CN111812316B - 一种甲氰菊酯人工抗原在酶联免疫试剂盒中的应用 - Google Patents
一种甲氰菊酯人工抗原在酶联免疫试剂盒中的应用 Download PDFInfo
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Abstract
本发明提供了一种检测甲氰菊酯的酶联免疫试剂盒,它包括:包被有包被原的酶标板、甲氰菊酯标准品溶液、甲氰菊酯抗体、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,所述包被原为甲氰菊酯偶联抗原,所述酶结合物为酶标记的甲氰菊酯抗体,所述甲氰菊酯抗体是由免疫原免疫动物得到的。本发明还公开了一种应用上述酶联免疫试剂盒检测甲氰菊酯的方法,它包括:首先进行样品前处理,然后用试剂盒进行检测,最后分析检测结果。本发明提供的酶联免疫试剂盒可用于检测蔬菜和水果样本中甲氰菊酯的含量,其操作简便、费用低廉、灵敏度高、能够现场监控且适合大量样本的筛查。
Description
技术领域
本发明涉及一种甲氰菊酯人工抗原在酶联免疫试剂盒中的应用技术,具体涉及一种甲氰菊酯人工抗原分子结构以及用于检测甲氰菊酯的酶联免疫试剂盒,可定性、定量检测蔬菜和水果中甲氰菊酯药物的残留量。
背景技术
农药在现代农业生产中发挥了极其重要的作用,拟除虫菊酯类农药因具有性质稳定,杀虫谱广,杀虫活性高,残效期短等特点,其中甲氰菊酯是应用较多的一种。甲氰菊酯长期大规模生产和使用,严重危害了生态环境安全及农产品品质。
国内外常用的甲氰菊酯残留检测的标准分析方法是GC和HPLC法,与这两种方法相比,免疫化学检测法对药物检测具有快速、特异、灵敏、准确、可以批量监测、样品处理简单、能实现自动化操作等优点。而免疫化学法检测甲氰菊酯的前提是需要具备针对甲氰菊酯的抗体,本申请发明了甲氰菊酯人工抗原制备方法,并将其应用于酶联免疫试剂盒中,用时短,操作简单,成本较低,适用于基层单位大批量地检测样品。
发明内容
本发明的目的在于提供一种能够检测蔬菜和水果中甲氰菊酯药物残留量的酶联免疫试剂盒,并提供一种高效、准确、简便、适于大批量样品筛选的定性、定量检测方法。
本发明试剂盒,它包括:包被有包被原的酶标板、甲氰菊酯标准品溶液、甲氰菊酯抗体、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,所述包被原为甲氰菊酯偶联抗原,所述酶结合物为酶标记的甲氰菊酯抗体。
所述甲氰菊酯特异性抗体是以一种甲氰菊酯人工抗原作为免疫原制备获得,所述甲氰菊酯特异性抗体可为甲氰菊酯单克隆抗体或甲氰菊酯多克隆抗体,其中优选甲氰菊酯单克隆抗体。
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氰菊酯抗体偶联得到的。
为了更方便现场监控和大量样本筛查,所述试剂盒还包括甲氰菊酯标准品溶液、底物显色液、终止液、洗涤液。
所述甲氰菊酯标准品溶液6瓶,浓度分别为0µg/L,1µg/L,3µg/L,9µg/L,27µg/L,81µg/L。
当标记酶为辣根过氧化物酶时,所述底物显色液由底物液A液和底物液B液组成,A为过氧化氢或过氧化脲,B液为邻苯二胺或四甲基联苯胺,所述终止液为1~2mol/L的硫酸溶液或盐酸缓冲液;当标记酶为细菌提取碱性磷酸酯酶时,所述底物显色液为对硝基磷酸盐缓冲液,所述终止液为1~2mol/L氢氧化钠溶液。
所述洗涤液优选为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。
其中在酶标板制备过程中所用到的包被缓冲液为pH值为9.6,0.05mol/L的碳酸盐缓冲液,封闭液为pH值为7.1~7.5,含有1%~3%酪蛋白、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。
本发明中酶标板的制备过程为: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或4℃过夜,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150~200μl封闭液,25℃避光孵育1~2h,倾去孔内液体拍干,干燥后用铝膜真空密封保存。
