CN110684110A - 一种甲基嘧啶磷单克隆抗体的制备及其应用 - Google Patents
一种甲基嘧啶磷单克隆抗体的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种甲基嘧啶磷单克隆抗体的制备方法，并将其应用于甲基嘧啶磷残留检测试纸条和酶联免疫试剂盒。试纸条包括：样品吸收垫(1)、结合物释放垫(2)、反应膜(3)、吸水垫(4)和底板(7)，所述反应膜上具有包被有甲基嘧啶磷半抗原‑载体蛋白偶联物的检测线(5)和包被有羊抗鼠抗抗体的质控线(6)，所述结合物释放垫(2)喷涂有甲基嘧啶磷单克隆抗体‑胶体金标记物。试剂盒包括：包被有包被原的酶标板、甲基嘧啶磷标准品溶液、甲基嘧啶磷抗体、酶结合物浓缩液、酶结合物稀释液、底物显色液、终止液、洗涤液，所述包被原为甲基嘧啶磷偶联抗原，所述酶结合物为酶标记的甲基嘧啶磷抗体。本发明所提供的试纸条和酶联免疫试剂盒具有操作简单、灵敏度高、检测速度快、成本低等特点，适合大量样本的筛查和现场监控。
Description
技术领域
本发明涉及一种甲基嘧啶磷单克隆抗体的制备及其应用，具体涉及一种甲基嘧啶磷单克隆抗体的制备方法，其特别适用于蔬菜、水果、谷物等农作物中甲基嘧啶磷残留的检测。
背景技术
甲基嘧啶磷又名安得利，是一种有机磷速效、广谱杀虫剂、杀螨剂，具有胃毒和熏蒸作用。随着甲基嘧啶磷农药的广泛使用，国家粮食卫生标准GB2715-2005增加了甲基嘧啶磷的最大残留限量标准，规定小麦、谷类中最大残留限量为5mg/kg。因此建立快速灵敏的甲基嘧啶磷残留检测方法显得尤为重要。
目前检测甲基嘧啶磷的方法大都是仪器方法，仪器成本高，检测时间长，操作复杂，不符合基层实验室的高效、便捷的检测要求，因此，本发明制备出一种甲基嘧啶磷的单克隆抗体，应用于胶体金试纸条、酶联免疫试剂盒等快速检测产品的制备，能够适应基层实验室的检测需求，有利于提高监管手段和监督水平。
发明内容
本发明的目的是提供一种甲基嘧啶磷单克隆抗体的制备方法，并将其应用于甲基嘧啶磷残留检测试纸条和酶联免疫试剂盒。
本发明所提供的甲基嘧啶磷单克隆抗体是以甲基嘧啶磷半抗原-载体蛋白偶联物作为免疫原制备获得，是由甲基嘧啶磷单克隆抗体杂交瘤细胞株分泌获得；所述羊抗鼠抗抗体是将鼠源抗体免疫羊得到；所述甲基嘧啶磷半抗原-载体蛋白偶联物是由甲基嘧啶磷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、人血清白蛋白。
本发明所提供的检测甲基嘧啶磷残留的试纸条，包括样品吸收垫(1)、结合物释放垫(2)、反应膜(3)、吸水垫(4)和底板(7)；所述反应膜上具有包被有甲基嘧啶磷半抗原-载体蛋白偶联物的检测线(5)和包被有羊抗鼠抗抗体的质控线(6)，所述结合物释放垫(2)喷涂有甲基嘧啶磷单克隆抗体-胶体金标记物。
所述样品吸收垫(1)、结合物释放垫(2)、反应膜(3)、吸水垫(4)依次粘贴在底板(7)上，所述结合物释放垫1/3～1/2被覆盖于样品吸收垫下。
所述底板可为PVC底板或其他硬质不吸水的材料；所述样品吸收垫可为吸滤纸或滤油纸；所述结合物释放垫可为玻璃棉或聚酯材料；所述吸水垫为吸水纸；所述反应膜可为硝酸纤维素膜或醋酸纤维素膜。
本发明所提供的检测甲基嘧啶磷残留的酶联免疫试剂盒，包括：包被有包被原的酶标板、甲基嘧啶磷标准品溶液、甲基嘧啶磷抗体、酶结合物浓缩液、酶结合物稀释液、底物显色液、终止液、洗涤液，所述包被原为甲基嘧啶磷偶联抗原，所述酶结合物为酶标记的甲基嘧啶磷抗体。
