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

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.