本发明的检测原理为:
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,样本中残留的甲氰菊酯和酶标板微孔条上预包被的偶联抗原竞争抗甲氰菊酯的酶结合物,用TMB底物显色,样本吸光度值与其所含残留物甲氰菊酯的含量成负相关,与标准曲线比较,再乘以其对应的稀释倍数,即可得出样本中甲氰菊酯的残留量。
本发明还提供了一种应用上述酶联免疫试剂盒检测甲氰菊酯的方法,它包括步骤:
(1)用试剂盒进行检测;
(2)分析检测结果。
本发明检测甲氰菊酯的酶联免疫试剂盒主要采用ELISA方法定性或定量检测样品中甲氰菊酯的含量;对样品的前处理要求低,样品前处理过程简单,能同时快速检测大批量样品;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。本发明的酶联免疫试剂盒,结构简单、使用方便、价格便宜、携带便利、检测方法高效、准确、简便、适于大批量样品筛选的定性、定量。
附图说明
图1:甲氰菊酯半抗原的分子结构式
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 试剂盒组分的制备
1、甲氰菊酯半抗原的制备
取乙烯基甲氰菊酯3.61g,加乙腈30ml溶解,加1mol/L HCl 1ml,充分搅拌,加含1.58g高锰酸钾水溶液3ml,50℃反应3h,停止反应,加水200ml,加乙酸乙酯100ml×3萃取三次,合并有机相,无水硫酸钠干燥,蒸干,得到红色油状物,上硅胶柱,石油醚/乙酸乙酯(v/v,5/1)洗脱,分离,得到羧基甲氰菊酯半抗原产物3.4g,收率89.7%。
2、抗原的制备
免疫原制备——甲氰菊酯半抗原与牛血清白蛋白(BSA)偶联得到免疫原。
取羧基甲氰菊酯半抗原产物14mg,加DMF 1ml溶解,加HOBT12mg, EDC14mg,室温反应3h,得到半抗原溶液A液;取牛血清白蛋白(BSA)50mg,加0.05M CB缓冲液溶解,得到B液,将A液滴加到B液中,4℃反应12h, 0.02M PBS透析纯化3天,每天换液三次,得到甲氰菊酯-BSA偶联物,即为免疫原,-20℃保存,备用。
包被原制备——甲氰菊酯半抗原与卵清蛋白(OVA)偶联得到免疫原。
取羧基甲氰菊酯半抗原产物8.4mg,加DMSO 1ml溶解,加NHS6.8mg,EDC7.7mg,室温反应3h,得到半抗原溶液A液;取卵血清白蛋白(OVA)50mg,加0.05M CB缓冲液溶解,得到B液,将A液滴加到B液中,4℃反应12h, 0.02M PBS透析纯化3天,每天换液三次,得到甲氰菊酯-OVA偶联物,即为包被原,-20℃保存,备用。
通过用乙烯基甲氰菊酯,在酸性溶液中,在高锰酸钾的作用下,氧化碳碳双键,得到羧基甲氰菊酯,从而引入羧基基团,最大程度保留药物原有结构,再与载体蛋白偶联,得到免疫原及包被原。
3、甲氰菊酯单克隆抗体的制备
动物免疫:将上述步骤得到的免疫原注入到Balb/c小鼠体内,免疫剂量为150μg/只,使其产生抗血清。
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(数量配比)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,直到得到分泌甲氰菊酯单克隆抗体的杂交瘤细胞株。
细胞冻存和复苏:将单克隆杂交瘤细胞株用冻存液制成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。
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天后腹腔注射稳定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。
4、酶结合物的制备
以山羊作为免疫动物,以甲氰菊酯单克隆抗体为免疫原对无病原体山羊进行免疫,得到甲氰菊酯抗体。将甲氰菊酯抗体与辣根过氧化物酶(HRP)进行偶联得到酶结合物。
5、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。
