Abstract
Stanozolol is one of the most widely used synthetic anabolic steroids but also presents a host of negative side effects. It is necessary to develop a rapid and sensitive method for stanozolol screening to monitor its abuse. In this study, the polyclonal antibody against stanozolol was prepared and a novel indirect competitive chemiluminescence enzyme immunoassay (CLEIA) for stanozolol was developed. The stanozolol hapten was conjugated to proteins to prepare immunogen and coating antigens, firstly. To improve the performances of the method, the chemiluminescent detection system was optimized. The optimal concentration of 4-iodophenol, hydrogen peroxide and luminol was studied and the proper detection time was also investigated. Under the optimal conditions, the IC50 value and the limit of detection (IC20) of the chemiluminescence immunoassay is 0.34 and 0.07 ng/mL in phosphate-buffered saline (PBS), respectively. The accuracy performances of the proposed CLEIA were compared with that of ELISA and HPLC. Different nutraceuticals, foodstuffs, and biological matrices spiked with stanozolol were applied to study the application of the proposed assays, and the average recoveries locate at 92–113 % with the coefficients of variation of 8–13.5 %. The polyclonal antibody-based chemiluminescence immunoassay formats are proved to be a feasible and reliable quantificational method for stanozolol.
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Acknowledgements
This work was supported by International Science & Technology Cooperation Program of China (no. 2015DFR40460); the financial support of National Natural Science Foundation (nos. 81173017, 31101277, 81402896); the National High-Tech Research and the Development Program of China (863 Program, no. 2014AA022303); and Tianjin Research Program of Application Foundation and Advanced Technology (13JCZDJC29700). We also appreciate the support from State Key Laboratory of Medicinal Chemical Biology (201503013), Nankai University.
Conflict of Interest
All of the authors (including Jing Wang, Lei Zheng, Yaqing Dong, Zhaorui Song, Yufen Wang, Meng Meng, Linlin Ren, Sergei A. Eremin, Chuan Deng, Yongmei Yin, Rimo Xi) declare that they have no conflict of interest.
Compliance with Ethical Standards
This article does not contain any studies with human subjects. Studies on animals were performed following all institutional and national guidelines for the care and use of laboratory animals, which were also mentioned in the Methods sections.
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Jing Wang and Lei Zheng contributed equally to this work.
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Wang, J., Zheng, L., Dong, Y. et al. Establishment of Enhanced Chemiluminescent Immunoassay Formats for Stanozolol Detection in animal-derived foodstuffs and Other Matrices. Food Anal. Methods 9, 1284–1292 (2016). https://doi.org/10.1007/s12161-015-0307-3
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DOI: https://doi.org/10.1007/s12161-015-0307-3