Immunoaffinity screening with capillary electrochromatography

Highly efficient capillary electrochromatog. sepns. of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatog. (CEC) with octadecyl silica particles. On-column focusing with a preconcn. factor of 180 was performed and sepn. efficiencies of up to 240 000 plates per m were obtained. Using label-free std. UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixts. of steroids, CEC was combined with immunoaffinity extn. using immobilized polyclonal anti-digoxigenin antibodies and Fab fragments. Simply adding small amts. of antibody carrying particles to the samples and comparing chromatograms before and after antibody addn. allowed screening for high affinity antigens in mixts. with moderate nos. of compds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC sepn. and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concns. in an untreated urine sample. [on SciFinder (R)]

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Electrophoresis, 23, 9, 1255-1262
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 Record created 2006-02-27, last modified 2018-03-17

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