHS)6 mg,室温反应1 h,得到半抗原活化液A液;取OVA 50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02mol/L PB透析纯化3天,每天换液3次,得到包被原,分装,-20℃保存。
采用所述啶虫脒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啶虫脒的快速检测。
本发明中合成的啶虫脒半抗原既最大程度的保留了啶虫脒的化学结构,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啶虫脒抗原决定簇,使得筛选出高特异性的啶虫脒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啶虫脒的快速检测。
附图说明
图1:啶虫脒半抗原合成路线图。
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 啶虫脒半抗原的制备
1、啶虫脒半抗原的合成
取E-N-(4-(6-氯吡啶-3-基)2-丁烯-2-基)氰胺0.50 g,加甲醇50 mL溶解,加氨基丙酸0.30 g,搅拌,加37%甲醛水溶液0.37 mL,搅拌,混匀,80℃反应4 h;停止反应,旋蒸除去甲醇,加水50 mL,加100 mL乙酸乙酯萃取,萃取三次,合并有机相,旋蒸蒸干,上硅胶柱,二氯甲烷/甲醇(V/V,10/1)洗脱分离,得到半抗原产物羧基啶虫脒0.69 g,收率92.81%。
2、啶虫脒半抗原的鉴定
取上述半抗原经核磁共振氢谱鉴定,1H NMR(CDCl3, 300MHZ)δ:11.0 (1H, -COOH), 8.53 (1H, s, ArH), 7.86 (1H, d, ArH), 7.21 (1H, d, ArH), 4.16 (1H, dd,=CH-), 3.91 (1H, s, -CH2-), 3.21 (2H, d, -CH2-), 2.82 (2H, t, -CH2-), 2.49(2H, t, -CH2-), 2.26 (3H, s, -CH3), 2.0 (1H, s, -NH-)。
化学位移δ=11的为间隔臂上羧基氢的共振吸收峰,δ=2.82, 2.49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 啶虫脒抗原的制备
1、啶虫脒免疫原的合成
啶虫脒半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取半抗原羧基啶虫脒11 mg,加二甲基亚砜1 mL溶解,加氯甲酸异丁酯0.18 mL,三乙胺0.1 mL,0~4℃反应1 h,得到半抗原活化液A液;取BSA 50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02 mol/L PB透析纯化3天,每天换液3次,得到免疫原,分装,-20℃保存。
2、啶虫脒包被原的合成
啶虫脒半抗原与卵清蛋白(OVA)偶联得到包被原。
取半抗原羧基啶虫脒5 mg,加二甲基亚砜1 mL溶解,加二环己基碳二亚胺(DCC)9mg,N-羟基琥珀酰亚胺(NHS)6 mg,室温反应1 h,得到半抗原活化液A液;取OVA 50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02 mol/L PB透析纯化3天,每天换液3次,得到包被原,分装,-20℃保存。
3、啶虫脒抗原的鉴定
按合成啶虫脒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 nm ~ 400 nm)扫描测定,通过比较三者分别在260 nm和280 nm的吸光值计算其结合比。偶联物啶虫脒半抗原-载体蛋白的最大吸收峰与啶虫脒半抗原、载体蛋白的最大吸收峰相比发生了明显的变化,表明啶虫脒半抗原-载体蛋白的合成是成功的。经计算,半抗原与BSA的结合比为14:1,与OVA的结合比为9:1。
实施例3 啶虫脒单克隆抗体的制备
1、杂交瘤细胞的获得
1)首次免疫:将啶虫脒半抗原-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啶虫脒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:取出啶虫脒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,得到啶虫脒单克隆抗体溶液(-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:用啶虫脒半抗原-OVA偶联物包被酶标板,加入啶虫脒标准品溶液、啶虫脒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。
本实验将烟碱类杀虫剂(啶虫脒、吡虫啉、烯啶虫胺、噻虫啉、噻虫嗪、噻虫胺、呋虫胺)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:
结果显示各类似物的交叉反应率为:啶虫脒100%、吡虫啉<1%、烯啶虫胺<1%、噻虫啉<1%、噻虫嗪<1%、噻虫胺<1%、呋虫胺<1%。本发明抗体对吡虫啉、烯啶虫胺、噻虫啉、噻虫嗪、噻虫胺、呋虫胺等其他烟碱类杀虫剂无交叉反应,只针对啶虫脒有特异性结合。
Claims (7)
2.如权利要求1所述方法制备的啶虫脒半抗原的应用,其特征在于:所述啶虫脒半抗原用于制作动物免疫的抗原体系原料。
3.一种啶虫脒抗原的制备方法,其特征在于:是由权利要求1制得的啶虫脒半抗原与载体蛋白偶联得到。
4.如权利要求3所述的啶虫脒抗原的制备方法,其特征在于:所述载体蛋白为牛血清白蛋白、人血清白蛋白、卵清蛋白或血蓝蛋白。
5.如权利要求3或4所述的啶虫脒抗原的制备方法,其特征在于:具体方法如下:取啶虫脒半抗原11 mg,加二甲基亚砜1 mL溶解,加氯甲酸异丁酯0.18 mL,三乙胺0.1 mL,0~4℃反应1 h,得到半抗原活化液A液;取牛血清白蛋白50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02 mol/L PB透析纯化3天,每天换液3次,得啶虫脒抗原;分装,-20℃保存。
6.如权利要求3或4所述的啶虫脒抗原的制备方法,其特征在于:具体方法如下:取啶虫脒半抗原5 mg,加二甲基亚砜1 mL溶解,加二环己基碳二亚胺9 mg,N-羟基琥珀酰亚胺6mg,室温反应1 h,得到半抗原活化液A液;取卵清蛋白50 mg,加0.8%盐水3 mL溶解,得到B液;将A液滴加到B液中,继续搅拌反应5 h,0.02 mol/L PB透析纯化3天,每天换液3次,得啶虫脒抗原;分装,-20℃保存。
7.如权利要求3所述方法制备的啶虫脒抗原的应用,其特征在于:采用啶虫脒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啶虫脒的快速检测。
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