CN110646605A - 一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用 - Google Patents
一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用 Download PDFInfo
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Abstract
一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用,属于食品检测技术领域。本发明PVC底板两端分别设有样品垫和吸水垫;在PVC底板中部设有硝酸纤维素膜检测层,在硝酸纤维素膜检测层与样品垫之间设有荧光微球标记垫;所述荧光微球标记垫一端与样品垫互相叠加,另一端与硝酸纤维素膜检测层互相叠加;所述硝酸纤维素膜检测层上依次设有质控线和检测线。本发明检测速度快,全过程只需要5min即可,可以实施大批量样品的快速检测;本发明灵敏度高,大米样本检测限为5ng/mL;本发明过程简单,直接上样检测,样品前处理简单,无需经过专业培训,易于推广,不需要仪器,适合现场检测。
Description
技术领域
本发明涉及一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用,属于食品检测技术领域。
背景技术
近年来,随着科技的发展,人们的生活质量越来越好,但是伴随着出现的环境问题也困扰着人们的生活。工业污染、化肥的大量使用,导致粮食中的重金属含量超标。铅,作为一种人体难以排除的重金属元素,进入人的体内之后很难完全排除,会伤害人的脑细胞,影响人脑正常活动,另外,铅金属严重影响胎儿的神经系统,造成智力低下等问题。
建立食品样品中铅快速准确的检测方法势在必行。目前,传统的重金属检测方法概括起来主要包括以下几种:原子吸收光谱法(AAS)、原子发射光谱法(AES)、电感耦合等离子体质谱法(ICP-MS)、原子荧光光谱法(AFS)、紫外可见分光光度法(UV-VIS)、阳极溶出伏安法(ASV)等。但这些方法专业性要求高、分析时间长、分析价格昂贵,不利于推广和现场检测。而免疫分析方法与以上分析方法相比具有特异性强、操作简便、仪器化程度和分析成本低的优点,荧光定量检测试纸条可以进行定量或半定量的测定,具有相当高的灵敏度,能满足不同样品的快速检测要求。
发明内容
本发明的目的是克服现有技术的不足,提供一种重金属铅离子荧光定量检测试纸条及其制备方法与应用,可大规模地对粮食及其加工产品残留重金属铅进行快速、方便、准确的检测。
本发明的技术方案,一种重金属铅离子的荧光定量快速检测试纸条,包括样品垫、荧光微球标记垫、检测线、质控线、吸水垫、硝酸纤维素膜检测层和PVC底板;
所述PVC底板两端分别设有样品垫和吸水垫;在PVC底板中部设有硝酸纤维素膜检测层,在硝酸纤维素膜检测层与样品垫之间设有荧光微球标记垫;所述荧光微球标记垫一端与样品垫互相叠加,另一端与硝酸纤维素膜检测层互相叠加;所述硝酸纤维素膜检测层上依次设有质控线和检测线。
一株高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob,巳保藏于中国微生物菌种保藏管理委员会普通微生物中心,分类命名为单克隆细胞株,保藏编号为CGMCCNo.17387,保藏日期为2019年3月7日。
所述重金属铅离子的荧光定量快速检测试纸条的制备方法,具体步骤如下:
(1)铅抗原的制备:首先,将10mg ITCBE溶于1mL无水DMSO中,震荡混匀,-20℃保存。称取BSA 20mg,加入5mLHBS(0.01M PH=9)溶液溶解,滴加66μL ITCBE溶液,室温搅拌反应8h,然后滴加596μL(1mg/mL)的铅标准品溶液,用0.5M NaOH调节PH,使溶液的PH维持在8-9,反应1h即可。反应完毕用截留量3000的Amicon Ultra-4 Ultracel-3K 超滤离心管离心6500rpm,时间20min,每次超滤完毕后用3mL HBS溶液重悬。反复超滤三次,加入10mL HBS溶液使蛋白的终浓度为2mg/mL,-20℃冻存保存。
