Nanobody-based fluorescent immunoassay for SARS-CoV-2 spike detection via site-specific microbial transglutaminase...

Nanobodies-single-domain antibody fragments derived from camelids-are ideal for diagnostic applications due to their small size, thermal stability, and ease of recombinant expression. However, traditional chemical conjugation methods often result in heterogeneous labeling, which can impair performance and reproducibility. Site-specific conjugation strategies, such as microbial transglutaminase (MTGase)-mediated labeling, offer precise and consistent modification. MTGase catalyzes the formation of stable isopeptide bonds at specific glutamine residues and can be harnessed for nanobody labeling via engineered C-terminal c-Myc-tag containing reactive glutamines. In this study, we established a nanobody-based fluorescent sandwich immunoassay for detecting the SARS-CoV-2 Spike protein. An immune VHH library constructed from an immunized alpaca yielded numerous unique nanobodies, ten of which exhibited high binding affinity. These nanobodies were site-specifically conjugated to biotin or Alexa Fluor 488 using MTGase and employed in a directional capture-detection format. The optimized assay, using NB-5F11 for capture and NB-8B5 for detection, demonstrated a broad dynamic range and high sensitivity. Our work highlights the utility of MTGase-mediated site-specific conjugation as a robust platform for engineering nanobody-based biosensors with superior performance and reproducibility.