CN104140396A - 苯并咪唑类药物半抗原及其制备方法和应用 - Google Patents
苯并咪唑类药物半抗原及其制备方法和应用 Download PDFInfo
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- CN104140396A CN104140396A CN201310162837.1A CN201310162837A CN104140396A CN 104140396 A CN104140396 A CN 104140396A CN 201310162837 A CN201310162837 A CN 201310162837A CN 104140396 A CN104140396 A CN 104140396A
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Abstract
本发明公开了一种苯并咪唑类药物半抗原及其制备方法和应用。本发明提供的苯并咪唑类药物半抗原,结构式如式Ⅰ所示:
Description
技术领域
本发明涉及一种苯并咪唑类药物半抗原及其制备方法和应用。
背景技术
苯并咪唑类药物(Benzimidazoles,BMZs)具有广泛的生物活性,对于动物体内各种寄生线虫、绦虫具有很强的驱杀作用,目前在兽医临床上应用广泛。常用的苯并咪唑类药物包括阿苯达唑、奥芬达唑、芬苯达唑、氟苯咪唑、甲苯咪唑、噻苯咪唑和丙氧苯咪唑。但由于苯并咪唑类药物在实验动物显示致畸和致突变作用,且在人体内转化的代谢产物亦有毒理作用,是食品残留中重要的监控对象。中国、美国、欧盟、日本等国家或地区将苯并咪唑类药物列入限制使用的药物和动物源性食品残留检测的监控对象,并制订了各种苯并咪唑类药物(包括其某些代谢物)在不同动物体内(包括肌肉、组织等)的最大残留限量(MRLs),在不同的动物和不同的组织中最大残留限量从10μg/kg到5mg/kg不等,大多数限量是100μg/kg。
目前对于动物组织中苯并咪唑类药物的分析方法较多,动物组织和饲料中苯并咪唑类药物的主要检测方法有气相色谱-质谱法(GC-MS)、高效液相色谱法(HPLC)、高效液相色谱串联质谱法(HPLC-MS/MS)、酶联免疫吸附法(ELISA)及高效毛细管电泳法(HPCE)等。气-质联用法需要衍生化后测定,液-质联用仪设备较昂贵,反相高效液相色谱法则作为主要的分析技术,然而理化方法存在复杂、繁琐、需要专业技术人员和专业技能、检测成本高等问题,不能实现大批量样品的快速检测分析;酶联免疫吸附法(ELISA)以其灵敏度高、特异性好、操作简便、成本低等优点适合于大量样品的快速筛选。
发明内容
本发明的目的是提供一种苯并咪唑类药物半抗原及其制备方法。
本发明提供的苯并咪唑类药物半抗原分子结构式如式Ⅰ所示:
式Ⅰ。
本发明提供的苯并咪唑类药物半抗原的制备方法,包括如下步骤:
取阿苯达唑0.5g,加甲醇100ml搅拌,加KOH 0.2g搅拌,60℃加热反应3h。TLC检测,原料基本消失,反应完成。停止反应,冷却到室温,旋蒸除去甲醇,加水溶解,抽滤,除去浑浊,滤液加稀盐酸调节pH值到5,有大量白色沉淀析出,抽滤,加乙醇洗涤,抽滤,得到白色固体,真空干燥,得到半抗原产物。
本发明的另一个目的是上述苯并咪唑类药物半抗原在免疫检测中的应用,具体包括由所述苯并咪唑类药物半抗原与载体蛋白偶联得到的苯并咪唑类药物抗原,及由所得苯并咪唑类药物抗原免疫动物制备得到的苯并咪唑类药物抗体,所述抗体为苯并咪唑类药物单克隆抗体。
其中所述载体蛋白可为牛血清白蛋白、甲状腺蛋白、卵清白蛋白、人血清蛋白、鼠血清蛋白、兔血清蛋白、血蓝蛋白或纤维蛋白原。
本发明还提供由上述苯并咪唑类药物半抗原或苯并咪唑类药物抗原在检测苯并咪唑类药物或制备检测苯并咪唑类药物的产品中的应用,具体涉及制备的酶联免疫试剂盒在检测动物源性食品中苯并咪唑类药物残留的应用,苯并咪唑类药物包括氟苯咪唑、丙氧咪唑、甲苯咪唑、阿苯达唑、奥芬达唑、丙硫咪唑亚砜。
本发明提供的苯并咪唑类药物半抗原、苯并咪唑类药物抗原合成方法简单,通过免疫动物产生了针对苯并咪唑类药物的特异性抗体,抗体的效价、特异性都比较好;用所得的抗体制备酶联免疫试剂盒,检测成本低,使用方便,检测方法准确、快速、可同时检测大批量的样本,适于动物源性食品中苯并咪唑类药物残留的现场监控和大量样本的筛查。本发明的苯并咪唑类药物半抗原在苯并咪唑类药物的检测中发挥重要作用。
