CN109336779B - 一种甲霜灵半抗原与抗原的制备及应用 - Google Patents
一种甲霜灵半抗原与抗原的制备及应用 Download PDFInfo
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- CN109336779B CN109336779B CN201811104602.6A CN201811104602A CN109336779B CN 109336779 B CN109336779 B CN 109336779B CN 201811104602 A CN201811104602 A CN 201811104602A CN 109336779 B CN109336779 B CN 109336779B
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Abstract
一种甲霜灵半抗原与抗原的制备及应用,其特征在于:所述甲霜灵半抗原是由甲霜灵与4‑醛基丁酰氯在氯化铝的催化下进行取代反应得到的;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及应用。属于农药免疫化学技术领域。
背景技术
甲霜灵 (Metalaxyl) 是酰胺类内吸性杀菌剂,具有预防和治疗作用,用于防治瓜果、蔬菜霜霉病、疫病,马铃薯早、晚疫病,烟草黑胫病等。虽然甲霜灵对人畜的毒性不高,但属于内吸性杀菌,在食品和蔬菜中的残留量季节性超标情况较多。我国针对不同作物,制定了甲霜灵的最大残留限量标准,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,其他谷物的最大残留限量为0.05 mg/kg。美国、加拿大、日本、欧盟等国均制定了本国植物产品中甲霜灵的最大残留限量。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。
目前,甲霜灵残留检测的方法主要有LC-UV、LC-MS 、LC-MS/MS、GC-MS/MS等仪器方法,传统仪器分析方法具备检测灵敏度高、特异性强等优势,但样品前处理繁琐、耗时,同时检测需要昂贵的大型仪器和设备,并需配备专业的检测技术人员,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、***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。
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,是由甲霜灵与4-醛基丁酰氯在氯化铝的催化下进行取代反应得到的,其分子结构式为:
具体步骤如下:
取4-醛基丁酰氯0.53 g,加30 mL 1,2-二氯乙烷溶解,加0.44 g 氯化铝,室温搅拌3 h,滴加含有0.6 g甲霜灵的1,2-二氯乙烷溶液20 mL,回流反应4 h,冷却到室温,加40mL水,0.5 mL稀盐酸,振荡分层,分去水相,有机相饱和食盐水洗涤,干燥,浓缩,上硅胶柱,用体积比为1:1的石油醚-乙酸乙酯洗脱分离,得到甲霜灵半抗原产物-4醛丁酰化甲霜灵0.71 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,是由所述甲霜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、卵清蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白。
具体步骤如下:
免疫抗原的制备:取16 mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中,搅拌澄清,记为反应液A;称取牛血清白蛋白(BSA)30 mg,使之充分溶解在2.7 mL 0.1 mol/L柠檬酸盐缓冲液(CB,pH值为9.6)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3 M的硼氢化钠水溶液 0.5 mL还原反应4 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。
包被抗原的制备:取12 mg半抗原,溶解于1 mL DMF中溶解澄清,记为反应液A;称取卵清蛋白(OVA)30 mg,使之充分溶解在2.7 mL 0.1 mol/L CB(pH值为9.6)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3 M的硼氢化钠水溶液 0.5 mL还原反应4 h,用0.01 mol/L 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,得到包被原。
制备的甲霜灵抗原的应用:采用所述甲霜灵抗原免疫动物得到的单克隆抗体,可用于快速检测烟草及食品中的甲霜灵残留。
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 为甲霜灵半抗原合成路线图(该图为摘要附图);
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 甲霜灵半抗原的制备
1、甲霜灵半抗原的合成(合成路线见附图1)
取4-醛基丁酰氯0.53 g,加30 mL 1,2-二氯乙烷溶解,加0.44 g 氯化铝,室温搅拌3 h,滴加含有0.6 g甲霜灵的1,2-二氯乙烷溶液20 mL,回流反应4 h,冷却到室温,加40mL水,0.5 mL稀盐酸,振荡分层,分去水相,有机相饱和食盐水洗涤,干燥,浓缩,上硅胶柱,用体积比为1:1的石油醚-乙酸乙酯洗脱分离,得到甲霜灵半抗原产物-4醛丁酰化甲霜灵0.71 g,收率89.7%。
2、甲霜灵半抗原的鉴定
取上述半抗原经核磁共振氢谱鉴定,1H NMR(CDCl3,300 HZ)δ: 4.735 (4,1H,q, J=7.072),3.661 (5,3H),1.403 (7,3H,d,J=7.072),4.046 (13,2H),2.351 (15,3H),7.367(16,1H,d,J=8.304),2.110 (17,3H),7.011 (20,1H,d,J=8.304),3.206 (21,3H),2.608(23,2H,t,J=7.426),2.944 (24,2H,td,J=7.426,J=6.840),9.659 (25,1H,t,J=6.840)。
图谱中化学位移δ=2.9、2.6的为间隔臂上亚甲基氢的共振吸收峰,δ=9.6的是间隔臂上醛基共振吸收峰,这些氢的吸收峰的存在,证明半抗原合成成功。
实施例2 甲霜灵抗原的制备
1、甲霜灵免疫原的制备
甲霜灵半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取16 mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中,搅拌澄清,记为反应液A;称取BSA 30 mg,使之充分溶解在2.7 mL 0.1 mol/L柠檬酸盐缓冲液(CB,pH值为9.6)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3 M的硼氢化钠水溶液0.5 mL还原反应4 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。
