Related Experiment Video
Updated: May 17, 2025
07:30
Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion
Published on: May 10, 2018
9.1K
Modulating the Kinetics of a Fluorescence Anisotropy Immunoassay Using Tracer Point Mutations to Measure Human
Yufeng Wang1,2, Nitya Gulati1,2, Romario Regeenes1,2
1Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto M5G 1L7, Canada.
ACS Omega
|March 31, 2025
Summary
Researchers optimized fluorescence anisotropy immunoassays (FAIAs) for measuring human C-peptide in microfluidic devices. They enhanced assay speed and dynamic range, enabling precise C-peptide detection from stem cell-derived islets.
Area of Science:
- Biochemistry
- Assay Development
- Microfluidics
Background:
- Fluorescence anisotropy immunoassays (FAIAs) quantify protein concentrations via competitive binding with antibodies and tracers.
- A microfluidic device (InsC-chip) was previously developed for mouse C-peptide measurement.
- Adapting FAIA for human C-peptide presented challenges with dynamic range and assay kinetics.
Purpose of the Study:
- To develop and optimize a FAIA for quantifying human C-peptide secretion in real-time using a microfluidic device.
- To overcome limitations of low dynamic range and slow kinetics in antibody selection for human C-peptide detection.
- To validate the optimized FAIA for measuring C-peptide secretion from human stem cell-derived islets.
Main Methods:
- Reengineered a tracer molecule by comparing human and mouse C-peptide sequences to improve assay kinetics for antibody 1 (Ab1).
- Employed partial epitope mapping and targeted point mutations to enhance the dynamic range for antibody 2 (Ab2).
- Integrated optimized assays into a continuous-flow microfluidic device (InsC-chip) for real-time measurements.
Main Results:
- Achieved a >30-fold reduction in the time-to-reach equilibrium for Ab1 by reengineering the tracer.
- Increased the dynamic range for Ab2 by 45% through epitope mapping and targeted mutations.
- Successfully validated both optimized FAIAs by measuring C-peptide secretion from individual human stem cell-derived islets on the InsC-chip.
Conclusions:
- Demonstrated a successful strategy for optimizing FAIA kinetics for microfluidic applications.
- The developed method allows for enhanced real-time measurement of human C-peptide secretion.
- This approach is crucial for advancing diagnostics and research in diabetes and related conditions.
