Preparation of a monoclonal antibody against testosterone and its use in development of an immunochromatographic assay

ABSTRACT

A monoclonal antibody against testosterone was produced and used to construct an indirect enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay. As testosterone could not be linked to the protein directly, testosterone and methyltestosterone were first derived by using carboxymethoxylamine hemihydrochloride (CMO) to introduce a carboxyl group at the carbonyl group position. Then the resulting testosterone-3-CMO was coupled to the carrier protein to form the immunogen, using the 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) method. A cell line obtained by cell fusion secreted an antibody which showed high affinity with testosterone. Based on methyltestosterone-3-CMO-ovalbumin (OVA) as the competitive antigen, the ELISA we developed showed high sensitivity to testosterone, with IC₅₀ of 0.11 ng/mL. The results of cross-reactivity testing showed that the antibody was specific to testosterone. Based on this antibody, an immunochromatographic assay was developed and used to detect testosterone in milk samples. This method could be used as a fast and cost-effective alternative tool for screening for endocrine-disrupting compounds.

KEYWORDS:

• Monoclonal antibody
• testosterone
• immunochromatographic assay

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Zhongxing Wang obtained his bachelor's degree from Zhaozhuang University, Zhaozhuang, China in 2014 and his master's degree in food science from Jiangnan University (Wuxi, China). His research interest includes immunoassay development for food safety.

Shuzhen Zou obtained her bachelor's degree from Shandong Agricultural University, Qingdao, China in 2014 and her master’s degree in food science from Jiangnan University (Wuxi, China). Her research interest includes immunoassay development for water safety.

Changrui Xing obtained her bachelor’s degree from Northeast Agricultural University Harbin, China in 2010 and his Ph.D degree in food science from Jiangnan University (Wuxi, China). Her research interest includes immunoassay development for food safety.

Shanshan Song received her Master’s degree in Food science in 2012 from Jiangnan University, Wuxi, China and then became a research assistant in college of Food science and technology of Jiangnan University. Her research interests are monoclonal antibody development.

Liqiang Liu earned his Ph.D. in Food science in 2014 from Jiangnan University, Wuxi, China and then became a faculty in college of Food science and technology of Jiangnan University. His research interests include immunochromatographic strip design and application.

Chuanlai Xu is a full professor of Food science and technology of Jiangnan University. He earned his Ph.D. in food science in 2002. His research interests are fast detection technology and food safety evaluation.

Additional information

Funding

This work is financially supported by the Key Programs from MOST [2012BAK08B01], and grants from Natural Science Foundation of Jiangsu Province, MOF and MOE [201310135].