A fluorescence polarization immunoassay for cadmium(II) Original Research Article

A target for antibody production was prepared by a 1 : 1 reaction of the bicyclic dianhydride of diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) with (Z)-2-amino-α-(1-tert-butoxycarbonyl)-1-methylethoxyimino)-4-thiazoleacetic acid to give a reactive intermediate that was used to derivatize (a) bovine serum albumin (BSA), (b) fluoresceinamine and (c) n-butylamine. The BSA conjugate was complexed with cadmium(II) and used as an immunogen to produce polyclonal antibodies in rabbits. The fluorescein conjugate was complexed with cadmium(II) to provide a fluorescent analog of the immunizing chelate structure, 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 that selectively bound to the cadmium(II) chelate and the chelate structure in the fluorescein and butylamine derivatives successfully mirrored that against which the antibody had been raised, enabling the displacement of tracer from the antibody binding site to be monitored by fluorescence polarization. Competitive binding fluorescence polarization immunoassay (FPIA) standard curves were constructed for the cadmium chelate, both alone and in the presence of a fixed excess of either the metal-free butylamine derivative or of its chelates with copper(II), zinc(II) or mercury(II). For the pure cadmium 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 presence of 250 nM concentrations of either the free chelating agent or the non-target chelate.