A fluorescence polarization immunoassay for cadmium(II)

A target for antibody production was prepared by a 1 : 1 reaction of the bi cyclic dianhydride of diethylenetriamine-N,N, N',N ",N "-pentaacetic acid ( DTPA) with (Z)-2-amino-alpha-(1-tert-butoxycarbonyl)-4-thiazoleacetic acid to give a reactive intermediate that was used to derivatize (a) bovine seru m albumin (BSA), (b) fluoresceinamine and (c) n-butylamine. The BSA conjuga te was complexed with cadmium(II) and used as an immunogen to produce polyc lonal antibodies in rabbits. The fluorescein conjugate was complexed with c admium(II) to provide a fluorescent analog of the immunizing chelate struct ure, while the butylamine derivative was used to convert ionic cadmium(II) into a form recognized by the antibody. The immunogen proved to be sufficiently stable in vivo produce antibodies t hat selectively bound to the cadmium(IT) chelate and the chelate structure in the fluorescein and butylamine derivatives successfully mirrored that ag ainst which the antibody had been raised, enabling the displacement of trac er from the antibody binding site to be monitored by fluorescence polarizat ion. Competitive binding fluorescence polarization immunoassay (FPIA) stand ard curves were constructed for the cadmium chelate, both alone and in the presence of a fixed excess of either the metal-free butylamine derivative o r of its chelates with copper(II), zinc(II) or mercury(rt). For the pure ca dmium chelate, the dynamic range of the assay was 0-100 nM and the limit of detection was below 1.0 nM. Cross-reactivity was sufficiently low so that standard curves for 0-100 nM cadmium chelate could be constructed in the pr esence of 250 nM concentrations of either the free chelating agent or the n on-target chelate. (C) 1999 Elsevier Science B.V. All rights reserved.