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 (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.
Zhang
X X
XX
Department of Chemistry, Peking University, Beijing, China. zxx@chem.pku.edu.cn
Li
J
J
Gao
J
J
Sun
L
L
Chang
W B
WB
D000178:Acrylamides; D000941:Antigens; D002021:Buffers; D004300:Doping in Sports; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005456:Fluorescent Dyes; D006358:Hot Temperature; D006801:Humans; D020100:Hydrogels; D007118:Immunoassay; D007834:Lasers; D008777:Methyltestosterone / Q000097:blood*; D009994:Osmolar Concentration
D000178:Acrylamides; D000941:Antigens; D002021:Buffers; D005456:Fluorescent Dyes; D020100:Hydrogels; C067295:N-isopropylacrylamide; D016650:Fluorescein-5-isothiocyanate; D008777:Methyltestosterone