Open flower fluoroimmunoassay: a general method to make fluorescent protein-based immunosensor probes

Area of Science:

• Biotechnology and Biomedical Engineering
• Fluorescence Spectroscopy
• Immunoassay Development

Background:

• Fluorescence-based probes, particularly Förster resonance energy transfer (FRET) using fluorescent proteins (FPs), are crucial for monitoring biological processes, often detecting ligand-induced conformational changes.
• Antibodies offer specific receptor recognition but their potential in FRET probes is limited, especially for large proteins like serum albumin (SA), as antigen binding may not significantly alter VH-VL interactions.

Purpose of the Study:

• To develop a novel FRET-based probe strategy, termed open flower fluoroimmunoassay (OF-FIA), for detecting large biomolecules like serum albumin.
• To create a probe capable of measuring clinically relevant concentrations of serum albumin for potential diagnostic use.

Main Methods:

• Designed an OF-FIA probe using donor-fused VH and acceptor-fused VL antibody fragments linked by a disulfide bond (CyPet/YPet-dsFv).
• Investigated the probe's FRET efficiency in relation to serum albumin concentration.
• Assessed the probe's ability to detect serum albumin within a clinically relevant range.

Main Results:

• The CyPet/YPet-dsFv probe exhibited high FRET efficiency due to the inherent dimerization of the FP pair.
• Increased serum albumin concentration led to a decrease in FRET efficiency, likely caused by dimer disruption.
• The OF-FIA probe successfully detected serum albumin in a range relevant for clinical diagnostics.

Conclusions:

• The novel OF-FIA strategy provides a sensitive method for detecting large proteins like serum albumin.
• The developed probe demonstrates significant potential as a diagnostic reagent for serum albumin quantification.