A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection.

Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain the manufacturing of traditional diagnostic platforms in low-resource settings. To address these challenges, we describe the Rapid and Modular Nanobody Assay (RAMONA), a versatile antigen detection platform that leverages nanobody-coiled coil fusion proteins for modular integration with downstream readout methods. RAMONA merges the portability of LFAs with the benefits of nanobodies such as their smaller size, improved solubility, and compatibility with cell-free protein synthesis systems, enabling on-demand biomanufacturing and rapid adaptation for diverse targets. We demonstrate assay generalizability through the detection of three distinct protein targets, robustness across various temperatures and incubation periods, and compatibility with saliva samples and cell-free synthesis. Detection occurs in under 30 minutes, with results strongly and positively correlating to ELISA data while requiring minimal resources. Moreover, RAMONA supports multiplexed detection of three antigens simultaneously by using orthogonal capture probes. By overcoming several limitations of traditional immunoassays, RAMONA represents a significant advancement in rapid, adaptable, and field-deployable antigen detection technologies.