附图说明
图1:苯并咪唑类药物半抗原合成路线图
图2:苯并咪唑类药物半抗原核磁共振图
图3:苯并咪唑类药物ELISA标准曲线
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。
下述实施例中所使用的实验方法、材料、试剂等,如无特殊说明,均为常规方法和常规材料。
实施例1:苯并咪唑类药物半抗原的合成和鉴定(合成路线如图1)
取阿苯达唑0.5g,加甲醇100ml搅拌,加KOH 0.2g搅拌,60℃加热反应3h。TLC检测,原料基本消失,反应完成。停止反应,冷却到室温,旋蒸除去甲醇,加水溶解,抽滤,除去浑浊,滤液加稀盐酸调节pH值到5,有大量白色沉淀析出,抽滤,加乙醇洗涤,抽滤,得到白色固体,真空干燥,得到半抗原产物。
上述苯并咪唑类药物半抗原分子结构式如式Ⅰ所示:
式Ⅰ。
取上述产物用核磁共振确定结构,如图2所示, 1HNMR鉴定,1HNMR(DMSO,300M);δ0.9(t, J=7.2Hz,3H CH3); δ1.35(q, J=7.2Hz 2H CH2);δ2.94(d, J=7.2Hz,2H SCH2); δ7.18(d, J=7.42Hz 1H CH);δ7.49(d, J=7.42Hz 1H CH); δ7.62(s, 1H CH); δ5.0(s, 1H NH); δ9.15(s, 1H NH); δ11.0(s, 1H OH)。
实施例2:苯并咪唑类药物抗原的制备
将苯并咪唑类药物半抗原与载体蛋白偶联得到苯并咪唑类药物抗原,分子结构式如式Ⅱ所示:
式Ⅱ。
一、免疫原制备
取12.3mg苯并咪唑类药物半抗原用1ml N,N-二甲基甲酰胺(DMF)溶解,取15mg碳化二亚胺(EDC)用0.2ml水充分溶解后加入上述溶液中,室温下搅拌24h,即可得到反应液A。称取牛血清白蛋白(BSA)40mg,使之充分溶解在3ml 水中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24h。用0.01mol/L的PBS溶液在4℃透析3天,每天换3次透析液,以除去未反应的小分子物质。分装,于-20℃保存备用。
二、包被原制备
取15.5mg苯并咪唑类药物半抗原用1.5ml DMF溶解冷却至10℃得到溶液B,取氯甲酸异丁酯10μl加入溶液B中,10℃搅拌反应30min得到溶液C;取25mg卵清蛋白(OVA)用2.2ml 50mmol/L Na2CO3溶解,和上述溶液C在10℃反应4h,然后4℃过夜;用0.01mol/L的PBS溶液在4℃透析3天,每天换3次透析液,以除去未反应的小分子物质。分装,于-20℃保存备用。
实施例3:苯并咪唑类药物单克隆抗体的制备
动物免疫:将实施例2中得到的免疫原注入到Balb/c小鼠体内,免疫剂量为150μg/只,使其产生多克隆抗体。
细胞融合和克隆化:小鼠血清测定结果较高后,取其脾细胞,按8:1比例与SP2/0骨髓瘤细胞融合,采用间接竞争ELISA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,直到得到分泌单克隆抗体的杂交瘤细胞株。
细胞冻存和复苏:将苯并咪唑类药物的单克隆杂交瘤细胞株用冻存液制成5×107个/ml的细胞悬液,在液氮中长期保存。复苏时取出冻存管,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。
单克隆抗体的生产与纯化:将Balb/c小鼠腹腔注入灭菌石蜡油0.5ml/只,7天后腹腔注射苯并咪唑类药物的单克隆杂交瘤细胞株5×105个/只,7天后采集腹水。用辛酸-饱和硫酸铵法进行腹水纯化,-20℃保存。该单克隆抗体特异性好,检测灵敏度可达0.5μg/L。
单克隆抗体的特异性
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。
本实验将氟苯咪唑、丙氧咪唑、甲苯咪唑、阿苯达唑、奥芬达唑、丙硫咪唑亚砜、芬苯达唑、多菌灵8种药物做系列稀释,制作标准曲线,分析得到IC50,然后按下式计算交叉反应率,结果见表1。
表1 抗体特异性
实施例4:由苯并咪唑类药物单克隆抗体制备的酶联免疫试剂盒
一、酶联免疫试剂盒的组成
(1)包被苯并咪唑类药物偶联抗原的酶标板;
(2)酶结合物工作液;
