Abstract The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to a pandemic threat in the form of vaccines and antigen tests. The causative agent of COVID-19 is SARS-CoV-2. The spike protein on the virus surface interacts with the human angiotensin-converting enzyme (ACE2) via the so-called receptor binding domain (RBD), facilitating virus entry. The RBD thus represents a prime target for vaccines, therapeutic antibodies, and antigen test systems. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive materials and permitting quick detection based on components produced in the microbial model Ustilago maydis . In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. In this study, we produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the viral RBD as Cts1 fusions and screened their RBD binding affinity in vitro and in vivo . Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of an inexpensive antigen test utilizing unconventionally secreted nanobodies as RBD trap and a matching ubiquitous and biogenic surface for immobilization.

Efficient SARS-CoV-2 detection utilizing chitin-immobilized nanobodies synthesized inUstilago maydis