CN113698394B - 一种丙环唑半抗原及其制备方法和应用 - Google Patents
一种丙环唑半抗原及其制备方法和应用 Download PDFInfo
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- CN113698394B CN113698394B CN202111125286.2A CN202111125286A CN113698394B CN 113698394 B CN113698394 B CN 113698394B CN 202111125286 A CN202111125286 A CN 202111125286A CN 113698394 B CN113698394 B CN 113698394B
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Classifications
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- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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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
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Abstract
本发明公开了一种丙环唑半抗原及其制备方法和应用。丙环唑半抗原采用从头合成法,在丙环唑的苯环上连接含有4个C原子的连接臂,并在连接臂末端引入可以和载体蛋白偶联的羧基基团。利用该半抗原制备的单克隆抗体灵敏度高,特异性强。用间接竞争酶联免疫测定(ic‑ELISA)的方法测得其灵敏度(IC50)为2.33μg/L,检测范围(IC10‑IC90)为0.26‑21.28μg/L。该单克隆抗体与丙环唑类似物均无交叉反应(CR<0.1%)。此外,利用该单克隆抗体制备了检测灵敏度高、特异性强、简便快速、成本低的丙环唑胶体金快速检测试纸条。该试纸条可用于检测农产品中丙环唑的残留,其检测限达5μg/L。
Description
技术领域
本发明属于生物技术领域,具体涉及一种丙环唑半抗原及其制备方法和应用。
背景技术
丙环唑是一种兼具保护和治疗作用的内吸性三唑类杀菌剂,它被广泛用于防治谷类、蔬菜、水果上白粉菌、锈菌、炭疽病菌等真菌的危害。其作用机理是通过影响真菌麦角甾醇的生物合成和抑制真菌类固醇的代谢从而抑制真菌的生长发育。此外,丙环唑作为一种植物生长调节剂具有较好的控旺压苗作用,常被用于控制菜心、普通白菜、芥蓝等叶菜的外形,促进茎叶粗壮和叶色深绿,增加产量。
丙环唑的广泛应用引发了人类对其是否会产生健康危害的思考。许多研究报道了丙环唑的过量施用损伤实验动物的肝脏功能,影响神经行为,内分泌调节及细胞增殖。因此,丙环唑的不合理使用引起食品中农药残留的超标严重影响人类的身体健康。
我国于2021年在国标中规定,丙环唑在农产品上最低的最大残留限量为0.02mg/kg(菠萝,甘蔗,黑麦等)。2005年,欧盟规定了农产品中丙环唑最低的最大残留限量为0.05mg/kg(叶菜类等)。因此,为了保障食品安全及对外贸易的发展,建立灵敏、准确、方便、省时的丙环唑检测方法是非常必要的。
当前,丙环唑的测定方法主要为仪器检测法,包括气相色谱法、气相色谱-串联质谱法、液相色谱法及液相色谱-串联质谱法等。仪器检测法虽然具有高精密度和准确度的优点,但是仪器价格昂贵,需要专业技术性人员操作等缺点使其不能被广泛应用。免疫检测法对仪器设备及专业人员的要求低,适用于大量样品的检测,能够克服仪器检测法的不足。目前,用于免疫检测的抗体主要为多克隆抗体和单克隆抗体,其中单克隆抗体因批次差异小、可大量制备等优点,是建立免疫检测方法和开发免疫检测产品的首选抗体。酶联免疫免疫吸附分析(Enzyme Linked Immunosorbent Assay,ELISA)和胶体金试纸条是常用的免疫检测方法,ELISA具有灵敏度高、准确性好、成本低等优点,且可对大量样品进行高通量的定量检测;试纸条具有操作简便、快速、经济等优点,且检测结果呈现直观。然而,免疫检测法的灵敏度、特异性取决于抗体的质量,而半抗原的结构决定了是否能够获得高质量的抗体。因此,半抗原的结构对于免疫方法的建立起着决定性的作用。
