Microsphere-based flow cytometry protease assays for use in protease activity detection and high-throughput screening

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protease activity is crucial in biological processes and disease.
• Accurate measurement of protease activity is essential for drug discovery and diagnostics.
• Existing methods may lack the throughput or adaptability for diverse protease targets.

Purpose of the Study:

• To describe a novel microsphere-based assay for protease activity.
• To enable high-throughput screening (HTS) of protease activity and inhibitors.
• To provide a versatile platform adaptable to various protease specificities.

Main Methods:

• Utilizing fluorescently labeled protease substrates immobilized on microspheres.
• Measuring loss of fluorescence upon substrate cleavage by target proteases.
• Employing flow cytometry for real-time and endpoint fluorescence measurements.
• Adapting endpoint assays to microplate formats for HTS.

Main Results:

• Demonstrated successful detection of protease activity via fluorescence loss.
• Validated the assay's adaptability for different protease types.
• Showcased real-time and endpoint measurement capabilities.
• Confirmed suitability for high-throughput microplate-based screening.

Conclusions:

• Microsphere-based protease assays offer a robust and adaptable platform.
• The assay facilitates efficient high-throughput screening of proteases and inhibitors.
• This method enhances the study of protease function in various biological contexts.