(2)抗铅离子单抗的制备:用双功能螯合剂ITCBE与KLH偶联之后加入重金属铅离子进行螯合,作为完全免疫原,以此完全免疫原与等量弗氏佐剂混合乳化后,对BALB/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫采用重金属铅离子的完全抗原与完全弗氏佐剂混合,剂量为100μg/只;多次加强免疫采用重金属铅离子的完全抗原与不完全弗氏佐剂混合,剂量为50μ只;最后一次用重金属铅离子完全抗原与生理盐水混合冲刺免疫。采用腹腔注射,剂量为25μg/只。首次免疫与第二次加强免疫之间间隔一个月,多次加强免疫之间间隔21天,冲刺免疫与最后一次加强免疫之间间隔18-21天。通过间接竞争酶联免疫法(ic-ELISA)观测小鼠免疫效果即检测小鼠血清的效价和抑制。
(3)细胞融合与细胞株建立:通过聚乙二醇(PEG 4000)法将小鼠脾细胞和小鼠骨髓瘤细胞进行融合,采用选择性培养基(HAT培养基)筛选出杂交瘤细胞,并用HT培养基进行细胞培养。融合一周后利用ic-ELISA法检测阳性细胞孔,并进一步利用ic-ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对抑制较好的阳性细胞孔进行亚克隆,一周后再次检测、挑孔、亚克隆。按上述方法进行三次亚克隆后获得重金属铅离子的高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob。
(4)重金属铅离子抗体纯化:将高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob进行扩大培养,注射杂交瘤细胞进小鼠体内诱生腹水;取1mL腹水,加入等体积的醋酸钠缓冲液1mL,于室温搅拌下逐滴加入辛酸33.3μL,震荡30min,8000rpm离心5min,得到上清液;加入饱和硫酸铵1.5-2mL,4℃静置1-2h;8000rpm离心5min,弃上清液,将沉淀溶于PBS溶液中透析2-3天,得到抗铅离子的单克隆抗体。
(5)重金属铅离子的荧光定量快速检测试纸条的制备:
a、荧光微球储备液的制备:取0.05M pH为8的硼酸缓冲液400μL于2mL离心管中,加入100-120μL荧光微球,漩涡震荡,混匀备用;
b、荧光微球标记抗体的制备:取10mg/mL的EDC溶液 20μL,室温震荡活化15min,2000rpm 10℃离心10min,弃上清,用0.5mL 0.05M pH=8 硼酸缓冲液复溶,超声分散;加入重金属铅离子抗体,使蛋白终浓度为30-50μg/mL,置于250r摇床上2h;加入封闭液,即最终浓度为1%-2%BSA,置于摇床封闭1-2h,2000rpm离心10min,弃上清,用0.5mL硼酸缓冲液复溶,洗涤离心后用偶联储存缓冲溶液复溶,超声分散后得到荧光标记的重金属铅离子抗体,4℃保存;
c、荧光微球标记垫的制备:将0.2mg/mL步骤b制备所得标记好荧光的重金属铅离子抗体均匀喷在玻璃纤维膜上,喷液量0.5-1μL/cm,37℃烘干过夜,封袋备用;
d0.2mg/mL的重金属铅离子抗原均匀喷在硝酸纤维素膜检测层上,得到检测线;将稀释好的羊抗鼠IgG均匀喷在硝酸纤维素膜上,得到质控线,37℃烘干过夜,封袋备用;
e、组合:将PVC底板、样品垫、荧光微球标记垫、涂有检测线、和质控线、的硝酸纤维素膜检测层、和吸水垫、组合,即得产品重金属铅离子的荧光定量快速检测试纸条。
将上述重金属铅离子的荧光定量快速检测试纸条应用于食品中重金属铅离子的快速检测,适用于海关、企业、检验检疫单位等,可实现大米样本中重金属铅离子的快速检测。
本发明的有益效果:本发明检测速度快,全过程只需要5min即可,可以实施大批量样品的快速检测;本发明灵敏度高,大米样本检测限为5ng/mL;
本发明过程简单,直接上样检测,样品前处理简单,无需经过专业培训,易于推广,不需要仪器,适合现场检测。
生物材料样品保藏:一株高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.17387,分类命名为单克隆细胞株,保藏日期为2019年3月7日。
附图说明
图1是本发明检测试纸条结构示意图。
图2是大米样本铅离子检测标准曲线。
附图标记说明:1、样品垫;2、荧光微球标记垫;3、检测线;4、质控线;5、吸水垫;6、硝酸纤维素膜检测层;7、PVC底板。
具体实施方式
实施例1
