A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

Area of Science:

• Biochemistry
• Molecular Biology
• Immunotechnology

Background:

• Protein-fragment complementation assays are used to monitor protein interactions.
• Current systems fuse reporter fragments directly to proteins, limiting native interaction studies.
• Existing antibody-based complementation systems lack sensitivity and practicality for immunoassays.

Purpose of the Study:

• To develop a novel, sensitive, and practical complementation system for antibody-based protein detection.
• To overcome limitations of existing methods for monitoring endogenous protein interactions.
• To create a versatile "add-and-read" homogeneous immunoassay platform.

Main Methods:

• Development of a first-in-class, tri-part split luciferase system.
• Utilizing two 11-residue peptides as antibody appendages.
• Fusion of antibody-peptide constructs for protein quantification.

Main Results:

• Demonstrated proof-of-concept using antibody-peptide fusions.
• Quantified picogram/mL concentrations of soluble or cell-bound HER2.
• Validated the system's sensitivity and practicality for homogeneous immunoassays.

Conclusions:

• The novel dual-peptide split luciferase system overcomes previous limitations in antibody-based complementation.
• This system provides a rapid, simple, and sensitive "add-and-read" homogeneous immunoassay platform.
• The technology is broadly adaptable as an alternative to traditional immunoassays and can monitor endogenous protein interactions.