Related Experiment Video
Updated: Aug 10, 2026
Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion
Published on: August 15, 2016
Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions
Kimberly A Kelly1, Fred Reynolds, Ralph Weissleder
1Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.
Insights
A new fluorescein isothiocyanate (FITC)-hapten immunoassay quantifies FITC-labeled peptide binding to cells. This sensitive method accurately measures peptide-receptor interactions and allows for receptor visualization using fluorescence techniques.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Peptide-receptor interactions are crucial in cellular signaling.
- Quantifying these interactions often requires sensitive and versatile assays.
- Fluorescence-based methods offer potential for both quantification and visualization.
Purpose of the Study:
- To develop and validate a novel fluorescein isothiocyanate (FITC)-hapten immunoassay for quantifying peptide-receptor binding.
- To assess the assay's sensitivity and versatility for various peptide-receptor systems.
- To explore the utility of FITC-labeled peptides for receptor visualization.
Main Methods:
- Development of a competitive immunoassay using a FITC-labeled peptide and an antifluorescein-horseradish peroxidase conjugate.
- Assay principle relies on measuring solid-phase peroxidase activity inversely proportional to bound FITC-peptide.
- Validation using FITC-labeled bombesin-like peptide interaction with gastrin-releasing peptide receptors on PC-3 and HT-29 cells.
Main Results:
- The FITC-hapten immunoassay demonstrated comparable results to a reference radioreceptor assay for displacement curves and receptor binding site quantification.
- The assay proved sensitive and versatile, applicable to diverse peptide-receptor interactions.
- FITC-labeled peptides were successfully used for receptor visualization via fluorescent microscopy and flow cytometry.
Conclusions:
- The FITC-hapten immunoassay is a sensitive, versatile, and valuable tool for studying peptide-receptor interactions.
- This method provides a cost-effective alternative to radioligands and enables direct visualization of receptors.
- The assay's adaptability makes it suitable for a wide range of biological research applications.
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.
