A "one to two" novel sandwich immunoassay based on nanobodies for detection of staphylococcal enterotoxin A in food samples

Staphylococcal enterotoxin A (SEA), a virulence factor produced by Staphylococcus aureus (S. aureus), poses a serious threat to human health. The rapid and sensitive detection of SEA is crucial for preventing the spread of contaminated food and safeguarding public health. The conventional enzyme-linked immunosorbent assay (ELISA) has encountered limitations in detecting SEA due to the false-positives caused by the combination of the Fc-terminus of the antibodies and staphylococcal protein A (SpA) and its insufficient sensitivity that fails to meet current detection requirements. Therefore, nanobodies (Nbs) without Fc-termini can replace traditional antibodies to prevent false-positives. We developed a "one to two" sandwich ELISA based on identical capture Nbs and double detection phage-displayed Nbs (OtTNb ELISA), which achieved detection with an LOD of 0.43 ng/mL in a linear range of 0.5–512 ng/mL. The sensitivity was over 3.4 times higher than that of the sandwich ELISA based on mAbs, and the linear range exceeded over 8 times. The OtTNb ELISA was successfully applied to the cross-reactivity test and the actual recovery of the sample. This study provides a sensitive tool for detecting staphylococcal enterotoxins (SEs) in food and offers insights into developing detection methods for various foodborne pathogens and toxins.