A rapid on-filter immunoassay screen for dexamethasone in equine urine

A simple on-tilter immunoassay screen has been developed for the detection and semi-quantification of dexamethasone in equine urine samples. The assay consists of an indirect competitive ELISA in which dexamethasone in standards or samples competes with an immobilised dexamethasone-protein conjugate for binding to a complex of sheep anti-dexamethasone antibodies complexed with alkaline phosphatase-labelled second antibody. The drug-protein conjugate is immobilised as a spot on the surface of a cellulose nitrate filter and the assay is performed with the filter retained within a filtration port. The assay involves two incubation steps, firstly a ten minute competitive step and secondly a five minute step to generate via an enzyme-catalysed reaction an insoluble coloured dye as a spot on the filter at the site of the immobilised drug-protein conjugate. Rapid washing is achieved through use of the filtration system. The assay has a limit of detection of 10(-6) M (390 ng ml(-1)) for a visual end point in which the colour intensity of spots developed in the presence of samples is compared with those of standards. Twelve filters can be processed in a single batch consisting of two standards and ten samples. The assay has been optimised for equine samples and dexamethasone in the urine of a race horse following injection of 20 mg of drug has been detected for 23 h post administration.