Antibodies conjugated with new highly luminescent Eu and Tb chelates as markers for time resolved immunoassays. Application to simultaneous determination of clenbuterol and free cortisol in horse urine

Highly luminescent Eu³⁺ and Tb³⁺ complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu³⁺ ⊂ 1 and Tb³⁺ ⊂ 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 μg L⁻¹ and 40 μg L⁻¹ sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds.