Environmental Science and Pollution Research

, Volume 24, Issue 8, pp 7042–7050 | Cite as

Investigation of diethylstilbestrol residue level in human urine samples by a specific monoclonal antibody

  • Dai Zhang
  • Lifang Zhou
  • Yajing Lei
  • Zhan Zhou
  • Jie ZhouEmail author
  • Shuqing ChenEmail author
Research Article


Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people’s health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3.33 ng/mL. The LOD and LOQ were 0.16 and 0.54 ng/mL. Linear range of the standard curve was from LOD to 12.50 ng/mL. There was no cross-reactivity with two kinds of estrogens and two structural analogs with DES. Five hundred seventy-six urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. The mean concentration and geometric mean were 4.70 and 3.50 ng/mL. The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of DES in human urine samples. The results warned us to pay more attention to human health and food safety.


Diethylstilbestrol Monoclonal antibody ELISA Human urine samples Residue level Human health 



This work was supported by the State Key Program of National Natural Science of China (Grant No. 81430081), the grant (No. 2012ZX09506001-004) from the National Major Projects for Science and Technology Development of Ministry Science and Technology of China, and the Fundamental Research Funds for the Central Universities.

Compliance with ethical standards

All experiments on mice were performed according to the ethics committee of the Experimental Animal Center of Zhejiang University.


