基于双功能螯合剂NOTA的重金属铜人工抗原的合成与鉴定
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摘要
为研究杂环类双功能螯合剂在重金属铜人工抗原制备方面的效果,以2-S-(4-氨基苯)-1,4,7三氮杂环壬烷-1,4,7-三乙酸(P-NH_2-Bn-NOTA,简写为NOTA)为双功能螯合剂,采用戊二醛法将重金属铜离子分别与载体蛋白BSA、OVA偶联制备免疫原和包被原,并用合成的免疫原免疫制备免多克隆抗体,建立间接竞争ELISA(ic-ELISA)。结果表明,所建立的ic-ELISA线性回归方程为y=7.8632Ln(x)+9.6765(R2=0.9934),IC50和IC20值分别为168.70、3.72 ng·m L-1。交叉反应试验结果显示,除与锌交叉反应率达16%外,该抗体与其他重金属铁、铅和镉的交叉反应率均小于5.6%。本研究成功合成了重金属铜人工抗原并制备了兔多克隆抗体,为杂环类双功能螯合剂应用于重金属快速检测技术奠定了理论基础。
In order to study the effect of heterocyclic bifunctional chelators on the preparation of heavy metal copper artifical antigen. Using 2-S-(4-aminobenzene)-1,4,7 nitrogen heterocyclic nonane-1,4,7-triacetic acid(P-NH_2-Bn-NOTA or NOTA) as a bifunctional chelator,the heavy metal copper ions were conjugated with vector proteins BSA and OVA respectively to prepare immunogen and pactin,and the rabbit polyclonal antibodies were prepared by synthesis of immunogenic preparation,establishing indirect competition ELISA(ic-ELISA). The results showed that the established linear regression equation of ic-ELISA was y = 7. 8632 Ln(x) + 9. 6765(R2= 0. 9934),and the values of IC50 and IC20 were 168. 70 and 3. 72 ng·ml-1,respectively. The cross-reaction test results showed that except the cross-reaction rate with zinc reached 16%,the cross-reaction rate between the antibody and other heavy metals iron,lead and cadmium was less than 5. 6%. In this study,heavy metal copper antigen was synthesized successfully and rabbit polyclonal antibody was prepared,which laid a thororetical foundation for the application to the rapid detection of heavy metal by heterocyclic bifunctional chelators.
In order to study the effect of heterocyclic bifunctional chelators on the preparation of heavy metal copper artifical antigen. Using 2-S-(4-aminobenzene)-1,4,7 nitrogen heterocyclic nonane-1,4,7-triacetic acid(P-NH_2-Bn-NOTA or NOTA) as a bifunctional chelator,the heavy metal copper ions were conjugated with vector proteins BSA and OVA respectively to prepare immunogen and pactin,and the rabbit polyclonal antibodies were prepared by synthesis of immunogenic preparation,establishing indirect competition ELISA(ic-ELISA). The results showed that the established linear regression equation of ic-ELISA was y = 7. 8632 Ln(x) + 9. 6765(R2= 0. 9934),and the values of IC50 and IC20 were 168. 70 and 3. 72 ng·ml-1,respectively. The cross-reaction test results showed that except the cross-reaction rate with zinc reached 16%,the cross-reaction rate between the antibody and other heavy metals iron,lead and cadmium was less than 5. 6%. In this study,heavy metal copper antigen was synthesized successfully and rabbit polyclonal antibody was prepared,which laid a thororetical foundation for the application to the rapid detection of heavy metal by heterocyclic bifunctional chelators.
