Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products

Abstract

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.

Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection

“…This result suggests that these beads have a higher capture efficiency. To the best of our knowledge, there is no reported immunosensor using nanobodies against the E. coli strains, but similar results have been obtained with a sandwich immunoassay based on anti-staphylococcal enterotoxin C nanobodies for the detection of S. aureus in milk (LOD = 10 CFU/mL) [26,27]. Furthermore, our current assay showed satisfactory analytical performance compared to other recent conventional immunoplatforms used for the detection of pathogenic E. coli (Table 1).…”

Tunable Preparation of SERS-Active Au–Ag Janus@Au NPs for Label-Free Staphylococcal Enterotoxin C Detection

“…This confirmed that the SERS aptasensor had good accuracy and could be used in practical applications. − In addition, the SERS aptasensor had the advantage of sensitive detection with a lower detection limit than that of the reported SEC detection methods (Table S2). ,,, …”

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects

“…A significant breakthrough in botulism treatment involves coupling Nbs to poisoned botulinum toxin, enabling their delivery to nerve cells. This strategy has led to remarkable recovery in animals from botulism, showcasing the transformative potential of Nbs in disease treatment. , Expanding beyond the aforementioned bacteria, the development of Nbs against toxins produced by Staphylococcus aureus ,− and Listeria monocytogenes , has also shown promising progress. However, as of now, the in vivo neutralization effects of these Nbs remain untested.…”