Related Experiment Video
Updated: Jul 27, 2026
Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System
Published on: May 22, 2020
Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric
J Mellentin-Michelotti1, L T Evangelista, E E Swartzman
1PE Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. micheljm@pebio.com
Abstract:
We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput.
Insights
A novel fluorescence assay quantifies receptor-ligand interactions using Cy5-labeled peptides and a no-wash fluorometric microvolume assay technology (FMAT) scanner. This method accurately determines IC(50) values and enables high-throughput screening of seven-transmembrane receptors.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Seven-transmembrane receptors play crucial roles in cellular signaling and are targets for drug development.
- Accurate quantification of receptor-ligand binding is essential for understanding these interactions and for drug discovery.
- Existing methods for receptor-ligand binding assays can be time-consuming and require multiple steps.
Purpose of the Study:
- To develop and validate a novel fluorescence-based, mix-and-read assay for quantitative determination of receptor-ligand binding interactions.
- To assess the utility of this assay for high-throughput screening (HTS) of peptide ligands for endogenous seven-transmembrane receptors.
- To demonstrate the capability of the assay for multiplexing, allowing simultaneous testing of multiple receptor targets.
Main Methods:
- Development of a fluorescence-based assay using Cy5-labeled peptide ligands (Substance P, neurokinin A, galanin) for endogenous receptors.
- Quantification of cell-associated fluorescence using a fluorometric microvolume assay technology (FMAT) scanner, a no-wash, multiwell plate-compatible system.
- Determination of IC(50) values for peptide ligands and validation against radioligand binding data.
- Demonstration of HTS capability by competing Cy5-labeled Substance P binding with unlabeled Substance P.
- Development of a two-color multiplex assay combining cells expressing different receptors (neuropeptide Y and Substance P receptors).
Main Results:
- The fluorescence-based assay accurately quantified receptor-ligand binding and determined IC(50) values for Substance P, neurokinin A, and galanin, which correlated well with established radioligand binding data.
- The fluorometric microvolume assay technology (FMAT) scanner facilitated high-throughput screening, successfully identifying competed binding in individual wells.
- A two-color multiplex assay was successfully developed, enabling simultaneous testing of two distinct seven-transmembrane receptor targets (neuropeptide Y and Substance P receptors) in a single well.
- The multiplex assay demonstrated specific binding of fluorescent ligands to their respective cell types and specific competition, minimizing reagent consumption and increasing throughput.
Conclusions:
- A robust and efficient fluorescence-based, mix-and-read assay has been developed for quantitative determination of receptor-ligand binding interactions.
- The assay, utilizing fluorometric microvolume assay technology (FMAT), is suitable for high-throughput screening of seven-transmembrane receptors.
- The developed multiplexing capability offers a significant advantage for drug discovery by allowing simultaneous assessment of multiple targets, thereby reducing costs and increasing screening efficiency.