实施例2 检测甲氰菊酯的酶联免疫试剂盒的组建
组建检测甲氰菊酯的酶联免疫试剂盒,使其包含下述组分:
(1)包被甲氰菊酯偶联抗原的酶标板;
(2)甲氰菊酯标准品溶液6瓶,浓度分别为0µg/L,1µg/L,3µg/L,9µg/L,27µg/L,81µg/L;
(3)用辣根过氧化物酶标记的甲氰菊酯抗体;
(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;
实施例3 蔬菜和水果中甲氰菊酯的检测
1、用试剂盒检测
将样本和标准品对应微孔按序编号,每个样本和标准品做2孔平行,并记录标准孔和样本孔所在的位置。加入标准品/样本50ml到对应的微孔中,然后加入酶结合物工作液50ml/孔, 25℃避光环境中反应30min。将孔内液体甩干,洗涤4-5次,拍干。加入底物液A液50ml /孔,再加入底物液B液50ml/孔,混匀,25℃避光环境中反应15min。加入终止液50ml/孔,混匀,设定酶标仪于450nm处,测定每孔OD值。
2、检测结果分析
标准品或样本的百分吸光率等于标准品或样本的吸光度值的平均值(双孔)除以第一个标准品(0标准)的吸光度值的平均值,再乘以100%,得到标准品或样本的百分吸光度值。以标准品百分吸光率为纵坐标,以甲氰菊酯标准品浓度(µg/L)的对数为横坐标,绘制标准曲线图。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甲氰菊酯的实际浓度。
实施例4 甲氰菊酯技术参数的确定试验
1、试剂盒灵敏度和检测限
按照常规方法测定试剂盒灵敏度,标准曲线的范围为1~81µg/L,IC50(50%抑制浓度)浮动范围为5.1~7.5μg/L;对20份样品进行检测,从标准曲线上查出对应于各百分吸光度值的浓度,以20份样本浓度的平均值加上3倍标准差表示检测限,结果显示,该方法对蔬菜和水果中甲氰菊酯的检测限分别为10μg/kg、20μg/kg。
2、样本精密度和准确度试验
以回收率作为准确度评价指标,重复测定某一浓度样品的检测结果相对标准偏差(RSD%)作为精密度评价指标。计算公式为:回收率(%)=实际测定值/理论值×100%,其中理论值为样品的添加浓度;相对标准偏差RSD%=SD/X×100%,其中SD为标准偏差,X为测定数据的平均值。
按10μg/kg、20μg/kg两个浓度的甲氰菊酯对蔬菜样品进行添加回收测定,按20µg/kg、40µg/kg两个浓度的甲氰菊酯对水果样品进行添加回收测定,每个样品做4个平行,用三批不同试剂进行测定,计算样品的平均回收率及精密度结果见下表。
表1 蔬菜和水果样本精密度及准确度试验
按10μg/kg、20μg/kg两个浓度的甲氰菊酯对蔬菜样品进行添加回收测定,按20µg/kg、40µg/kg两个浓度的甲氰菊酯对水果样品进行添加回收测定,平均回收率分别为70.3%~76.1%,65.4%~69.7%;批内、批间相对标准偏差均小于10%。
3、试剂盒稳定性试验
试剂盒保存条件为2~8℃,经过12个月的测定,试剂盒的最大吸光度值(零标准)、50%抑制浓度、甲氰菊酯添加实际测定值均在正常范围之内。考虑在运输和使用过程中,会有非正常保存条件出现,将试剂盒在37℃保存条件下放置7天,进行加速老化实验,结果表明该试剂盒各项指标完全符合要求。考虑到试剂盒冷冻情况发生,将试剂盒放入-20℃冰箱冷冻7天,测定结果也表明试剂盒各项指标完全正常。从以上结果可得出试剂盒可以在2~8℃至少保存12个月以上。
Claims (3)
2.如权利要求1所述的试剂盒,其特征在于所述免疫原的制备方法如下:
取羧基甲氰菊酯半抗原产物14mg,加DMF 1ml溶解,加HOBT 12mg,EDC 14mg,室温反应3h,得到半抗原溶液A液;取牛血清白蛋白BSA 50mg,加0.05M CB缓冲液溶解,得到B液,将A液滴加到B液中,4℃反应12h,0.02M PBS透析纯化3天,每天换液三次,得到甲氰菊酯-BSA偶联物,即为免疫原,-20℃保存,备用。
3.一种检测样品中甲氰菊酯含量的方法,包括步骤:
(1)用权利要求1~2任一项所述的试剂盒进行检测;
(2)分析检测结果。
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