(3)标准品溶液:浓度分别为0μg/L、0.5μg/L、1μg/L、2μg/L、4μg/L、8μg/L,高浓度标准品溶液浓度为1mg/L;
(4)底物显色液由A液和B液组成,A液为过氧化脲,B液为四甲基联苯胺溶液;
(5)终止液为2mol/L的硫酸溶液;
(6)浓缩洗涤液为含0.1%的吐温-20的0.1~0.2mol/L pH7.4的磷酸盐缓冲液,所述百分比为重量体积百分比;
(7)浓缩复溶液为pH7.2~7.6,0.1~0.3mol/L的磷酸盐缓冲液,所述百分比为重量体积百分比。
本试剂盒的主要试剂以工作液的形式提供,检验方法方便易行,具有特异性高、灵敏度高、精确度高、准确度高等特点。
二、酶联免疫试剂盒检测实际样本的应用
(一)样本前处理
1. 成品奶样本前处理方法
移取50μl成品奶样本;加入950μl 成品奶稀释液(用去离子水将4×浓缩复溶液按1:9体积比进行稀释)中,用涡旋仪涡动至均匀;取50μl用于分析。
2. 原奶样本前处理方法
移取50μl原奶样本;加入950μl 原奶稀释液(用去离子水将4×浓缩复溶液按1:4体积比进行稀释)中,用涡旋仪涡动至均匀;取50μl用于分析。
3. 鸡肉、猪肉样本的前处理
称取(1.0±0.05)g均质物至50ml 聚苯乙烯离心管中,加入7.7ml乙酸乙酯和0.3ml甲醇,用涡旋仪涡动至混匀;3000g以上,室温(20-25℃/68-77℉)离心5min;移取1ml 上层有机相至10ml干净玻璃试管中,于50~60℃水浴氮气/空气流下吹干;加入2ml 复溶工作液(用去离子水将4×浓缩复溶液按1:3体积比进行稀释)涡动30s,取50μl用于分析。
4. 牛肉样本前处理方法
称取(1.0±0.05)g用均质器均质的样本至50ml聚苯乙烯离心管中;加入7.3ml乙酸乙酯和0.7ml甲醇,用涡旋仪涡动至均匀。3000g以上,室温(20-25℃/68-77℉)离心5min;移取1ml上层有机相至10ml 干净玻璃试管中,于50~60℃水浴氮气/空气流下吹干;加入2ml复溶工作液,涡动30s;取50μl用于分析。
5. 鸡蛋样本前处理方法
称取(1±0.05)ml均匀样本至50ml聚苯乙烯离心管中;加入8ml乙酸乙酯,用涡旋仪涡动至均匀。3000g以上,室温(20-25℃/68-77℉)离心5min;移取1ml上层有机相至10ml 干净玻璃试管中,于50~60℃水浴氮气/空气流下吹干;加入2ml复溶工作液,涡动30s;取50μl用于分析。
2. 用试剂盒进行检测
向包被有包被原的酶标板微孔中加入50μl标准品/样本,再加入50μl酶结合物工作液,轻轻振荡混匀,盖上盖板膜,25℃恒温箱中避光反应30min。倒出孔中的液体,用洗涤工作液(用去离子水将20×浓缩洗涤液按1:19体积比进行稀释)250μl/孔充分洗涤4-5次,每次间隔10s,用吸水纸拍干以保证完全除去孔中的液体,加入底物液A液50μl,再加入底物液B液50μl,轻轻振荡混匀,用盖板膜盖板后25℃恒温箱中避光显色15min。加入50μl终止液,轻轻振荡混匀,设定酶标仪于450nm 处测量每孔的吸光度值。
3. 检测结果分析
用所获得的标准品或样本的吸光度值的平均值(双孔)除以第一个标准(0标准)的吸光度值,再乘以100%,即得到标准品或样本的百分吸光率。以苯并咪唑类药物标准品百分吸光率为纵坐标,以苯并咪唑类药物标准品浓度的对数为横坐标,绘制标准曲线图,如图3所示。将样本的百分吸光率代入标准曲线中,从标准曲线上读出样本所对应的浓度,乘以其对应的稀释倍数即为样本中苯并咪唑类药物实际浓度。
三、酶联免疫试剂盒技术参数的确定
最低检测限:分别对20份空白牛奶、鸡肉、猪肉、牛肉、鸡蛋样本进行检测,从标准曲线上查出对应于各百分吸光率的浓度,以20份样本苯并咪唑类药物浓度的平均值加上3倍标准差表示检测限,结果得该方法对牛奶样本检测限为10μg/kg,对鸡肉、猪肉、牛肉样本检测限为8μg/kg,对鸡蛋样本检测限为8μg/kg。
准确度和精密度:ELISA测定的准确度以回收率表示,精密度以变异系数表示。分别取空白牛奶样本,以10、20、40μg/kg三个添加浓度,取空白鸡肉、猪肉、牛肉、鸡蛋样本,以8、16、32μg/kg三个浓度的氟苯咪唑药物对其进行添加回收试验,结果得该方法对牛奶样本的回收率为90%±15%,对鸡肉样本的回收率为90%±15%,对猪肉样本的回收率为90%±15%,对牛肉样本的回收率为90%±15%,对鸡蛋样本的回收率为90%±15%,批内变异系数<15%,批间变异系数<25%。