引文
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[2]蔡艳,周亦君,吴晓艺,吴哲铭,梁兴维,郑安然,钟莺莺,俞雪钧,杨震峰.3种海洋贝类重金属污染及食用风险评价研究[J].核农学报,2016,30(6):1126-1134
[3]朱国念,周新文,孙锦荷.混合重金属离子诱导的鲫鱼DNA损伤与基因表达[J].核农学报,2003,17(3):199-202
[4]张波,孟紫强.镍和汞对人血淋巴细胞细胞转化及DNA合成抑制的研究[J].中国环境科学,1996,16(6):452-455
[5]周新文,孙锦荷.应用3H-TdR研究混合重金属对鲫鱼DNA合成的影响[J].核农学报,2001,15(2):115-120
[6]余自力,程光磊.金属离子分析技术[M].北京:化学工业出版社,2004
[7]冯雷,张承聪,扬艳,杨金辉.流动注射-冷蒸汽发生AAS法测定烟草样品中的痕量汞[J].云南大学学报(自然科学版),2002,24(6):454-456
[8]Tokalioglu S,Livkebabci A.A new solid-phase extraction method was developed for the determination of Cu(Ⅱ)and Fe(Ⅲ)in various samples by flame atomic absorption spectrometry using Nbenzoyl-N-phenylhydroxylamine[J].Microchimica Acta,2009,164(3/4):471-477
[9]Baezzat M R,Maleki H,Moghadamhosseyni E,Tabandeh M.Determination of trace amounts of zinc,iron and copper by flame atomic absorption after their preconcentration using sodium dodecyl sulfate(SDS)coated alumina nanoparticles modified with 3-Mercapto-D-Valin from environmental samples[J].Amercan Journal of Analytical Chemistry,2014,5(17):1228-1238
[10]Shahab S,Yadollah Y,Mohammad F,Abolfazl S.On-line solid phase extraction coupled to ICP-OES for simultaneous preconcentration and determination of some transition elements[J].Microchimica Acta,2009,165(1/2):65-72
[11]Ioannidou M D,Zachariadis G A,Anthemidis A N,Stratis J A.Direct determination of toxic trace metals in honey and sugars using inductive Iy coupled plasma atomic emission spectmmetry[J].Talanta,2005,65(1):92-97
[12]王玉萍,庞艳华,李军,董振林,周历.微波消解-电感耦合等离子体质谱法测定调味品中砷的含量[J].食品科学,2008,29(1):231-234
[13]毛红,张妮娜.应用电感耦合等离子体质谱法测定紫菜、海带中铅、镉含量[J].中国卫生检验杂志,2008,18(11):2290-2293
[14]Faraji M,Yamini Y,Saleh Y,Rezaee M.A nanoparticle based solid-phase extraction procedure followed by flow injection inductively coupled plasma-optical emission spectrometry to determine some heavy metal ions in water samples[J].Analytica Chimica Acta,2010,659(1):172-177
[15]中华人民共和国环境保护部.水和废水监测分析方法[M].第4版.北京:中国环境科学出版社,2002:334-338
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[17]吴献花,林洪,李海涛,胡秋芬,杨光宇.用快速分离柱高效液相色谱法测定食品中的重金属元素的研究[J].食品科学,2005,26(6):218-220
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[19]刘丹.抗重金属锌单克隆抗体的制备[D].兰州:甘肃农业大学,2009
[20]李鹏,刘凤权,杨慧,赵丽,李霞.锌螯合物抗体可变区的克隆鉴定、真核表达及重组抗体的三维建模[J].生物技术通报,2012(1):108-115
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[23]孙勇,付世杰,秦保亮,王亚楠,姜金庆,王自良.抗重金属铜离子单克隆抗体的制备及其免疫学特性[J].西北农业学报,2016,25(6):836-840
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[25]王建华,唐勇,向军险,王宏,杨宏宇,凌钦婕.隔离子多克隆抗体ELISA竞争法的初步建立[J].中国生物制品学杂志,2008,21(1):56-59
[26]孙勇,付世杰,秦保亮,王亚楠,王自良.铜离子螯合物人工抗原的合成与鉴定[J].现代畜牧兽医,2012(12):12-16
[27]韩盈盈,李小兵,刘国文,孔涛,郎广平.重金属镉离子人工抗原的合成与鉴定[J].吉林农业大学学报,2013,35(2):188-191
[28]蒋芸,严文静,刘丽强,匡华,陈伟.重金属汞螯合物人工抗原的合成与鉴定[J].食品科学,2011,32(7):244-246
[29]刘功良,王菊芳,李志勇,梁世中.铅螯合物人工抗原的制备与鉴定[J].生物技术通报,2009(11):158-162
[30]黄雅丽.氯霉素酶联免疫分析方法及试剂盒研究[D].杭州:浙江大学,2005