发明内容
本发明提供了一种丙环唑半抗原及其制备和应用。
第一方面,该发明设计的丙环唑半抗原结构式如式I。
第二方面,本发明提供了一种上述丙环唑半抗原的制备方法,其制备步骤如下:
步骤1)1.52g N-溴代琥珀酰亚胺与2g 2-氯-4-溴-苯乙酮加入12mL乙腈中,在12℃的条件下搅拌反应10分钟。随后2.95g对甲苯磺酸加入上述混合液中,并在15-80℃的条件下搅拌反应1.17小时。反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物1。
步骤2)4g产物1与2g 1,2-戊二醇加入24mL甲苯中,然后加入12mL正丁醇和1.1g对甲苯磺酸,上述混合物在135℃下搅拌反应15h。反应结束后其产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物2。
步骤3)1.39g KOH和0.73g 1H-1,2,4-苯三唑溶于14mL DMSO中,将2.8g产物2在45℃条件下加入上述混合物中,该混合物在135℃搅拌反应15h。反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物3。
步骤4)1.77g产物3溶于7.5mL四氢呋喃中,5.56g三乙胺和0.16g双(三苯基膦)氯化钯(II)加入上述溶液。随后0.087g CuI也加入上述溶液中(在CuI加入前后需用氮气净化混合溶液三次)。然后1.03g 4-戊炔酸甲酯加入到混合溶液中并用氮气净化三次。15℃搅拌10min后,0.087g CuI加入到混合溶液中并于75℃条件下持续搅拌12h。反应结束后,产物通过硅胶柱纯化,用体积比为50∶1到1∶1的石油醚-乙酸乙酯洗脱分离,得产物4。
步骤5)1.77g产物4和0.096g二氧化铂加入20mL乙酸乙酯中,用氢气净化三次后,上述混合物在30℃,氢气压强50psi条件下搅拌反应6h。反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物5。
步骤6)1.2g产物5与0.12g单水氢氧化锂加入6.5mL四氢呋喃并于15-25℃条件下搅拌反应15h。反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,获得无色油状的丙环唑半抗原。
第三方面,本发明提供了一种丙环唑人工抗原,其通过上述的丙环唑半抗原与载体蛋白偶联获得。丙环唑人工抗原包括丙环唑免疫原和包被原,丙环唑免疫原是丙环唑半抗原与牛血清白蛋白(BSA)偶联获得,丙环唑包被原是丙环唑半抗原与鸡卵白蛋白(OVA)偶联制得。
丙环唑人工抗原的制备包括如下步骤:
步骤1)半抗原的活化。取0.082g如权利要求1所述丙环唑半抗原溶于1.5mLN,N-二甲基甲酰胺中,向上述溶液中加入0.023g N-羟基琥珀酰亚胺后,室温下搅拌15分钟,随后向混合物中加入1mL含0.041g N,N-二环己基碳二亚胺的N,N-二甲基甲酰胺溶液,搅拌反应过夜。
步骤2)半抗原与载体蛋白的偶联。将步骤1反应后的产物4000rpm离心5分钟,取上清1.25mL缓慢滴加到5mL 10mg/mL牛血清白蛋白的磷酸缓冲液(0.01mol/L,pH=8)中,室温下避光搅拌反应4h后得到丙环唑免疫抗原;另取步骤1离心后的产物1.25mL缓慢滴加到5mL10mg/mL鸡卵白蛋白的磷酸缓冲液(0.01mol/L,pH=8)中,室温下避光搅拌反应4h即得到丙环唑包被抗原。
