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^–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.

References

1. P. H. Gaignage, G. Logany, M. Marlier, M. Severin and P. H. Dreze, Chromatographia, 1989, 28, 623.
2. M. I. R. M. Santoro, E. B. Giovato and E. R. M. Hackmann, Anal. Lett., 1993, 26, 925.
3. S. W. I. Schalm, W. H. J. Summers Skill and V. L. W. Gio, Mayo Clin. Proc., 1979, 51, 761.
4. M. L. Rodriguez, I. McConnell, J. Lamont, J. Cambell and S. P. Fitzgerald, Analyst, 1994, 119, 2631.
5. C. J. Roberts and L. S. Jackson, J. Immunol. Methods, 1995, 181, 157.
6. S. S. Hassan, F. J. Rowell and L. S. Jackson, Anal. Commun., 1996, 33, 299.
7. S. S. Hassan, F. J. Rowell and L. S. Jackson, Pakistan J. Med. Res., 1997, 36, 75.
8. D. Courtheyn, J. Vercammen, H. De-Brabander, I. Vandenreyt, P. Batjoens, K. Vanoosthuyze and C. Van Peteghem, Analyst, 1994, 119, 2557.
9. C. S. Creaser, S. J. Feely, E. Houghton, M. Seymour and P. Teale, Anal. Commun., 1996, 33, 5.
10. M. G. Pappas, Vet. Parasitol., 1988, 29, 105.
11. M. G. Pappas, R. Hajkowski and W. T. Hockmeyer, J. Immunol. Methods, 1983, 64, 205.
12. S. M. Burt, T. J. N. Carter and L. J. Kricka, J. Immunol. Methods, 1979, 31, 231.
13. J. Ara, Z. Gans, R. Sweeney and B. Wolf, J. Clinical Lab. Analysis, 1995, 9, 320.
14. P. Sithigorngul, A. O. W. Streton and C. Cowden, J. Immunol. Methods, 1991, 141, 23.
15. F. J. Rowell, Z. F. Miao, R. N. Reeve and R. H. Cumming, Analyst, 1997, 122, 1505.
16. R. H. Cumming, C. Farrell, I. Nitescu, F. J. Rowell and L. X. Tang, Anal. Chim. Acta, 1995, 311, 377.