Green fluorescent protein mutant as label in homogeneous assays for biomolecules

The green fluorescent protein (GFP) and its mutants have been extensively u sed to study various cellular processes and, more recently, as labels in bi nding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstra te the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin an d an antigen-antibody as the binding pairs. Biotin and cortisol were chemic ally conjugated to EGFP, A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentag e fluorescence quenching observed decreased as the concentration of free bi otin in the sample increased due to the fewer binding sites on avidin avail able for binding to the EGFP-biotin conjugate. A dose-response curve for bi otin was generated by relating percentage fluorescence quenched with free b iotin in the sample. To determine whether EGFP can undergo a similar type o f homogeneous change when used as a label for antigen-antibody type of bind ing, cortisol was selected as a model analyte. In the presence of an anti-c ortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has b een employed as a label in homogeneous assays, thus enhancing the versatili ty of employing GFP or its mutants in a number of bioanalytical application s, such as clinical analysis and high-throughput screening systems. (C) 200 1 Academic Press.