如图1所示,一种重金属铅离子的荧光定量快速检测试纸条,包括样品垫1、荧光微球标记垫2、检测线3、质控线4、吸水垫5、硝酸纤维素膜检测层6和PVC底板7;
所述PVC底板7两端分别设有样品垫1和吸水垫5;在PVC底板7中部设有硝酸纤维素膜检测层6,在硝酸纤维素膜检测层6与样品垫1之间设有荧光微球标记垫2;所述荧光微球标记垫2一端与样品垫1互相叠加,另一端与硝酸纤维素膜检测层6互相叠加;所述硝酸纤维素膜检测层6上依次设有质控线4和检测线3。
所述重金属铅离子的荧光定量快速检测试纸条的制备方法,具体步骤如下:
(1)铅抗原的制备:首先,将10mg ITCBE溶于1mL无水DMSO中,震荡混匀,-20℃保存。称取BSA 20mg,加入5mLHBS(0.01M PH=9)溶液溶解,滴加66μL ITCBE溶液,室温搅拌反应8h,然后滴加596μL(1mg/mL)的铅标准品溶液,用0.5M NaOH调节PH,使溶液的PH维持在8-9,反应1h即可。反应完毕用截留量3000的Amicon Ultra-4 Ultracel-3K 超滤离心管离心6500rpm,时间20min,每次超滤完毕后用3mL HBS溶液重悬。反复超滤三次,加入10mL HBS溶液使蛋白的终浓度为2mg/mL,-20℃冻存保存。
(2)抗铅离子单抗的制备:用双功能螯合剂ITCBE与KLH偶联之后加入重金属铅离子进行螯合,以此作为完全免疫原与等量弗氏佐剂混合乳化后,对BALB/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫采用重金属铅离子的完全抗原与完全弗氏佐剂混合,剂量为100μg/只;多次加强免疫采用重金属铅离子的完全抗原与不完全弗氏佐剂混合,剂量为50μg/只;最后一次用重金属铅离子完全抗原与生理盐水混合冲刺免疫。采用腹腔注射,剂量为25μg/只。首次免疫与第二次加强免疫之间间隔一个月,多次加强免疫之间间隔21天,冲刺免疫与最后一次加强免疫之间间隔18-21天。通过间接竞争酶联免疫法(ic-ELISA)观测小鼠免疫效果即检测小鼠血清的效价和抑制。
(3)细胞融合与细胞株建立:通过聚乙二醇(PEG 4000)法将小鼠脾细胞和小鼠骨髓瘤细胞进行融合,采用选择性培养基(HAT培养基)筛选出杂交瘤细胞,并用HT培养基进行细胞培养。融合一周后利用ic-ELISA法检测阳性细胞孔,并进一步利用ic-ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对抑制较好的阳性细胞孔进行亚克隆,一周后再次检测、挑孔、亚克隆。按上述方法进行三次亚克隆后获得重金属铅离子的高分泌特异抗体的单克隆杂交瘤细胞株Rob。
(4)重金属铅离子抗体纯化:将高分泌特异抗体的单克隆杂交瘤细胞株Rob进行扩大培养,注射杂交瘤细胞进小鼠体内诱生腹水;取1mL腹水,加入等体积的醋酸钠缓冲液1mL,于室温搅拌下逐滴加入辛酸33.3μL,震荡30min,8000rpm离心5min,得到上清液;加入饱和硫酸铵1.5-2mL,4℃静置1-2h;8000rpm离心5min,弃上清液,将沉淀溶于PBS溶液中透析2-3天,得到抗铅离子的单克隆抗体。
(5)重金属铅离子的荧光定量快速检测试纸条的制备:
a、荧光微球储备液的制备:取0.05M pH为8的硼酸缓冲液400μL于2mL离心管中,加入100μL荧光微球,漩涡震荡,混匀备用;
b、荧光微球标记抗体的制备:取10mg/mL的EDC溶液 20μL,室温震荡活化15min,20000rpm 10℃离心10min,弃上清,用0.5ml 0.05M PH=8 硼酸缓冲液复溶,超声分散;加入重金属铅离子抗体,使蛋白终浓度为30μg/mL,置于250r摇床上2h;加入封闭液,即最终浓度为1% BSA,置于摇床封闭2h,2000rpm离心10min,弃上清,用0.5ml硼酸缓冲液复溶,洗涤离心后用偶联储存缓冲溶液复溶,超声分散后得到荧光标记的重金属铅离子抗体,4℃保存;
c、荧光微球标记垫的制备:将0.2mg/mL步骤b制备所得标记好荧光的重金属铅离子抗体均匀喷在玻璃纤维膜检测层上,喷液量1μL/cm,37℃烘干过夜,封袋备用;
d、硝酸纤维素膜检测层的制备:将稀释好的0.2mg/mL的重金属铅离子抗原均匀喷在硝酸纤维素膜检测层上,得到检测线;将稀释好的羊抗鼠IgG均匀喷在硝酸纤维素膜上,得到质控线,37℃烘干过夜,封袋备用;
