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

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 (kd = 109) 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 4oC, 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.