Abstract
We have developed a fluorescein isothiocyanate (FITC)–hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein–horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC–hapten immunoassay and a reference radioreceptor assay. The FITC–hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.
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