重金属铜化学发光酶联免疫快速检测方法的建立
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摘要
目的:用自制重金属铜单抗和合成铜完全抗原,建立检测重金属铜离子的间接竞争化学发光酶联免疫(CLEIA)法,为植物中草药和土壤环境中铜的快速定量测定提供简便、可靠的方法。方法:使用本实验室前期制备并鉴定合格的优质铜单抗和合成铜抗原,建立铜间接竞争CLEIA法,并对其准确度、精密度、特异性和稳定性进行评价;采集植物中草药和土壤样品共31份,通过微波消解联合沉淀法进行前处理并制备待测样,由间接竞争CLEIA法进行铜含量测定,以ICP-AES仪器法所测结果为对照评价其可靠性。结果:该方法检测灵敏度为17. 9 ng/ml,检测限为1. 9 ng/ml,线性范围为3. 0～200 ng/ml;准确度试验回收率为98. 8%～105. 5%,精密度试验RSD≤5. 0%;特异性试验仅与Mg~(2+)、Zn~(2+)有13. 3%、3. 3%的交叉反应;酶标板置-20℃避光保存6周内其检测结果准确性未出现明显改变。对植物中草药和土壤样品进行前处理并制样检测,对比ICP-AES法检测结果,相关系数分别为0. 972和0. 989,两种方法具有较好相关性,表明间接竞争CLEIA法可应用于一般环境样品中铜的简便快速定量测定。结论:成功建立铜间接竞争CLEIA检测法,其灵敏度及各参数均符合国家法定相关样品铜离子检测技术指标的要求,可应用于一般环境样品中铜含量的快速测定。
Objective: Use the prepared anti copper McAb and the synthesized copper antigen to establish the indirect competitive chemiluminescence linked enzyme immunoassay( CLEIA) for simple,fast and quantitative detection of copper content in herbal plants and soil samples. Methods: Use the anti copper McAb and antigen to establish the indirect competitive CLEIA,and evaluate its accuracy,precision,specificity and stability performance. A total of 31 samples include herbal plants and soil were collected and pretreated by microwave digestion combined with precipitation. The copper content was determined by indirect competition CLEIA and ICPAES after sample preparation,and the relevance of the two methods was evaluated. Results: The copper detection sensitivity was 17. 9 ng/ml,the low detection limit was 1. 9 ng/ml,and the linear range was 3. 0 to 200 ng/ml. The recovery rate of accuracy test was 98. 8%to 105. 5%,and the RSD was less or equal to 5%. Specific test showed that the anti copper McAb had a slight cross reactivity rate of13. 3%and 3. 3%to Mg~(2+)and Zn~(2+). Storage the CLEIA microplates at-20℃ away from light for 2 to 8 weeks,the stability test showed no significant change in 6 weeks. Herbal plants and soil samples were pre-treated and prepared into test samples detected by indirect competitive CLEIA and ICP-AES for copper content. The two methods showed good relativity with the correlation coefficient value of0. 972 and 0. 989. The above results indicated that the indirect competitive CLEIA method can be applied to the detection of copper content in various environmental samples. Conclusion: Successfully established the indirect competitive CLEIA for simple,fast and quantitative detection of copper content in various environmental samples,and the detection sensitivity and other parameters are in line with the requirements of national inspection technical.