步骤3)人工抗原的纯化。将步骤2得到的丙环唑免疫抗原和丙环唑包被抗原用0.01mol/L PBS缓冲液透析纯化3天,取出后4000rpm离心5min,取上清测浓度并分装冻存。
第五方面,本发明提供了一种单克隆抗体,所述的单克隆抗体特异性识别上述的丙环唑半抗原或上述的丙环唑抗原。
第六方面,本发明提供了一种检测环境样品和农产品中丙环唑残留的胶体金试纸条。
本发明具有以下有益效果:
本发明提供的丙环唑半抗原不仅最大程度保留了丙环唑的特征结构,使其具有强的免疫原性,而且具有偶联载体蛋白的羧基,使丙环唑抗原易于合成;用本发明丙环唑免疫抗原免疫小鼠,更有利于刺激机体免疫应答,产生特异性更强、灵敏度更高的抗体,为建立丙环唑的免疫检测方法提供基础。
采用本发明的丙环唑免疫抗原得到的丙环唑单克隆抗体的效价、特异性、亲和力都较好,应用该抗体建立的ELISA的抑制中浓度(IC50)为2.33μg/L,线性范围为0.26-21.28μg/L,与丙环唑类似物的交叉反应率低于0.1%;基于该单克隆抗体制备的胶体金试纸条,其最低检测限可达5μg/L,具有较好的敏感性和特异性,且操作简单、使用方便,能够满足食品和环境样品中丙环唑的快速检测。
附图说明
图1为丙环唑半抗原合成路线图。
图2为丙环唑ELISA标准曲线。
图3为丙环唑胶体金试纸条俯瞰结构示意图。
图4为丙环唑胶体金试纸条剖面结构示意图;图中,1:衬板,2:样品垫,3:金标结合垫,4:纤维素膜,5:隐形检测线,6:隐形对照线,7:吸水垫,8-1:样品浸入端保护膜,8-2:手柄端保护膜,9:标识线。
图5为丙环唑胶体金试纸条结果判定示意图;图中,A、B为阴性样品检测结果,C、D为强阳性样品检测结果,E、F为试纸条失效。
具体实施方式
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:丙环唑半抗原的合成
步骤1)1.52g N-溴代琥珀酰亚胺与2g 2-氯-4-溴-苯乙酮加入12mL乙腈中,在12℃的条件下搅拌反应10分钟。随后2.95g对甲苯磺酸加入上述混合液中,并在15-80℃的条件下搅拌反应1.17小时。反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物1。
步骤2)4g产物1与2g 1,2-戊二醇加入24mL甲苯中,然后加入12mL正丁醇和1.1g对甲苯磺酸,上述混合物在135℃下搅拌反应15h。反应结束后其产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物2。
步骤3)1.39g KOH和0.73g 1H-1,2,4-苯三唑溶于14mL DMSO中,将2.8g产物2在45℃条件下加入上述混合物中,该混合物在135℃搅拌反应15h。反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物3。
步骤4)1.77g产物3溶于7.5mL四氢呋喃中,5.56g三乙胺和0.16g双(三苯基膦)氯化钯(II)加入上述溶液。随后0.087g CuI也加入上述溶液中(在CuI加入前后需用氮气净化混合溶液三次)。然后1.03g 4-戊炔酸甲酯加入到混合溶液中并用氮气净化三次。15℃搅拌10min后,0.087g CuI加入到混合溶液中并于75℃条件下持续搅拌12h。反应结束后,产物通过硅胶柱纯化,用体积比为50∶1到1∶1的石油醚-乙酸乙酯洗脱分离,得产物4。
步骤5)1.77g产物4和0.096g二氧化铂加入20mL乙酸乙酯中,用氢气净化三次后,上述混合物在30℃,氢气压强50psi条件下搅拌反应6h。反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物5。
步骤6)1.2g产物5与0.12g单水氢氧化锂加入6.5mL四氢呋喃并于15-25℃条件下搅拌反应15h。反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,获得无色油状的丙环唑半抗原。
实施例2:丙环唑半抗原的鉴定
