CN109265364B - 一种二甲戊灵半抗原与抗原的制备及应用 - Google Patents
一种二甲戊灵半抗原与抗原的制备及应用 Download PDFInfo
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Abstract
一种二甲戊灵半抗原与抗原的制备及应用,其特征在于:所述二甲戊灵半抗原是由3‑(1‑乙基丙氨)‑6‑甲基‑2,4‑二硝基苯甲醛与3‑肼基丙酸反应得到;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及应用,属于农药免疫化学技术领域。
背景技术
二甲戊灵是一种旱田作物选择性除草剂,可以广泛应用于玉米、大豆、花生、棉花、直播旱稻、马铃薯、烟草、蔬菜等多种作物田除草。目前,二甲戊灵是世界第三大除草剂,销售额仅次于灭生性除草剂草甘膦、百草枯,也是世界上销售额最大的选择性除草剂。因此,在我国农业生产中的应用非常广泛。同时,二甲戊灵在农作物中的残留问题也受到了高度关注,我国规定二甲戊灵在稻谷中的最大残留限量(MRL)为0.2 mg/kg,在糙米、玉米、棉籽中的MRL为0.1 mg/kg,在韭菜、结球甘蓝、菠菜、芹菜、大白菜等蔬菜中的MRL为0.2 mg/kg,在大蒜、莴苣中的MRL为0.1 mg/kg;美国规定二甲戊灵在豆类、茶叶、大蒜中的MRL为0.1mg/kg,在韭菜中的MRL为0.2 mg/kg,在胡萝卜中的MRL为0.5 mg/kg;欧盟规定二甲戊灵在豆类、胡萝卜中的MRL为0.2 mg/kg,在芹菜中的MRL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(GRL)为5.00 mg/kg。
目前常用的二甲戊灵检测方法主要是仪器方法,如液相色谱串联质谱法、气相色谱串联质谱法等,采用这些分析方法需要昂贵的仪器和专门的技术人员,样品前处理过程复杂且花费高、费时长,难以满足大量样品和现场样品快速检测的需要。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。这一技术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。
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。免疫分析研究的关键是半抗原的分子设计、合成和人工抗原的制备。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。
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。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及应用。
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N3-(1-乙基丙氨)-6-甲基-2,4-二硝基苯甲醛与3-肼基丙酸反应得到,其分子结构式为:
具体步骤如下:
取0.60 g 3-(1-乙基丙氨)-6-甲基-2,4-二硝基苯甲醛,加50 mL乙腈溶解,澄清后,滴加溶解0.32 g 3-肼基丙酸的甲醇溶液5 mL,室温搅拌3 h;停止反应,旋蒸除去有机溶剂,加水,加氢氧化钠 0.45 g,溶解澄清后,加80 mL乙酸乙酯萃取,分去有机相,水相加稀盐酸调节pH=4,加50 mL氯仿萃取,水洗,浓缩,用体积比1:1的乙醇/石油醚重结晶,得到半抗原产物0.70 g。
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。
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,
其分子结构式为:
所述载体蛋白可为牛血清白蛋白、卵清蛋白、血蓝蛋白、甲状腺蛋白、人血清白蛋白。
免疫抗原制备:取15 mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中;加8 mg碳化二亚胺(EDC),室温下搅拌24 h,得到反应液A;称取牛血清白蛋白(BSA)30 mg,使之充分溶解在4 mL 0.1 mol/L磷酸盐缓冲液(PB,pH 7.0)中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L 磷酸盐缓冲液(PBS)4 ℃透析3 d,每天换3次透析液,以除去未反应的小分子物质,得到免疫原。
包被抗原制备:取20 mg半抗原,溶解于1 mL DMF中;加10 mg二环已基碳化二亚胺(DCC),室温下搅拌24 h,抽滤,除去不溶固体,得到反应液A;称取卵清蛋白(OVA)30 mg,使之充分溶解在6 mL 0.1 mol/L PB(pH 7.0)中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L PBS 4 ℃透析3d,每天换3次透析液,以除去未反应的小分子物质,得到包被原。
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为二甲戊灵半抗原合成路线图(该图作为摘要附图);
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。另外,本领域的技术人员在所附权利要求书限定的范围内可能会对本发明进行各种改动或修饰,这些改动或修饰同样应落入发明的保护范围。
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)
取0.60 g 3-(1-乙基丙氨)-6-甲基-2,4-二硝基苯甲醛,加50 mL乙腈溶解,澄清后,滴加溶解0.32 g 3-肼基丙酸的甲醇溶液5 mL,室温搅拌3 h;停止反应,旋蒸除去有机溶剂,加水,加氢氧化钠 0.45 g,溶解澄清后,加80 mL乙酸乙酯萃取,分去有机相,水相加稀盐酸调节pH=4,加50 mL氯仿萃取,水洗,浓缩,用体积比1:1的乙醇/石油醚重结晶,得到半抗原产物0.70 g,收率90.1%。
2、二甲戊灵半抗原的鉴定
取上述半抗原经核磁共振氢谱鉴定,1H-NMR(CDCl3,300 MHZ)δ:8.505 (10,1H),4.363 (11,1H,quint,J=6.864),8.326 (13,1H),2.247 (14,3H),1.525 (16,2H,qd,J=7.141,J=6.864),1.525 (17,2H,qd,J=7.141,J=6.864),0.822 (21,3H,t,J=7.141),0.822(22,3H,t,J=7.141),3.680 (23,2H,t,J=6.869),2.679 (24,2H,t,J=6.869)。化学位移δ=3.68、2.67为半抗原间隔臂上的氢的共振吸收峰,这两个峰的存在及位置表明半抗原合成成功。
实施例2 二甲戊灵抗原的制备
1、二甲戊灵免疫原的合成
二甲戊灵半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取15 mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中;加8 mg碳化二亚胺(EDC),室温下搅拌24 h,得到反应液A;称取BSA 30 mg,使之充分溶解在4 mL 0.1 mol/L磷酸盐缓冲液(PB,pH 7.0)中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L 磷酸盐缓冲液(PBS)4 ℃透析3 d,每天换3次透析液,以除去未反应的小分子物质,得到免疫原,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20 mg半抗原,溶解于1 mL DMF中;加10 mg二环已基碳化二亚胺(DCC),室温下搅拌24 h,抽滤,除去不溶固体,得到反应液A;称取OVA 30 mg,使之充分溶解在6 mL 0.1mol/L PB(pH 7.0)中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L PBS 4 ℃透析3d,每天换3次透析液,以除去未反应的小分子物质,得到包被原,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BSA的结合比为18:1,与OVA的结合比为10:1。