  1. Almeida C, Nogueira JM (2006) Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection. J Pharm Biomed Anal 41:1303–1311CrossRefGoogle Scholar
  2. Aman CS, Pastor A, Cighetti G, de la Guardia M (2006) Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC-MS/MS. Anal Bioanal Chem 386:1869–1879CrossRefGoogle Scholar
  3. Cooper J, Currie W, Elliott CT (2001) Comparison of the efficiences of enzymatic and chemical hydrolysis of (nortestosterone and diethylstilboestrol) glucuronides in bovine urine. J Chromatogr B Biomed Sci Appl 757:221–227CrossRefGoogle Scholar
  4. Dodds EC, Goldberg L, Lawson W, Robinson R (1938) Oestrogenic activity of certain synthetic compounds. Nature 141:247–248CrossRefGoogle Scholar
  5. Fernandez-Arauzo L, Pimentel-Trapero D, Hernandez-Carrasquilla M (2014) Simultaneous determination of resorcylic acid lactones, beta and alpha trenbolone and stilbenes in bovine urine by UHPLC/MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 973C:89–96CrossRefGoogle Scholar
  6. Ganan J, Morante-Zarcero S, Perez-Quintanilla D, Marina ML, Sierra I (2016) One-pot synthesized functionalized mesoporous silica as a reversed-phase sorbent for solid-phase extraction of endocrine disrupting compounds in milks. J Chromatogr A 1428:228–235CrossRefGoogle Scholar
  7. Gore AC, Chappell VA, Fenton SE, Flaws JA, Nadal A, Prins GS, Toppari J, Zoeller RT (2015) Executive Summary to EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals. Endocr Rev 36:593–602CrossRefGoogle Scholar
  8. Han H, Kim B, Lee SG, Kim J (2013) An optimised method for the accurate determination of zeranol and diethylstilbestrol in animal tissues using isotope dilution-liquid chromatography/mass spectrometry. Food Chem 140:44–51CrossRefGoogle Scholar
  9. Hatch EE, Troisi R, Palmer JR, Wise LA, Titus L, Strohsnitter WC, Ricker W, Hyer M, Hoover RN (2015) Prenatal diethylstilbestrol exposure and risk of obesity in adult women. J Dev Orig Health Dis 6:201–207CrossRefGoogle Scholar
  10. Hatch EE, Herbst AL, Hoover RN, Noller KL, Adam E, Kaufman RH, Palmer JR, Titus-Ernstoff L, Hyer M, Hartge P, Robboy SJ (2001) Incidence of squamous neoplasia of the cervix and vagina in women exposed prenatally to diethylstilbestrol (United States). Cancer Causes Control 12:837–845CrossRefGoogle Scholar
  11. He X, Mei X, Wang J, Lian Z, Tan L, Wu W (2016) Determination of diethylstilbestrol in seawater by molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography. Mar Pollut Bull 102:142–147CrossRefGoogle Scholar
  12. Huo T, Wang L, Liu L, Chu X, Xu C (2006) Rapid time-resolved fluoroimmunoassay for diethylstilbestrol residues in chicken liver. Anal Biochem 357:272–276CrossRefGoogle Scholar
  13. Iparraguirre A, Navarro P, Rodil R, Prieto A, Olivares M, Etxebarria N, Zuloaga O (2014) Matrix effect during the membrane-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry for the determination of a variety of endocrine disrupting compounds in wastewater. J Chromatogr A 1356:163–170CrossRefGoogle Scholar
  14. Jiang X, Chen HQ, Cui ZH, Yin L, Zhang WL, Liu WB, Han F, Ao L, Cao J, Liu JY (2016) Low-dose and combined effects of oral exposure to bisphenol A and diethylstilbestrol on the male reproductive system in adult Sprague-Dawley rats. Environ Toxicol Pharmacol 43:94–102CrossRefGoogle Scholar
  15. Kumirska J, Plenis A, Lukaszewicz P, Caban M, Migowska N, Bialk-Bielinska A, Czerwicka M, Stepnowski P (2013) Chemometric optimization of derivatization reactions prior to gas chromatography-mass spectrometry analysis. J Chromatogr A 1296:164–178CrossRefGoogle Scholar
  16. Laronda MM, Unno K, Butler LM, Kurita T (2012) The development of cervical and vaginal adenosis as a result of diethylstilbestrol exposure in utero. Differentiation 84:252–260CrossRefGoogle Scholar
  17. Leena H-C (2014) Maternal exposure to diethylstilbestrol during pregnancy and increased breast cancer risk in daughters. Breast Cancer Res 16:208–217Google Scholar
  18. Lei Y, Zhang Q, Fang L, Akash MS, Rehman K, Liu Z, Shi W, Chen S (2014) Development and comparison of two competitive ELISAs for estimation of cotinine in human exposed to environmental tobacco smoke. Drug testing and analysis 6:1020–1027CrossRefGoogle Scholar
  19. Lv X, Xiao S, Zhang G, Jiang P, Tang F (2016) Occurrence and removal of phenolic endocrine disrupting chemicals in the water treatment processes. Sci Rep 6:1–10CrossRefGoogle Scholar
  20. Tedeschi CA, Rubin M, Krumholz BA (2005) Six cases of women with diethylstilbestrol in utero demonstrating long-term manifestations and current evaluation guidelines. J Low Genit Tract Dis 9:11–18CrossRefGoogle Scholar
  21. Wang X, Reisberg S, Serradji N, Anquetin G, Pham MC, Wu W, Dong CZ, Piro B (2014) E-assay concept: detection of bisphenol A with a label-free electrochemical competitive immunoassay. Biosens Bioelectron 53:214–219CrossRefGoogle Scholar
  22. Wu X, Wang L, Ma W, Zhu Y, Xu L, Kuang H, Xu C (2012) A simple, sensitive, rapid and specific detection method for bisphenol A based on fluorescence polarization immunoassay. Immunol Investig 41:38–50CrossRefGoogle Scholar
  23. Yang Q, Diamond MP, Al-Hendy A (2016) Early life adverse environmental exposures increase the risk of uterine fibroid development: role of epigenetic regulation. Front Pharmacol 7:40Google Scholar
  24. Yin L, Zheng LJ, Jiang X, Liu WB, Han F, Cao J, Liu JY (2015) Effects of low-dose diethylstilbestrol exposure on DNA methylation in mouse spermatocytes. PLoS One 10:1–18Google Scholar
  25. Zhang S, Lei Y, Xu X, Shi W, Chen S (2013) Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies. Zhejiang Da Xue Xue Bao Yi Xue Ban 42:25–31Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical SciencesZhejiang UniversityHangzhouChina
  2. 2.Hangzhou EPIE Bio-detection Technology LimitedHangzhouChina

Personalised recommendations