丙环唑半抗原核磁鉴定的结果:1H NMR(500MHz,DMSO-d6)δ8.36(d,J=7.1Hz,1H),7.84(d,J=16.3Hz,1H),7.41(dd,J=14.9,7.9Hz,1H),7.31(dd,J=6.9,1.6Hz,1H),7.13(ddd,J=18.7,8.0,1.7Hz,1H),4.77-4.6(m,2H),3.91-3.75(m,2H),3.15(q,J=6.6,5.5Hz,1H),2.58(td,J=7.5,3.3Hz,2H),2.23(t,J=7.3Hz,2H),2.07(s,1H),1.62-1.46(m,4H),1.27(tdt,J=25.7,14.3,7.1Hz,4H),0.92-0.72(m,3H)。
实施例3:丙环唑抗原的制备
步骤1)半抗原的活化。取0.082g如权利要求1所述丙环唑半抗原溶于1.5mLN,N-二甲基甲酰胺中,向上述溶液中加入0.023gN-羟基琥珀酰亚胺后,室温下搅拌15分钟,随后向混合物中加入1mL含0.041g N,N-二环己基碳二亚胺的N,N-二甲基甲酰胺溶液,搅拌反应过夜。
步骤2)半抗原与载体蛋白的偶联。将步骤1反应后的产物4000rpm离心5分钟,取上清1.25mL缓慢滴加到5mL 10mg/mL牛血清白蛋白的磷酸缓冲液(0.01mol/L,pH=8)中,室温下避光搅拌反应4h后得到丙环唑免疫抗原;另取步骤1离心后的产物1.25mL缓慢滴加到5mL10mg/mL鸡卵白蛋白的磷酸缓冲液(0.01mol/L,pH=8)中,室温下避光搅拌反应4h即得到丙环唑包被抗原。
步骤3)人工抗原的纯化。将步骤2得到的丙环唑免疫抗原和丙环唑包被抗原用0.01mol/L PBS缓冲液透析纯化3天,取出后4000rpm离心5min,取上清测浓度并分装冻存。
实施例4:丙环唑抗原免疫动物
用丙环唑免疫抗原(丙环唑半抗原与牛血清白蛋白的偶联物)对五只6-8周龄的BALB/c雌性小鼠进行五次免疫制备单克隆抗体。每次取100μg经过等体积弗氏完全佐剂或弗氏不完全佐剂乳化的免疫抗原对小鼠进行腹腔注射,具体的免疫程序如表1所示。
表1小鼠免疫程序
分别在三免,四免,五免后第七天取小鼠尾血,采用酶联免疫测定的方法(ELISA)测小鼠尾血效价,选择一只尾血血清效价最高的小鼠通过腹腔注射的方式注射100μg不经乳化的免疫抗原加强免疫,三天后进行细胞融合实验。
实施例5:细胞融合
步骤1)骨髓瘤细胞的制备:取三瓶生长状况良好的小鼠骨髓瘤细胞,用DMEM培养液吹下后移入50mL离心管中并定容到35mL。
步骤2)脾细胞的制备:取加强免疫后三天的BALB/c小鼠,采集眼眶血后脱臼处死,在75%酒精中消毒后取脾脏,去除结缔组织,用DMEM培养液制备脾细胞悬液,转移到50mL离心管中,定容至35mL。
步骤3)骨髓瘤细胞和脾细胞混合:将置于50mL离心管的骨髓瘤细胞和脾细胞悬液1000rpm离心10min,弃上清,分别用10mL DMEM将细胞悬起,取少量细胞悬液计数后按脾细胞和骨髓瘤细胞10∶1的比例混合,并吸打混匀。
步骤4)细胞融合:将上述混合细胞的的悬液1000rpm离心10min,弃上清,在桌面上轻轻敲击使细胞散开。置于37℃水中预热,60s内加完预热至37℃的1mL PEG1500,然后在4min内由慢至快加入30mL预热至37℃的DMEM培养液,静置10min,然后800rpm离心8min,弃上清,用HAT培养液重悬后加入适量白细胞介素和抗生素,吸打混匀后均匀滴加到96孔细胞培养板中并于CO2培养箱中培养。
实施例6:细胞株筛选
待细胞长至孔底的1/3时,取细胞上清进行抗体检测。检测时先用ELISA筛选出阳性细胞孔,再用丙环唑标准品和间接竞争ELISA对阳性细胞进行抑制效果测定。选出对丙环唑标准品抑制效果好的孔,采用有限稀释法进行亚克隆,用同样的方法进行检测。重复多次,直到得到能稳定分泌丙环唑单克隆抗体的细胞株。
实施例7:腹水和抗体制备
