免疫偶联物设计、合成及间接ELISA方法快速检测纯净水中双酚A的研究

谢桂勉; 杨培新; 郑燕丹; 郑锐东; 郑钢勇; 王珍苑

doi:10.13386/j.issn1002-0306.2013.20.040

免疫偶联物设计、合成及间接ELISA方法快速检测纯净水中双酚A的研究

揭阳职业技术学院

基金项目:

揭阳市科技计划项目(201126)；

详细信息

作者简介:
谢桂勉 (1982-) , 男, 硕士研究生, 讲师, 研究方向:食品质量与安全。;

中图分类号: TS275.1
计量
- 文章访问数: 119
- HTML全文浏览量: 13
- PDF下载量: 109
出版历程
- 收稿日期: 2013-05-05

Design and synthesis of immunoconjugates and development of an indirect ELISA for rapid detection of bisphenol A in purified water

Jieyang Vocational and Technical College

摘要

摘要: 设计并合成了双酚A（BPA）的免疫抗原和包被抗原,制备抗BPA多克隆抗体,并建立了间接ELISA方法用于检测瓶装纯净水中BPA的残留。首先,合成了免疫抗原DPA-BSA,并免疫新西兰大白兔得到抗BPA多克隆抗体。其次,合成结构与DPA-OVA有较大差别的包被抗原HPPA-OVA作为"次级结构包被抗原"以提高ELISA方法的灵敏度。基于"次级结构包被原"的策略,建立了检测BPA的间接ELISA方法。该方法的IC50值为1.88ng/mL,最低检测限为0.08ng/mL。最后,用于瓶装纯净水样品中BPA的检测,其平均回收率为89.6%,平均变异系数为8.8%。因此,该方法可用于水样中BPA的快速检测。
- 双酚A /
- 多克隆抗体 /
- 间接ELISA
Abstract: The immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to detect residues of bisphenol A ( BPA) in bottled purified water. The immunogen DPA-BSA was first synthesized and used to immunized to rabbits to produce a polyclonal antibody against BPA. Furthermore, HPPA-OVA structurally different from DPA-OVA was designed and used as a “substructural coating antigen” to improve the sensitivity of an indirect ELISA analysis for BPA detection. Based on the “substructural coating antigen” concept, an optimized indirect ELISA method was developed that exhibited good specificity and high sensitivity for detecting BPA, with IC50 of 1.88ng/mL and detection limit of 0.08ng/mL. Finally, simple and efficient analysis of BPA samples in bottled purified water showed that the average recovery rate of the ELISA analysis was 89.6%, with the average coefficient of variation 8.8%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of BPA residues in water.
- bisphenol A /
- polyclonal antibody /
- indirect ELISA

HTML全文

参考文献(14)

[1]	Watabe Y, Kondo T, Morita M, et al.Determination of bisphenol A in environmental water at ultra-low level by high-performance liquid chromatography with an effective on-line pretreatment device[J].Journal of Chromatography A, 2004, 1032 (1-2) :45-49.
[2]	Wu S Y, Xu Q, Chen T S, et al.Determination of bisphenol A in plastic bottled drinking water by high performance liquid chromatography with solid-membrane extraction based on electrospun nylon 6 nanofibrous membrane[J].Chinese Journal of Analytical Chemistry, 2010, 38 (4) :503-507.
[3]	Kuo H W, Ding W H.Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry[J].Journal of Chromatography A, 2004, 1027 (1-2) :67-74.
[4]	Olmo M D, Zafra A, Jurado A B, et al.Determination of bisphenol A (BPA) in the presence of phenol by first-derivative fluorescence following micro liquid-liquid extraction (MLLE) [J].Talanta, 2000, 50 (6) :1141-1148.
[5]	Yin H S, Cui L, Ai S Y, et al.Electrochemical determination of bisphenol A at Mg-Al-CO3layered double hydroxide modified glassy carbon electrode[J].Electrochimica Acta, 2010, 55 (3) :603-610.
[6]	Rodriguez-Mozaz S, Alda M L, BarcelóD.Analysis of bisphenol A in natural waters by means of an optical immunosensor[J].Water Research, 2005, 39 (20) :5071-5079.
[7]	石春红, 姚卫蓉, 孙秀兰, 等.兔抗双酚A多克隆抗体的制备[J].安徽农业科学, 2010, 38 (8) :3886-3888.
[8]	Lindholst C, Pedersen K L, Pedersen S N.Estrogenic response of bisphenol A in rainbow trou (tOncorbynchus mykiss) [J].Aquatic Toxicology, 2000, 48 (2-3) :87-94.
[9]	Kim A, Li C R, Jin C F, et al.A sensitive and reliable quantification method for bisphenol A based on modified competitive ELISA method[J].Chemosphere, 2007, 68 (7) :1204-1209.
[10]	Feng Y, Ning B A, Su P, et al.An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates[J].Talanta, 2009, 80 (2) :803-808.
[11]	Sashidhar R B, Capoor A K, Ramana D.Quantitation ofε-amino group using amino acids as reference standards by trinitrobenzenesulfonic acid:A simple spectrophotometric method for the estimation of hapten to carrier protein ratio[J].Journal of Immunological Methods, 1994, 167 (1-2) :121-127.
[12]	Liu Y H, Jin M J, Gui W J, et al.Hapten design and indirect competitive immunoassay for parathion determination:Correlation with molecular modeling and principal component analysis[J].Analytica Chimica Acta, 2007, 591 (2) :173-182.
[13]	Marvin H G, Bruce D H.Hapten design for compoundselective antibodies:ELISAS for environmentally deleterious small molecules[J].Analytica Chimica Acta, 1998, 376 (1) :83-91.
[14]	Zhang Q, Wang L B, Ahn K C, et al.Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion[J].Analytica Chimica Acta, 2007, 596 (2) :303-311.