A RAPID ON-FILTER IMMUNOASSAY SCREEN FOR DEXAMETHASONE IN EQUINE URINE

A simple on-tilter immunoassay screen has been developed for the detec tion and semi-quantification of dexamethasone in equine urine samples. The assay consists of an indirect competitive ELISA in which dexameth asone in standards or samples competes with an immobilised dexamethaso ne-protein conjugate for binding to a complex of sheep anti-dexamethas one antibodies complexed with alkaline phosphatase-labelled second ant ibody. The drug-protein conjugate is immobilised as a spot on the surf ace of a cellulose nitrate filter and the assay is performed with the filter retained within a filtration port. The assay involves two incub ation 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 d rug-protein conjugate. Rapid washing is achieved through use of the fi ltration system. The assay has a limit of detection of 10(-6) M (390 n g ml(-1)) for a visual end point in which the colour intensity of spot s developed in the presence of samples is compared with those of stand ards. 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 inje ction of 20 mg of drug has been detected for 23 h post administration.