所述甲基嘧啶磷特异性抗体是以甲基嘧啶磷偶联抗原作为免疫原制备获得，所述甲基嘧啶磷特异性抗体可为甲基嘧啶磷单克隆抗体或甲基嘧啶磷多克隆抗体，其中优选甲基嘧啶磷单克隆抗体。
所述酶结合物的标记酶为辣根过氧化物酶或细菌提取碱性磷酸酯酶，其中优选辣根过氧化物酶；酶结合物是由酶和甲基嘧啶磷抗体偶联得到的。
本发明的甲基嘧啶磷快速检测试纸条采用高度特异性的抗体抗原反应及竞争抑制免疫层析分析技术，将甲基嘧啶磷单克隆抗体-胶体金标记物固定于结合物释放垫上，样品中的甲基嘧啶磷在流动过程中，与结合物释放垫上的甲基嘧啶磷单克隆抗体-胶体金标记物结合，形成药物-抗体-胶体金标记物。样本中的药物与反应膜检测线上的甲基嘧啶磷半抗原-载体蛋白偶联物竞争结合甲基嘧啶磷单克隆抗体-胶体金标记物，根据检测线红色条带有无或颜色深浅来判断待测样品液中是否含有甲基嘧啶磷残留。
本试剂盒采用直接竞争ELISA方法，在酶标板微孔条上预包被偶联抗原，样本中残留的甲基嘧啶磷和酶标板微孔条上预包被的偶联抗原竞争抗甲基嘧啶磷的酶结合物，用TMB底物显色，样本吸光度值与其所含残留物甲基嘧啶磷的含量成负相关，与标准曲线比较，再乘以其对应的稀释倍数，即可得出样本中甲基嘧啶磷的残留量。
本发明的试纸条和试剂盒具有灵敏度高、特异性强、成本低、操作简单、检测时间短、适合各种单位使用、储存简单、保质期长的优点。用本发明试纸条和试剂盒检测甲基嘧啶磷残留的方法简便、快速、直观、准确、适用范围广、成本低、易推广使用。
附图说明
图1为甲基嘧啶磷半抗原合成图。
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甲基嘧啶磷单克隆抗体的制备
1、甲基嘧啶磷半抗原的制备
取2.26g化合物Ⅰ，1,2-二氯乙烷150ml溶解,加二甲基硫代磷酰氯1.82g，三乙胺0.8ml,室温搅拌反应12h，停止反应，加水100ml，震荡萃取，分去水相，有机相蒸干，得到红色油状物，加甲醇70ml溶解，加钯碳0.4g，充分搅拌，抽取空气，通入氢气，室温搅拌4h，停止反应，过滤，除去钯碳，减压蒸干，得到粗产物，无水乙醇10ml重结晶，得到化合物Ⅲ0.9g，收率28.1％。
化合物Ⅲ0.9g，加吡啶80ml溶解，加琥珀酸酐0.32g，油浴加热，80℃反应12h，停止反应，旋蒸除去吡啶，油状物上硅胶柱，二氯甲烷/甲醇体积比10:1洗脱分离，得到琥珀酸甲基嘧啶磷半抗原产物0.7g，收率59.32％。
2、免疫原的制备
取琥珀酸甲基嘧啶磷半抗原产物16mg，加DMF1ml，溶解澄清，加EDC14.2mg，加NHS9.7mg，充分混匀后，反应4h，得到半抗原活化液A液；取牛血清白蛋白(BSA)50mg,加0.05M PB溶解，得到B液，将A液滴加到B液中，4℃反应24h，0.02M PBS透析纯化三天，每天换液3次，得到甲基嘧啶磷-BSA偶联物，即为免疫原，分装，-20℃保存。
3、包被原的制备
取琥珀酸甲基嘧啶磷半抗原产物10mg，加DMF1ml，溶解澄清，加EDC11mg，加NHS8.45mg，充分混匀后，反应4h，得到半抗原活化液A液；取卵清白蛋白(OVA)50mg,加0.05MPB溶解，得到B液，将A液滴加到B液中，4℃反应24h，0.02M PBS透析纯化三天，每天换液3次，得到甲基嘧啶磷-OVA偶联物，即为免疫原，分装，-20℃保存。
4、甲基嘧啶磷单克隆抗体的制备
(1)动物免疫
将步骤2得到的免疫原注入Balb/c小鼠体内，免疫剂量为150μg/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，采用间接竞争ELISA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，直到得到稳定分泌单克隆抗体的杂交瘤细胞株。
