Immunoaffinity extraction of testosterone by antibody immobilized monolithic capillary with on-line laser-induced fluorescence detection

An immunoaffinity capillary column has been made with poly (2-vinyl-4, 4-dimethylazlactone-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (VDA-co-HEMA-co-EDMA) monolith as a support for the immobilized antibody. The monolith is prepared by UV-initiated in situ polymerization, followed by immobilizing anti-testosterone polyclonal antibody through the rapid reaction with VDA. Fluorescence labeled testosterone at C₃ is designed as a tracer to estimate the extraction ability of this immunoaffinity column, and to optimize the immunoextraction process, such as washing, eluting, incubation and injection. The performance in a more realistic application is then demonstrated successfully for the rapid extraction of testosterone (T) by competitive immunoassay and on-line laser-induced fluorescence (LIF) detection. This immunoaffinity monolith is easily prepared, with high mechanical strength and low flow resistance. It is a satisfactory material as an immunoextractor to detect small molecular compounds specifically and rapidly with high sensitivity (sub ng/mL level).