Evaluation of a fluorescein-labeled estradiol derivative for use in affinity capillary electrophoresis

A fluorescein-labeled estradiol derivative was assessed for use in affinity capillary electrophoresis (ACE) in a competitive immunoassay format, in which the fluorescently labeled estradiol competed with unlabeled estradiol for a mouse anti-estradiol antibody. The preparation of the labeled estradiol produced a mixture of fluorescein-containing compounds that led to multiple peaks in the electropherogram and to which the antibody responded differently. Two of the components of the mixture, towards which the mouse antibody showed most affinity, were isolated using fraction collection via capillary electrophoresis (CE). The two fractions of the labeled estradiol products isolated by CE were characterized using mass spectrometric methods. The two active fluorescein-conjugated products differed in the carboxylate on the fluorescein moiety, one having a methyl group instead of the acidic hydrogen for the other. The estradiol antibody showed a stronger binding for the conjugate containing the methyl group, as determined from the estimated binding constants using Scatchard analysis. The isolated fractions of labeled estradiol were shown to be applicable to the ACE immunoassay method.