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

Abstract

A simple on-filter 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 26 M (390 ng ml 21 ) 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.

Analysis of anabolic steroids in the horse: Development of a generic ELISA for the screening of 17α-alkyl anabolic steroid metabolites

“…The polyclonal antibodies raised were assessed against a panel of steroids to determine cross-reactivity and showed promise for the detection of a broad range of 16-hydroxylated D-ring steroid metabolites. Antibodies against the parent mestanolone (10) were also raised, and showed promise for the detection of metabolites retaining the non-oxidised parent D-ring structure. The enzyme-linked immunosorbent assay (ELISA) methods developed have application as a primary screening tool for the detection of new and known steroid metabolites.…”

“…In the main, these approaches rely on antibodies produced against antigens derived from the readily available parent drug. As a result, they are only effective in situations where there is minimal biotransformation of the parent substance [9][10][11][12][13][14][15].…”

Preparation of an anti-dexamethasone monoclonal antibody and its use in development of a colloidal gold immunoassay

“…Recently, various analytical methods have been developed for detecting DEX, such as high-performance liquid chromatography (Bhargava et al, 2016;Dési, Kovács, Palotai, & Kende, 2008;Lasić, Bobarević, & Nikolin, 1989;Tsuei, Ashley, Moore, & McBride, 1978), gas chromatography-mass spectrometry (Amendola, Garribba, & Botrè, 2003;Bagnati et al, 1996;Huetos Hidalgo, Jiménez López, Ajenjo Carazo, San Andrés Larrea, & Reuvers, 2003), liquid chromatography-mass spectrometry (LC-MS) (Chen et al, 2011;Creaser, Feely, Houghton, Seymour, & Teale, 1996), radioimmunoassay methods (Meikle, Lagerquist, & Tyler, 1973), and enzyme immunoassay (Hassan, Rowell, Hambleton, & Jackson, 1998;Vdovenko, Gribas, Vylegzhanina, & Sakharov, 2012;Yadav et al, 2013;Yoshino, Yoshiharu, Noriko, Kiyoshi, & Fukuko, 1992). Each method has its own strong and weak points.…”