Determination of doping methyltestosterone by capillary electrophoresis immunological analysis with thermally reversible hydrogel and laser-induced fluorescence.

Abstract

In this paper thermally reversible hydrogel used as a replaceable packed material for capillary electrophoresis was examined. A simple and rapid method of detecting doping methyltestosterone (MTS) was developed, which demonstrated the potential of strong affinity antibodies (k(d) = 10(9)) as a selector for immunologically based separations in serum, not in urine, by capillary electrophoresis. Polyclonal antibodies (Ab) were polymerized on the hydrogel and applied in the separation of fluorescein isothiocyanate (FITC)-labeled antigen and free antigen with laser-induced fluorescence detector (LIF). The results indicated that N-isopropylacrylamide (PNIPA) hydrogel is a steady, replaceable gel. The specific determination of MTS did not require reaching equilibrium of immunological reaction and the PNIPA hydrogel including antibodies can be stockpiled at 4 degrees C, in preparation for determining MTS at any time. It can be used to determine MTS with good precision with a sensitivity lower than 50 ng/mL. The theoretical plate numbers obtained could reach 168,449 for free-antigen. Details of the preparation of hydrogel cross-linked polyclonal antibody and of typical separations of bound and free antigen are presented.