经测定本发明试剂盒在2~8℃至少可以保存12个月。
Claims (8)
1.一种苯并咪唑类药物半抗原,其特征在于分子结构式如式Ⅰ所示:
式Ⅰ。
2.一种如权利要求1所述的苯并咪唑类药物半抗原的制备方法,其特征在于包括如下步骤:
取阿苯达唑0.5g,加甲醇100ml搅拌,加KOH 0.2g搅拌,60℃加热反应3h。TLC检测,原料基本消失,反应完成。停止反应,冷却到室温,旋蒸除去甲醇,加水溶解,抽滤,除去浑浊,滤液加稀盐酸调节pH值到5,有大量白色沉淀析出,抽滤,加乙醇洗涤,抽滤,得到白色固体,真空干燥,得到半抗原产物。
3.一种苯并咪唑类药物抗原,其特征在于由权利要求1所述的苯并咪唑类药物半抗原与载体蛋白偶联得到,分子结构式如式Ⅱ所示:
式Ⅱ。
4.一种如权利要求3所述的苯并咪唑类药物抗原的制备方法,其特征在于包括如下步骤:
取12.3mg苯并咪唑类药物半抗原用1ml DMF溶解,取15mg EDC用0.2ml水充分溶解后加入上述溶液中,室温下搅拌24h,即可得到反应液A。称取BSA 40mg,使之充分溶解在3ml 水中,将反应液A逐滴缓慢滴加到蛋白溶液中,并于室温下搅拌24h。用0.01mol/L的PBS溶液在4℃透析3天,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,于-20℃保存备用。
取15.5mg苯并咪唑类药物半抗原用1.5ml DMF溶解冷却至10℃得到溶液B,取氯甲酸异丁酯10μl加入溶液B中,10℃搅拌反应30min得到溶液C;取25mg OVA用2.2ml 50mmol/L Na2CO3溶解,和上述溶液C在10℃反应4h,然后4℃过夜;用0.01mol/L的PBS溶液在4℃透析3天,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,于-20℃保存备用。
5.由权利要求1所述的苯并咪唑类半抗原或权利要求3-4任一所述的苯并咪唑类药物抗原制备的抗体。
6.如权利要求5所述的抗体,其特征在于所述抗体为苯并咪唑类药物单克隆抗体。
7.权利要求1所述的苯并咪唑类药物半抗原或权利要求3-4中任一所述的苯并咪唑类药物抗原在检测苯并咪唑类药物或制备检测苯并咪唑类药物的产品中的应用。
8.如权利要求7所述的检测苯并咪唑类药物的产品,其特征在于:苯并咪唑类药物包括氟苯咪唑、丙氧咪唑、甲苯咪唑、阿苯达唑、奥芬达唑、丙硫咪唑亚砜。
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Cited By (7)
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CN106008361A (zh) * | 2015-07-01 | 2016-10-12 | 北京维德维康生物技术有限公司 | 一种阿苯达唑人工抗原及其制备方法与应用 |
CN106008361B (zh) * | 2015-07-01 | 2018-10-19 | 北京维德维康生物技术有限公司 | 一种阿苯达唑人工抗原及其制备方法与应用 |
CN105403703A (zh) * | 2015-12-23 | 2016-03-16 | 中国烟草总公司郑州烟草研究院 | 检测多菌灵的酶联免疫试剂盒及其应用 |
CN105403703B (zh) * | 2015-12-23 | 2017-06-30 | 中国烟草总公司郑州烟草研究院 | 检测多菌灵的酶联免疫试剂盒及其应用 |
CN109265404A (zh) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | 一种多菌灵半抗原与抗原的制备方法及应用 |
CN109824599A (zh) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | 一种阿苯达唑半抗原及其制备方法和应用 |
CN109824599B (zh) * | 2019-02-28 | 2020-10-30 | 中国农业大学 | 一种阿苯达唑半抗原及其制备方法和应用 |
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