Objective: Use the prepared anti copper McAb and the synthesized copper antigen to establish the indirect competitive chemiluminescence linked enzyme immunoassay( CLEIA) for simple,fast and quantitative detection of copper content in herbal plants and soil samples. Methods: Use the anti copper McAb and antigen to establish the indirect competitive CLEIA,and evaluate its accuracy,precision,specificity and stability performance. A total of 31 samples include herbal plants and soil were collected and pretreated by microwave digestion combined with precipitation. The copper content was determined by indirect competition CLEIA and ICPAES after sample preparation,and the relevance of the two methods was evaluated. Results: The copper detection sensitivity was 17. 9 ng/ml,the low detection limit was 1. 9 ng/ml,and the linear range was 3. 0 to 200 ng/ml. The recovery rate of accuracy test was 98. 8%to 105. 5%,and the RSD was less or equal to 5%. Specific test showed that the anti copper McAb had a slight cross reactivity rate of13. 3%and 3. 3%to Mg~(2+)and Zn~(2+). Storage the CLEIA microplates at-20℃ away from light for 2 to 8 weeks,the stability test showed no significant change in 6 weeks. Herbal plants and soil samples were pre-treated and prepared into test samples detected by indirect competitive CLEIA and ICP-AES for copper content. The two methods showed good relativity with the correlation coefficient value of0. 972 and 0. 989. The above results indicated that the indirect competitive CLEIA method can be applied to the detection of copper content in various environmental samples. Conclusion: Successfully established the indirect competitive CLEIA for simple,fast and quantitative detection of copper content in various environmental samples,and the detection sensitivity and other parameters are in line with the requirements of national inspection technical.
引文
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[10]Ouyang H,Shu Q,Wang WW,et al.An ultra-facile and label-free immunoassay strategy for detection of copper(II)utilizing chemiluminescence self-enhancement of Cu(II)-ethylenediaminetetraacetate chelate[J].Biosens Bioelectron,2016,85:157-163.
[11]Kim KM,Kim YH,Oh SH,et al.A chelate complex-enhanced luminol system for selective determination of Co(II),Fe(II)and Cr(III)[J].Luminescence,2013,28:372-377.
[12]Zhao HW,Nan TG,Tan GY,et al.Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals[J].Analytical Chimica Acta,2011,702:102-108.
[13]Xu LG,Xing CR,Liu LQ,et al.Quick,easy,cheap,effective,rugged and safe strategy for quantifying cadmium polluted rice[J].Food Agr Immunol,2016,27(6):783-795.
[14]Qi YY,Xiu FR,Li BX.One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles[J].Anal Biochem,2014,449:1-88.
[15]张赟,温伟红.细胞和分子免疫学实用实验技术[M].西安:第四军医大学出版社,2013:55-59.Zhang Y,Wen WH.Cellular and molecular immunology practical experimental techniques[M].Xi'an:Fourth Military Medical U-niversity Press,2013:55-59.
[16]郝代玲,黄建芳,杨浩,等.重金属铜的单抗的制备及免疫学检测方法的建立[J].食品工业科技,2017,38(19):245-248.Hao DL,Huang JF,Yang H,et al.Preparation of monoclonal antibody against copper and establishment of immunological detection methods[J].Food Ind Technol,2017,38(19):245-248.
[17]何恬叶,张颖红,胡子文.微波消解ICP-OES法测定土壤样品中22种元素[J].分析试验室,2018,37(1):84-87.He TY,Zhang YH,Hu ZW.Determination of 22 elements in soil samples by microwave digestion ICP-OES[J].Anal Lab,2018,37(1):84-87.
[18]周勇义,谷学新,马群.微波消解-石墨炉原子吸收法测定中药中铅、镉含量的研究[J].光谱实验室,2004,21(3):465-468.Zhou YY,Gu XX,Ma Q.Determination of lead and cadmium in chinese herbs by microwave digestion with GF-AAS[J].Spectrum Lab,2004,21(3):465-468.
[19]熊英禹,付忠田,黄戊生.化学沉淀法处理模拟含铜废水的研究[J].环境保护科学,2014,40(2):35-38.Xiong YY,Fu ZT,Huang WS.Study on chemical precipitation process for simulating waste water containing copper[J].Environ Protec Sci,2014,40(2):35-38.
[20]郭燕妮,方增坤,胡杰华,等.化学沉淀法处理含重金属废水的研究进展[J].工业水处理,2011,31(12):9-13.Guo YN,Fang ZK,Hu JH,et al.Research progress of treatment of waste water containing heavy metals by chemical precipitation[J].Industrial Water Treatment,2011,31(12):9-13.
[21]刘艳梅,钟辉,黄建芳,等.直接竞争ELISA检测大米样品中的重金属镉[J].免疫学杂志,2015,31(6):528-532.Liu YM,Zhong H,Huang JF,et al.Direct competitive ELISA for detection of heavy metals in rice samples[J].J Immunol,2015,31(6):528-532.