2、甲霜灵包被原的制备
甲霜灵半抗原与卵清蛋白(OVA)偶联得到包被原。
取12 mg半抗原,溶解于1 mL DMF中溶解澄清,记为反应液A;称取OVA 30 mg,使之充分溶解在2.7 mL 0.1 mol/L CB(pH值为9.6)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3 M的硼氢化钠水溶液 0.5 mL还原反应4 h,用0.01 mol/L 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,得到包被原。
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18:1,与OVA的结合比为13:1。
实施例3 甲霜灵单克隆抗体的制备
1、动物免疫
将得到的免疫原注入Balb/c小鼠体内,免疫剂量为150 μg/只,使其产生抗血清。
2、细胞融合和克隆化
取免疫Balb/c小鼠脾细胞,按8:1(数量配比)比例与SP2/0骨髓瘤细胞融合,筛选得到稳定分泌甲霜灵单克隆抗体的甲霜灵单克隆抗体杂交瘤细胞株。
3、细胞冻存和复苏
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。
4、单克隆抗体的制备与纯化
增量培养法:将杂交瘤细胞置于细胞培养基中,在37 ℃条件下进行培养,用辛酸-饱和硫酸铵法将得到的培养液进行纯化,得到单克隆抗体,-20 ℃保存。
所述细胞培养基为向RPMI1640培养基中添加小牛血清和碳酸氢钠,使小牛血清在细胞培养基中的终浓度为20%(质量分数),碳酸氢钠在细胞培养基中的终浓度为0.2%(质量分数);所述细胞培养基的pH值为7.4。
5、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。
6、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。
实施例4 甲霜灵胶体金试纸条的制备
1、甲霜灵单克隆抗体-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)甲霜灵单克隆抗体-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甲霜灵单克隆抗体,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甲霜灵单克隆抗体-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,得到冻干有甲霜灵单克隆抗体-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0.1 mol/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:用磷酸缓冲液将甲霜灵半抗原-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 检测甲霜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粉碎后的待测样品至50 mL离心管中,加入10 mL 50%甲醇水溶液,涡旋1 min,3000 rpm以上离心5 min;取100 μL上清液,加入400 μL样本复溶液,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***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,表示样品中甲霜灵浓度低于检测限。
阳性(+):T线显色比C线显色浅或T线不显色,表示样品中甲霜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 检测甲霜灵的胶体金试纸条技术参数的确定
1、检测限试验
取空白农产品及烟草样品,在其中分别添加甲霜灵至终浓度为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及烟草样品时,当其中甲霜灵添加浓度为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;当其中甲霜灵添加浓度为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甲霜灵的检测限为0.1 mg/kg。
2、假阳性率、假阴性率试验
取已知甲霜灵含量大于0.1 mg/kg的阳性样品20份,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。说明本发明的检测甲霜灵的试纸条可以对农产品及烟草中的甲霜灵进行快速检测。
3、特异性试验
将毒草胺、甲草胺、乙草胺、异丙甲草胺、丙草胺、丁草胺等其他甲霜灵结构类似物用pH值为 7.2、0.2 mol/L的磷酸盐缓冲液稀释至1 mg/L,用甲霜灵试纸条进行检测。结果显示,用该试纸条检测1 mg/L毒草胺、甲草胺、乙草胺、异丙甲草胺、丙草胺、丁草胺时,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,特异性良好。
Claims (4)
2.一种甲霜灵抗原的制备方法,其特征在于:是由权利要求1制备的甲霜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、卵清蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白。
3.如权利要求2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16 mg半抗原,溶解于1 mL N,N-二甲基甲酰胺(DMF)中,搅拌澄清,记为反应液A;称取牛血清白蛋白30 mg,使之充分溶解在pH值为9.6的2.7 mL 0.1 mol/L柠檬酸盐缓冲液(CB)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3 M的硼氢化钠水溶液 0.5 mL还原反应4 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,得甲霜灵抗原;分装, -20℃保存备用。
4.如权利要求2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12 mg半抗原,溶解于1 mL DMF中溶解澄清,记为反应液A;称取卵清蛋白30 mg,使之充分溶解在pH值为9.6的2.7 mL 0.1 mol/L CB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3 M的硼氢化钠水溶液 0.5 mL还原反应4 h,用0.01 mol/L 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,得甲霜灵抗原;分装, -20℃保存备用。
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