Development of homogeneous immunoassays based on protein fragment complementation

Area of Science:

• Biochemistry
• Assay Development
• Molecular Diagnostics

Background:

• Protein fragment complementation assays (PFCA) offer versatile detection strategies.
• TEM-1 beta-lactamase is a well-characterized enzyme suitable for developing novel PFCA systems.
• Homogeneous assays simplify detection by eliminating separation steps, enabling rapid diagnostics.

Purpose of the Study:

• To establish a functional in vitro proof-of-principle homogeneous assay using TEM-1 beta-lactamase protein fragment complementation.
• To demonstrate the assay's capability in detecting analytes across a wide size range (<1kDa to 150kDa).
• To explore the impact of functional mutations on assay performance, including enzymatic activity, fragment self-association, and inhibitor resistance.

Main Methods:

• Utilized complementary fragments of TEM-1 beta-lactamase that reconstitute upon analyte binding.
• Incorporated a substrate for colorimetric detection of reconstituted enzyme activity.
• Introduced and characterized functional mutations to modulate enzyme kinetics and reduce self-association.

Main Results:

• Achieved colorimetric detection within 10 minutes for a range of analyte sizes.
• Demonstrated that functional mutations can enhance enzymatic activity and inhibitor resistance post-complementation.
• Showcased the critical balance between increased enzyme activity and reduced fragment self-association for optimal signal-to-noise ratios.

Conclusions:

• TEM-1 beta-lactamase protein fragment complementation provides a viable platform for homogeneous immunoassays.
• The assay is adaptable for detecting various analytes, including antibodies, antigens, and biomarkers.
• Optimizing enzyme kinetics and fragment interactions is key to developing diagnostically relevant assays.