(3)细胞冻存和复苏
将杂交瘤细胞用冻存液制成1×106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。
(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为7.4。
5、羊抗鼠抗抗体的制备
以羊作为免疫动物，以鼠源抗体为免疫原对无病原体羊进行免疫，得到羊抗鼠抗抗体。
实施例2甲基嘧啶磷残留试纸条的制备
该试纸条的制备方法主要包括以下步骤：
1)制备喷涂有甲基嘧啶磷单克隆抗体-胶体金标记物的结合物释放垫；
2)制备具有包被有甲基嘧啶磷半抗原-载体蛋白偶联物的检测线和包被有羊抗鼠抗抗体的质控线的反应膜；
3)将1)和2)制备好的结合物释放垫、反应膜与样品吸收垫、吸水垫和PVC底板组装成试纸条。
1、甲基嘧啶磷单克隆抗体-胶体金标记物的制备
(1)胶体金的制备
用双蒸去离子水将1％氯金酸稀释成0.01％(质量分数)，取100ml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2.5ml 1％柠檬酸三钠，继续匀速搅拌加热至溶液呈透亮的红色时停止，冷却至室温后用去离子水恢复到原体积，4℃保存。制备好的胶体金外观纯净、透亮、无沉淀和漂浮物。
(2)甲基嘧啶磷单克隆抗体-胶体金标记物的制备
在磁力搅拌下，用0.2mol/L碳酸钾溶液调胶体金的pH值至7.0，按每毫升胶体金溶液中加入20～50μg的标准向胶体金溶液中加入甲基嘧啶磷单克隆抗体，继续搅拌混匀30min，加入10％BSA，使其在胶体金溶液中的终浓度为1％(体积分数)，静置10min。12000r/min、4℃离心40min，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：含酪蛋白0.02％～0.1％(质量分数)、吐温-80 0.05％～0.2％(质量分数)、pH7.2的0.02mol/L磷酸盐缓冲液。
2、结合物释放垫的制备
将结合物释放垫浸泡于含有牛血清白蛋白(牛血清白蛋白在缓冲液中的浓度为0.5％)、pH为7.2、0.5mol/L的磷酸盐缓冲液中，均匀浸湿1h，37℃烘3h备用。用Isoflow喷膜仪将制备好的甲基嘧啶磷单克隆抗体-胶体金标记物均匀喷涂在结合物释放垫上，每1cm结合物释放垫喷涂0.01ml甲基嘧啶磷单克隆抗体-胶体金标记物后，置于37℃环境中(湿度＜20％)60min后取出，置于干燥环境(湿度＜20％)中保存备用。
3、反应膜的制备
将甲基嘧啶磷半抗原-卵清蛋白偶联物包被到反应膜上构成检测线，将羊抗鼠抗抗体包被在反应膜上构成质控线。
包被过程：用磷酸缓冲液将甲基嘧啶磷半抗原-卵清蛋白偶联物稀释到10mg/ml，用Isoflow点膜仪将其包被于硝酸纤维素膜上的检测线(T线)，包被量为0.8μl/cm；用0.01mol/L、pH7.4的磷酸盐缓冲液将羊抗鼠抗抗体稀释到200μg/ml，用Isoflow点膜仪将其包被于硝酸纤维素膜上的质控线(C线)，包被量为1.0μl/cm。将包被好的反应膜置于37℃条件下干燥2h，备用。
4、样品吸收垫的制备
将样品吸收垫置于含0.5％牛血清白蛋白(体积分数)、pH7.2、0.1mol/L磷酸盐缓冲液中浸泡2h，37℃烘2h备用。
5、试纸条的组装
