Enzyme Immunoassay for Clenbuterol, an β2-Adrenergic Stimulant

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Abstract
A sensitive double antibody and heterologous enzyme immune-assay for the quantitation of clenbuterol is established. Specific antiserum to this agent was raised in rabbits by immunization with diazotized clenbuterol and human serum albumin conjugate. For competitive reactions, antibody was incubated with a mixture of diazotized clenbuterol analog (NA 1141) labelled with β-D-galactosidase and unlabelled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 0.5 pg/tube of clenbuterol. By use of this assay method, the time course of plasma levels of clenbuterol was examined after a single oral administration (20 μg) to 3 healthy volunteers. It was shown that the maximum level was achieved after 2–3 hr with approximately 10 ng clenbuterol/d1 of plasma.