A bifunctional protein RANbody based on nanobody facilitates dual-mode immunoassay of Staphylococcal enterotoxin B in food samples

Abstract

Staphylococcal enterotoxins B (SEB), the major virulence factor of Staphylococcus aureus (S. aureus), brings about grievous food poisoning by infiltrating the food supply chain at diverse phases. ELISA based on traditional antibody and single signal analysis is slightly less specific and stable in SEB detection. Here, we innovatively reported an immunoassay based on a bifunctional protein reconstructed by nanobody, greatly enhancing the specificity of ELISA. Meanwhile, this is complemented by the second signal response, quenching effect of oxidative chromogenic substances on fluorochrome, to heighten the accuracy with dual mode. Under optimal conditions, the dual-mode immunoassay showed a wide linear range from 0.31 to 2500ng/mL and a lower limit of detection (LOD) with 0.12ng/mL for colorimetric mode and 0.24ng/mL for fluorescence mode. There was no nonspecific binding occurred under the interference of S. aureus and other enterotoxins, demonstrating the splendid specificity. Moreover, the proposed assay was successfully employed in detecting SEB in food samples with satisfying practicability. This work integrated the nanobody modification technology with a dual signal sensing strategy, providing a reference for the development of a more specific and feasible platform for food safety.