e、组合:将PVC底板7、样品垫1、荧光微球标记垫2、涂有检测线3和质控线4的硝酸纤维素膜检测层6和吸水垫5组合,即得产品重金属铅离子的荧光定量快速检测试纸条。
实施例2
检测时按如下步骤处理样品:准确称量粉碎至合适粒径的大米样品1g于50mL离心管中,加入4mL 0.5M稀盐酸溶液和1mL 1M稀硝酸,于振荡器上震荡3min,8000rpm离心5min,取上清液500μL加1mL的正己烷,取下层溶液200μL用1M NaHCO3稀释4倍备用,同时做试剂空白。
取20μL稀释好的样本溶液滴加在样品垫上,5min之后将试纸条放入荧光定量检测仪,即可检测出重金属铅离子浓度,图2为大米样本铅离子检测标准曲线;如果试纸条质控线4不显色,则说明试纸条质量有问题。
Claims (6)
1.一种重金属铅离子的荧光定量快速检测试纸条,其特征在于:包括样品垫(1)、荧光微球标记垫(2)、检测线(3)、质控线(4)、吸水垫(5)、硝酸纤维素膜检测层(6)和PVC底板(7);
所述PVC底板(7)两端分别设有样品垫(1)和吸水垫(5);在PVC底板(7)中部设有硝酸纤维素膜检测层(6),在硝酸纤维素膜检测层(6)与样品垫(1)之间设有荧光微球标记垫(2);所述荧光微球标记垫(2)一端与样品垫(1)互相叠加,另一端与硝酸纤维素膜检测层(6)互相叠加;所述硝酸纤维素膜检测层(6)上依次设有质控线(4)和检测线(3)。
2.权利要求1所述重金属铅离子的荧光定量快速检测试纸条的制备方法,其特征在于步骤如下:
(1)荧光微球储备液的制备:取0.05M pH为8的硼酸缓冲液400μL于2mL离心管中,加入100 -120μL荧光微球,漩涡震荡,混匀备用;
(2)荧光微球标记抗体的制备:取10mg/mL的EDC溶液 20μL,室温震荡活化15min,10℃2000rpm离心10min,弃上清,用0.5mL 0.05M pH=8 硼酸缓冲液复溶,超声分散;加入重金属铅离子抗体,使蛋白终浓度为30-50μg/mL,置于250r摇床上2h;加入封闭液,即最终浓度为1%-2%BSA,置于摇床封闭1-2h,2000rpm离心10min,弃上清,用0.5mL硼酸缓冲液复溶,洗涤离心后再用硼酸缓冲液复溶,超声分散后得到荧光标记的重金属铅离子抗体,4℃保存;
(3)荧光微球标记垫的制备:将0.2mg/mL步骤(2)制备所得标记好荧光的重金属铅离子抗体均匀喷在玻璃纤维膜上,喷液量0.5-1μL/cm,37℃烘干过夜,封袋备用;
(4)硝酸纤维素膜检测层的制备:将稀释好的0.2mg/mL的重金属铅离子抗原均匀喷在硝酸纤维素膜检测层(6)上,得到检测线(3);将稀释好的羊抗鼠IgG均匀喷在硝酸纤维素膜检测层(6)上,得到质控线(4),37℃烘干过夜,封袋备用;
(5)组合:将PVC底板(7)、样品垫(1)、荧光微球标记垫(2)、涂有检测线(3)和质控线(4)的硝酸纤维素膜检测层(6)和吸水垫(5)组合,即得产品重金属铅离子的荧光定量快速检测试纸条。
3.根据权利要求2所述重金属铅离子的荧光定量快速检测试纸条的制备方法,其特征在于:所述重金属铅离子抗体是由高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob分泌所得。
4.一株高分泌抗铅离子特异抗体的单克隆杂交瘤细胞株Rob,保藏于中国微生物菌种保藏管理委员会普通微生物中心,分类命名为单克隆细胞株,保藏编号为CGMCC No.17387,保藏日期为2019年3月7日。
5.抗铅离子的单克隆抗体,其特征在于:用权利要求4所述单克隆杂交瘤细胞株Rob,对其进行扩大培养,注射细胞进小鼠体内诱生腹水;取1mL腹水,加入等体积的醋酸钠缓冲液1mL,于室温搅拌下逐滴加入辛酸33.3μL,震荡30min,8000rpm离心5min,得到上清液;加入饱和硫酸铵1.5-2mL,4℃静置1-2h;8000rpm离心5min,弃上清液,将沉淀溶于PBS溶液中透析2-3天,得到抗铅离子的单克隆抗体。
6.权利要求1所述重金属铅离子的荧光定量快速检测试纸条的应用,其特征在于:将其应用于食品中重金属铅离子的快速检测。
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