将样品吸收垫、结合物释放垫、反应膜、吸水垫依次按顺序粘贴在PVC底板上；结合物释放垫从起始端有1/3区域被样品吸收垫覆盖，结合物释放垫的末端与反应膜的始端连接，反应膜的末端与吸水垫的始端相连，样品吸收垫的始端与PVC底板的始端对齐，吸水垫的末端与PVC底板的末端对齐；所述反应膜上有检测线和质控线，检测线(T线)和质控线(C线)均为与所述试纸条的长相垂直的条状带；检测线位于靠近结合物释放垫的末端的一侧；质控线位于远离结合物释放垫的末端的一侧；将试纸条用机器切成3mm宽的小条，装在特制的塑料制卡中，4～30℃条件下可保存12个月。
实施例3甲基嘧啶磷残留酶联免疫试剂盒的制备
1、酶结合物的制备
以山羊作为免疫动物，以甲基嘧啶磷单克隆抗体为免疫原对无病原体山羊进行免疫，得到甲基嘧啶磷抗体。将甲基嘧啶磷抗体与辣根过氧化物酶(HRP)进行偶联得到酶结合物。
2、酶标板的制备
用包被缓冲液将包被原稀释成20μg/mL，每孔加入100μl，25℃避光孵育2h，倾去孔中液体，用洗涤液洗涤2次，每次30s，拍干，然后在每孔中加入200μl封闭液，25℃避光孵育2h，倾去孔内液体拍干，干燥后用铝膜真空密封保存。
3、酶联免疫试剂盒包含下述组分：
(1)包被甲基嘧啶磷偶联抗原的酶标板；
(2)甲基嘧啶磷标准品溶液6瓶；
(3)用辣根过氧化物酶标记的甲基嘧啶磷抗体；
(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；
(5)终止液为2mol/L硫酸；
(6)洗涤液为pH值为7.4，含有0.5％～1.0％吐温-20、0.01‰～0.03‰叠氮化钠防腐剂、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。
Claims (4)
1.一种甲基嘧啶磷单克隆抗体，其特征在于所述单克隆抗体由甲基嘧啶磷半抗原-载体蛋白偶联物作为免疫原制备获得，所述半抗原的制备方法为：取2.26g化合物Ⅰ，1,2-二氯乙烷150ml溶解,加二甲基硫代磷酰氯1.82g，三乙胺0.8ml,室温搅拌反应12h，停止反应，加水100ml，震荡萃取，分去水相，有机相蒸干，得到红色油状物，加甲醇70ml溶解，加钯碳0.4g，充分搅拌，抽取空气，通入氢气，室温搅拌4h，停止反应，过滤，除去钯碳，减压蒸干，得到粗产物，无水乙醇10ml重结晶，得到化合物Ⅲ0.9g，收率28.1％；化合物Ⅲ0.9g，加吡啶80ml溶解，加琥珀酸酐0.32g，油浴加热，80℃反应12h，停止反应，旋蒸除去吡啶，油状物上硅胶柱，二氯甲烷/甲醇体积比10:1洗脱分离，得到琥珀酸甲基嘧啶磷半抗原产物0.7g，收率59.32％。
2.如权利要求1所述的甲基嘧啶磷单克隆抗体，其特征在于所述甲基嘧啶磷半抗原的分子结构式为：
3.如权利要求1所述的甲基嘧啶磷单克隆抗体，其特征在于所述单克隆抗体用于制备检测甲基嘧啶磷的试纸条，所述试纸条包括：样品吸收垫(1)、结合物释放垫(2)、反应膜(3)、吸水垫(4)和底板(7)，其特征在于所述反应膜上具有包被有甲基嘧啶磷半抗原-载体蛋白偶联物的检测线(5)和包被有羊抗鼠抗抗体的质控线(6)，所述结合物释放垫(2)喷涂有甲基嘧啶磷单克隆抗体-胶体金标记物。
4.如权利要求1所述的甲基嘧啶磷单克隆抗体，其特征在于所述单克隆抗体用于制备检测甲基嘧啶磷的酶联免疫试剂盒，所述试剂盒包括：包被有包被原的酶标板、甲基嘧啶磷标准品溶液、甲基嘧啶磷抗体、酶结合物浓缩液、酶结合物稀释液、底物显色液、终止液、洗涤液，所述包被原为甲基嘧啶磷偶联抗原，所述酶结合物为酶标记的甲基嘧啶磷抗体。
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