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

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. To enhance the signal-to-background ratio of the immunoassay, a GUS switch mutant that exhibits reduced background activation was developed by screening several additional mutations at the diagonal interface residue H514. Moreover, we optimized the composition of the reaction buffer, including organic solvents, salt, and surfactant. Under optimal conditions, we customized OpenGUS immunoassays for Cry j 1, human C-reactive protein, and human lactoferrin, achieving around 10-20-fold maximum fluorescence (15 min) or colorimetric (2 h) responses with picomolar to low nanomolar level detection limit, simply by using commercially available IgGs. Additionally, the three analytes were successfully detected in complex matrices similar to those used in practical applications. We believe that this customizable OpenGUS immunoassay will pave the way for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and onsite environmental assessments.