Heterogeneous fluoroimmunoassays using fluorescein as label with measurement of the fluorescence signal directly onto...

A heterogeneous fluoroimmunoassay approach with measurement of the fluorescence signal directly onto the solid support is described. Fluorescein was employed as fluorescent label whereas plastic microtitration wells were used as solid carriers. The influence of the type of the plastic wells on the proposed approach was investigated. Different types of commercially available plastic microtitration wells (white-opaque, black, and transparent) were tested. The solid supports were judged on the grounds of the maximum fluorescence signal, the precision, and the sensitivity they provided in a model non-competitive assay for mouse IgG. Among the solid supports tested, the white-opaque wells provided the most promising results with respect to the parameters in question. This support was also used for the development of a competitive fluoroimmunoassay for the determination of total thyroxin in human serum samples in order to assess the validity of the proposed approach under real immunoassay conditions. The assay developed had appropriate analytical characteristics for the determination of thyroxin and the serum sample values obtained were well correlated with those determined by a commercial solid-phase radioimmunoassay.