将灭过菌的液体石蜡注射到6~8周龄BALB/c小鼠的腹腔中(每只小鼠500μL),7-14天后将处于对数生长期的杂交瘤细胞用完全培养液收集并注射到小鼠腹腔中(每只2×106个细胞)。一周内观察小鼠腹部情况,待小鼠腹部明显隆起后用灭过菌的16号针头收集腹水。将收集到的腹水10000rpm离心10min后收集上清,用ProteinA柱纯化后获得抗体。
实施例8:抗体交叉反应性测定
配制一系列不同浓度的三唑类杀菌剂(乙唑醇、三唑醇、苯醚甲环唑、多效唑、烯唑醇、粉唑醇、联苯三唑醇、戊唑醇)标准溶液,通过间接竞争ELISA分别拟合相应标准曲线,根据公式CR(交叉反应率)=IC50(丙环唑)/IC50(其他三唑类杀菌剂)×100%计算交叉反应率。结果证明丙环唑单克隆抗体对丙环唑类似物的交叉反应均<0.1%,特异性强。
实施例9:丙环唑ELISA标准曲线的制作
1、包被:将丙环唑包被原(丙环唑半抗原与鸡卵白蛋白的偶联物)用CBS稀释到125μg/L,每孔100μL加入到酶标板中,4℃过夜包被。
2、洗板:用PBST洗板五次并拍干。
3、封闭:向酶标板中加入5%脱脂奶粉(每孔200μL),37℃培养箱中温育1.5h。
4、洗板:用PBST洗板五次并拍干。
5、加入分析物和丙环唑单克隆抗体:将丙环唑标准品用PBS系列稀释,在酶标板每孔中加入50μL,然后每孔中再加入50μL,156.3μg/L的抗丙环唑单克隆抗体,37℃温育1h。
6、洗板:用PBST洗板五次并拍干。
7、加入酶标二抗:加入PBS稀释20000的HRP羊抗小鼠IgG,每孔加入100μL,37℃温育1h。
8、洗板:用PBST洗板八次并拍干。
9、显色:加入现配的TMB/H2O2溶液,每孔100μL,37℃温育15min。
10、终止:每孔加入50μL终止液终止反应,酶标仪检测OD450值。
以丙环唑标准品浓度为横坐标,B/B0(每个浓度的标准品溶液的吸光度值(B)除以对照孔(标准品浓度为0的孔)的吸光度值(B0)再乘以100%)为纵坐标绘制标准曲线,如图2所示。计算抑制中浓度(IC50)为2.33μg/L,线性范围(IC10-IC90)为0.26-21.28μg/L。
实施例10:丙环唑快速检测试纸条的制备
1、金标抗体的制备
步骤1)柠檬酸三钠还原法合成胶体金:将置于250mL锥形瓶中的超纯水加热至沸腾,将1mL 1%氯金酸水溶液加至锥形瓶中,在持续加热搅拌的条件下,快速向锥形瓶中加入1%柠檬酸钠水溶液1.8mL,当溶液由深蓝色变为明显的酒红色后继续煮沸5min,然后将溶液冷却至室温。
步骤2)胶体金与丙环唑抗体偶联:通过盐沉淀法确定丙环唑抗体的最佳标记量。取10mL胶体金用0.1M K2CO3调pH为8.2。加入138μL丙环抗体后室温震荡孵育1h,然后加入1300μL 10%BSA溶液,室温震荡孵育1h。将上述溶液10000rpm离心15min,弃上清,用含1%BSA,3%蔗糖的1000μL硼酸盐缓冲液重悬,并于4℃保存。
2、包被抗原和羊抗鼠包被纤维素膜
用XYZ-3000三维喷膜仪进行划膜。喷包被抗原为检测线,包被抗原的浓度为0.55mg/mL。喷羊抗鼠IgG为对照线,其浓度为0.25mg/mL,两线间隔5mm,划膜后置于37℃烘箱中烘干40min。
3、试纸条的组装
将已划膜并烘干的纤维素膜粘贴在衬板中间部位上,吸水垫粘在纤维素膜上侧和纤维素膜重叠1mm。金标结合物垫粘在纤维素膜下方重叠1mm。样品垫粘贴在进标结合物垫下方重叠2mm。组装好的试纸板用斩切机切成4.00mm宽的试纸条。
实施例11:丙环唑快速检测试纸条的测试
1、试纸条的敏感性试验
将1000mg/L的丙环唑标准溶液用丙环唑试纸条最优缓冲液稀释成100、50、25、10、5、2、1、0μg/L系列浓度梯度的标准溶液,各取100μL分别加入酶标孔中,将丙环唑试纸条的样品垫端插入酶标孔中,液面不超过标记线9。将试纸条水平放置,溶液自然扩散5-8min后读取结果。结果发现,100、50、25、10、5μg/L的标准溶液呈阳性(只有对照线或者检测线条带颜色弱于对照线);2、1、0μg/L呈阴性(即检测线条带颜色比对照线深或检测线条带颜色与对照线相似)。因此,丙环唑试纸条的灵敏度为5μg/L。
2、试纸条的特异性试验
用丙环唑试纸条最优缓冲液配制1000μg/L浓度的三唑类杀菌剂(己唑醇、三唑醇、苯醚甲环唑、多效唑、烯唑醇、粉唑醇、联苯三唑醇、戊唑醇)标准溶液,各取100μL加入酶标孔,液面不超过标记线9。将试纸条水平放置,溶液自然扩散5-8min后读取结果。结果发现,所有的试纸条均呈现阴性,即检测线条带比对照线深,所以本发明的试纸条对丙环唑类似物均无交叉反应性。
3、丙环唑快速检测试纸条检测菜心样品
取菜心空白样品,使用破壁料理机将其打成匀浆,准确称取5g菜心匀浆至50mL离心管中,添加丙环唑标准品使其终浓度为50mg/kg。向添加样品中加入10mL 50%的甲醇-PBS缓冲液,震荡5min,超声10min后静置5min,用离心机4000rpm离心5min。取上清液用试纸条最优缓冲液稀释一定的倍数后取100μL加至酶标孔中,将丙环唑试纸条的样品垫端插入酶标孔中,液面不超过标志线9,静置8min。检测结果显示为阳性,表明试纸条可以满足菜心样品中丙环唑的检测。
4、试纸条稳定性试验
将试纸条放入铝铂袋中真空包装,并且室温保存,3个月后取出检测其灵敏度,发现灵敏度达5μg/L,且显色深度均一,证明丙环唑试纸条稳定性好。
Claims (5)
1.一种丙环唑半抗原,其特征在于,其分子结构式如下式I所示:
2.一种制备如权利要求1所述的丙环唑半抗原的制备方法,其制备步骤如下:
步骤1)1.52g N-溴代琥珀酰亚胺与2g 2-氯-4-溴-苯乙酮加入12mL乙腈中,在12℃的条件下搅拌反应10分钟;随后2.95g对甲苯磺酸加入上述混合液中,并在15-80℃的条件下搅拌反应1.17小时;反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物1,
步骤2)4g产物1与2g 1,2-戊二醇加入24mL甲苯中,然后加入12mL正丁醇和1.1g对甲苯磺酸,上述混合物在135℃下搅拌反应15h;反应结束后其产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物2,
步骤3)1.39g KOH和0.73g三唑溶于14mL DMSO中,将2.8g产物2在45℃条件下加入上述混合物中,该混合物在135℃搅拌反应15h;反应得到的产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物3,
步骤4)1.77g产物3溶于7.5mL四氢呋喃中,5.56g三乙胺和0.16g双(三苯基膦)氯化钯加入上述溶液;随后0.087g CuI也加入上述溶液中,在CuI加入前后需用氮气净化混合溶液三次;然后1.03g 4-戊炔酸甲酯加入到混合溶液中并用氮气净化三次;15℃搅拌10min后,0.087g CuI加入到混合溶液中并于75℃条件下持续搅拌12h;反应结束后,产物通过硅胶柱纯化,用体积比为50∶1到1∶1的石油醚-乙酸乙酯洗脱分离,得产物4,
步骤5)1.77g产物4和0.096g二氧化铂加入20mL乙酸乙酯中,用氢气净化三次后,上述混合物在30℃,氢气压强50psi条件下搅拌反应6h;反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,得产物5,
步骤6)1.2g产物5与0.12g单水氢氧化锂加入6.5mL四氢呋喃并于15-25℃条件下搅拌反应15h;反应结束后产物通过硅胶柱纯化,用体积比为100∶1到10∶1的石油醚-乙酸乙酯洗脱分离,获得无色油状的丙环唑半抗原,
3.一种丙环唑的人工抗原,其特征在于,所述丙环唑人工抗原包括丙环唑免疫抗原和丙环唑包被抗原,所述丙环唑人工抗原是载体蛋白和权利要求1所述的丙环唑半抗原偶联得到的偶联物,所述载体蛋白为牛血清白蛋白或卵清蛋白。
4.一种丙环唑单克隆抗体,其特征在于,所述的单克隆抗体特异性识别权利要求1所述的丙环唑半抗原或权利要求3所述的丙环唑人工抗原。
5.一种权利要求4所述的丙环唑单克隆抗体在检测丙环唑残留中的应用。
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