[22]Cooper MS,Ma MT,Sunassee K,et al.Comparison of 64Cu-complexing bifunctional chelators for radioimmunoconjugation:labeling efficiency,specic activity,and in vitro/in vivo stability[J].Bioconjugate Chem,2012,23:1029-1039.
[23]白桥,任小菊.ICP-AES技术及其应用研究[J].广东化工,2017,44(17):97-98.Bai Q,Ren XJ.ICP-AES technology and its application research[J].Guangdong Chem Ind,2017,44(17):97-98.
[24]GB 15618-2009土壤环境质量标准[S].2009.GB 15618-2009 Environmental quality standard for soils[S].2009.
[25]Kong T,Liu GW,Li XB,et al.Synthesis and identification of artificial antigens for cadmium and copper[J].Food Chem,2010,123(4):1204-1209.
[26]梁书怀,蒙华毅,吴祖军,等.微波消解-电感耦合等离子体质谱法同时检测大米中的6种重金属元素[J].中国无机分析化学,2014,4(3):10-14.Liang SH,Meng HY,Wu ZJ,et al.Simultaneous determination of6 heavy metal elements in rice by microwave digestion with ICP-MS[J].Chin Inorg Anal,2014,4(3):10-14.
[2]周建军,周桔,冯仁国.我国土壤重金属污染现状及治理战略[J].中国科学院院刊,2014,29(3):315-320.Zhou JJ,Zhou J,Feng RG.The status and control strategy of heavy metal pollution in soil in China[J].Bulletin Chin Acad Sci,2014,29(3):315-320.
[3]Chen L,Zhou SL,Shi YX,et al.Heavy metals in food crops,soil,and water in the Lihe River Watershed of the Taihu Region and their potential health risks when ingested[J].Sci Total Environ,2018,615:141-149.
[4]朱达俊.中国重大环境案例回顾:紫金矿业水污染案[J].环境保护与循环经济,2013,2:28-31.Zhu DJ.China's major environmental case review:Zijin mining water pollution case[J].Environ Prot Circular Econ,2013,2:28-31.
[5]潘科,朱艾嘉,徐志斌,等.中国近海和河口环境铜污染的状况[J].生态毒理学报,2014,9(4):618-631.Pan K,Zhu AJ,Xu ZB,et al.The status of copper pollution in China's offshore and estuary environments[J].J Ecotoxicol,2014,9(4):618-631.
[6]Chen ZL,Huang L,Zhou CY,et al.Characteristics and evaluation of heavy metal pollution in vegetables in Guangzhou[J].Environ Sci,2017,38(1):389-398.
[7]杨德才,王哲,姜玉富.家畜铜中毒研究进展[J].动物医学进展,2005,26(4):27-30.Yang DC,Wang Z,Jiang YF.Research progress of copper poisoning in cattle[J].Prog Animal Med,2005,26(4):27-30.
[8]哈尔阿力·沙布尔,杨润,阿依夏木.铜、锌、砷过量对饲料安全性的影响[J].中国动物保健,2008,109:56-59.Halaly S,Yang R,Ayi X.The effect of Cu,Zn and As excess on feed safety[J].Chin Animal Health,2008,109:56-59.
[9]Murillo Pulgarín JA,García Bermejo LF,Carrasquero Durán A.An easy and fast IC-CL procedure for the monitoring of Cu(II)and Co(II)in environmental samples[J].Int J Environ Sci Te,2017,14:233-240.
[10]Ouyang H,Shu Q,Wang WW,et al.An ultra-facile and label-free immunoassay strategy for detection of copper(II)utilizing chemiluminescence self-enhancement of Cu(II)-ethylenediaminetetraacetate chelate[J].Biosens Bioelectron,2016,85:157-163.
[11]Kim KM,Kim YH,Oh SH,et al.A chelate complex-enhanced luminol system for selective determination of Co(II),Fe(II)and Cr(III)[J].Luminescence,2013,28:372-377.
