Increased sensitivity of heterogeneous fluoroimmunoassays employing fluorescein-labeled antibodies by simple treatment of the wells with glycerin solution

Abstract

Here we present a simple and rapid procedure that enhances and stabilizes the fluorescence signal determined directly onto the solid phase and increases the sensitivity of heterogeneous fluoroimmunoassays which employ fluorescein as label. The evaluation of the proposed procedure was performed through a heterogeneous immunofluorimetric assay for mouse γ-globulins in white-opaque microtitration wells. The proposed fluorescence enhancement and stabilization method consists of a 3-min treatment of the wells with a glycerin solution, after completion of the assay, followed by a 15-min incubation of the emptied wells at 37 °C. Following this protocol, an approximately 50% increase of the fluorescence signal was achieved compared with the signal determined without treatment of the wells (control assay). In addition, the proposed protocol resulted in considerable stabilization of the fluorescence signal. The assay sensitivity achieved following this protocol was improved by twofold compared to the control assay, whereas the precision and the dynamic range of the assay were unaffected.