Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody

Area of Science:

• Biochemistry
• Immunotechnology
• Assay Development

Background:

• Antibody modification is often required for immunoassay development.
• Existing immunoassays can have limitations in sensitivity, speed, and customization.

Purpose of the Study:

• To develop a customizable, label-free immunoassay for rapid and sensitive analyte detection.
• To create an immunoassay platform that does not require antibody modification.

Main Methods:

• Developed a fusion protein (OpenGUS probe) combining a Z domain (ZD) and a β-glucuronidase (GUS) switch mutant.
• Engineered a GUS switch mutant with reduced background activation by mutating residue H514.
• Optimized reaction buffer conditions (organic solvents, salt, surfactant).
• Customized the assay for detecting Cry j 1, human C-reactive protein, and human lactoferrin using commercially available IgGs.

Main Results:

• Achieved 10-20-fold signal enhancement in fluorescence (15 min) or colorimetric (2 h) assays.
• Demonstrated picomolar to low nanomolar detection limits for target analytes.
• Successfully detected analytes in complex biological matrices.

Conclusions:

• The customizable OpenGUS immunoassay offers a versatile platform for rapid, sensitive, and homogeneous detection.
• This technology facilitates prompt development of immunoassays for diverse applications, including diagnostics and environmental monitoring.
• The label-free, antibody-independent approach simplifies assay customization.