[12]Zhao HW,Nan TG,Tan GY,et al.Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals[J].Analytical Chimica Acta,2011,702:102-108.
[13]Xu LG,Xing CR,Liu LQ,et al.Quick,easy,cheap,effective,rugged and safe strategy for quantifying cadmium polluted rice[J].Food Agr Immunol,2016,27(6):783-795.
[14]Qi YY,Xiu FR,Li BX.One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles[J].Anal Biochem,2014,449:1-88.
[15]张赟,温伟红.细胞和分子免疫学实用实验技术[M].西安:第四军医大学出版社,2013:55-59.Zhang Y,Wen WH.Cellular and molecular immunology practical experimental techniques[M].Xi'an:Fourth Military Medical U-niversity Press,2013:55-59.
[16]郝代玲,黄建芳,杨浩,等.重金属铜的单抗的制备及免疫学检测方法的建立[J].食品工业科技,2017,38(19):245-248.Hao DL,Huang JF,Yang H,et al.Preparation of monoclonal antibody against copper and establishment of immunological detection methods[J].Food Ind Technol,2017,38(19):245-248.
[17]何恬叶,张颖红,胡子文.微波消解ICP-OES法测定土壤样品中22种元素[J].分析试验室,2018,37(1):84-87.He TY,Zhang YH,Hu ZW.Determination of 22 elements in soil samples by microwave digestion ICP-OES[J].Anal Lab,2018,37(1):84-87.
[18]周勇义,谷学新,马群.微波消解-石墨炉原子吸收法测定中药中铅、镉含量的研究[J].光谱实验室,2004,21(3):465-468.Zhou YY,Gu XX,Ma Q.Determination of lead and cadmium in chinese herbs by microwave digestion with GF-AAS[J].Spectrum Lab,2004,21(3):465-468.
[19]熊英禹,付忠田,黄戊生.化学沉淀法处理模拟含铜废水的研究[J].环境保护科学,2014,40(2):35-38.Xiong YY,Fu ZT,Huang WS.Study on chemical precipitation process for simulating waste water containing copper[J].Environ Protec Sci,2014,40(2):35-38.
[20]郭燕妮,方增坤,胡杰华,等.化学沉淀法处理含重金属废水的研究进展[J].工业水处理,2011,31(12):9-13.Guo YN,Fang ZK,Hu JH,et al.Research progress of treatment of waste water containing heavy metals by chemical precipitation[J].Industrial Water Treatment,2011,31(12):9-13.
[21]刘艳梅,钟辉,黄建芳,等.直接竞争ELISA检测大米样品中的重金属镉[J].免疫学杂志,2015,31(6):528-532.Liu YM,Zhong H,Huang JF,et al.Direct competitive ELISA for detection of heavy metals in rice samples[J].J Immunol,2015,31(6):528-532.
[22]Cooper MS,Ma MT,Sunassee K,et al.Comparison of 64Cu-complexing bifunctional chelators for radioimmunoconjugation:labeling efficiency,specic activity,and in vitro/in vivo stability[J].Bioconjugate Chem,2012,23:1029-1039.
[23]白桥,任小菊.ICP-AES技术及其应用研究[J].广东化工,2017,44(17):97-98.Bai Q,Ren XJ.ICP-AES technology and its application research[J].Guangdong Chem Ind,2017,44(17):97-98.
[24]GB 15618-2009土壤环境质量标准[S].2009.GB 15618-2009 Environmental quality standard for soils[S].2009.
[25]Kong T,Liu GW,Li XB,et al.Synthesis and identification of artificial antigens for cadmium and copper[J].Food Chem,2010,123(4):1204-1209.
[26]梁书怀,蒙华毅,吴祖军,等.微波消解-电感耦合等离子体质谱法同时检测大米中的6种重金属元素[J].中国无机分析化学,2014,4(3):10-14.Liang SH,Meng HY,Wu ZJ,et al.Simultaneous determination of6 heavy metal elements in rice by microwave digestion with ICP-MS[J].Chin Inorg Anal,2014,4(3):10-14.