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-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:(300000~700000)。
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和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。
实施例4 二甲戊灵胶体金试纸条的制备
1、二甲戊灵单克隆抗体-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100 mL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1.5 mL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)二甲戊灵单克隆抗体-胶体金标记物的制备
在磁力搅拌下,用0.2 mol/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20~50 μg抗体的标准向胶体金溶液中加入上述二甲戊灵单克隆抗体,搅拌混匀,室温静置10 min,加入10% BSA使其在胶体金溶液中的终质量分数为1%,静置10 min。12000 r/min,4 ℃离心40 min,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0.02 mol/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100 μL二甲戊灵单克隆抗体-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3 h后,再真空干燥15 h,即可取出,得到冻干有二甲戊灵单克隆抗体-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:用磷酸缓冲液将二甲戊灵半抗原-卵清蛋白偶联物稀释到1 mg/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1.0 μL/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200 μg/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1.0 μL/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 检测二甲戊灵的胶体金试纸条的应用
1、样品的前处理
称取1.0±0.05 g匀浆后的待测样品至50 mL离心管中,加入10 mL乙腈,用旋涡仪涡动1 min,室温(20~25 ℃)3000 rpm以上离心5 min;取上清液2 mL至10 mL离心管中,在40-50 ℃水浴中氮气吹干,加入500 μL样本复溶液,用涡旋仪涡动2 min,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3 min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10 min,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,表示样品中二甲戊灵浓度低于检测限。
阳性(+):T线显色比C线显色浅或T线不显色,表示样品中二甲戊灵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 检测二甲戊灵的胶体金试纸条技术参数的确定
1、检测限试验
取空白农产品样品(如,谷物、蔬菜,等),在其中分别添加二甲戊灵至终浓度为0.05、0.1、0.2 mg/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样品时,当其中二甲戊灵添加浓度为0.05 mg/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;当其中二甲戊灵添加浓度为0.1、0.2 mg/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中二甲戊灵的检测限为0.1 mg/kg。
2、假阳性率、假阴性率试验
取已知二甲戊灵含量大于0.1 mg/kg的阳性样品20份,已知二甲戊灵含量小于0.1mg/kg的阴性样品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。说明本发明的检测二甲戊灵的试纸条可以对农产品中的二甲戊灵进行快速检测。
3、特异性试验
将氟节胺、仲丁灵、氟乐灵、乙丁氟灵等其他二硝基苯胺类除草剂用pH值为 7.2、0.2 mol/L的磷酸盐缓冲液稀释至5 mg/L,用二甲戊灵试纸条进行检测。结果显示,用该试纸条检测5 mg/L氟节胺、仲丁灵、氟乐灵、乙丁氟灵时,试纸条T线显色比C线显色深或与C线显色一致,呈阴性。说明本试纸条对氟节胺、仲丁灵、氟乐灵、乙丁氟灵等二甲戊灵结构类似物无交叉反应。
Claims (8)
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:
取0.60 g 3-(1-乙基丙氨)-6-甲基-2,4-二硝基苯甲醛,加50 mL乙腈溶解,澄清后,滴加溶解0.32 g 3-肼基丙酸的甲醇溶液5 mL,室温搅拌3 h;停止反应,旋蒸除去有机溶剂,加水,加氢氧化钠 0.45 g,溶解澄清后,加80 mL乙酸乙酯萃取,分去有机相,水相加稀盐酸调节pH=4,加50 mL氯仿萃取,水洗,浓缩,用体积比1:1的乙醇/石油醚重结晶,得到半抗原产物0.70 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用于制作动物免疫的抗原体系原料。
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:所述载体蛋白为牛血清白蛋白、卵清蛋白、血蓝蛋白、甲状腺蛋白、人血清白蛋白。
6.如权利要求4或5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15mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中,加8 mg EDC,室温下搅拌24 h,得到反应液A;称取牛血清白蛋白30 mg,使之充分溶解在4 mL 0.1 mol/L、 pH 7.0的磷酸盐缓冲液中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L 磷酸盐缓冲液4 ℃透析3 d,每天换3次透析液,以除去未反应的小分子物质,得二甲戊灵抗原,分装,-20℃保存。
7.如权利要求4或5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20mg半抗原,溶解于1 mL DMF中;加10 mg二环已基碳化二亚胺(DCC),室温下搅拌24 h,抽滤,除去不溶固体,得到反应液A;称取卵清蛋白30 mg,使之充分溶解在6 mL 0.1 mol/L、pH7.0的磷酸盐缓冲液中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24 h,用0.01 mol/L的磷酸盐缓冲液 4 ℃透析3d,每天换3次透析液,以除去未反应的小分子物质,得二甲戊灵抗原,分装,-20℃保存。
8.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:采用二甲戊灵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二甲戊灵的快速检测。
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