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.