Selection of specific nanobodies to develop an immuno-assay detecting Staphylococcus aureus in milk

•The first report ever on nanobodies directed against intact S. aureus.•A Nb-mediated immuno-assay avoids false-positive detection of S. aureus.•Development of a sensitive sandwich ELISA against S. aureus.•The potential application to detect intact S. aureus in food matrix. The interaction between conventional immunoglobulins (Igs) and the Ig-binding surface proteins of Staphylococcus aureus (S. aureus) have obstructed the development of immuno-assays to detect these bacteria. The current study aimed to select nanobodies (Nbs) recognizing specifically S. aureus and to establish an immuno-assay to uncover S. aureus contaminations in foods. An alpaca was immunized with an inactivated S. aureus strain followed by the construction of a Nb library from which four target-specific Nbs were retrieved. Subsequently, a sandwich ELISA employing the Nb147 and biotinylated-Nb147 pair to capture and to detect S. aureus, respectively, was established to possess a detection limit of 1.4 × 105 colony forming units (CFU)/mL. The dedicated immuno-assay has been verified by detecting 10 CFU/mL of S. aureus in milk samples after an 8 h-enrichment step. This study provides the basis of an easy, reproducible and effective immuno-assay to screen for S. aureus contaminations in foods.