Nanobodies Based on a Sandwich Immunoassay for the Detection of Staphylococcal Enterotoxin B Free from Interference...

As one of the leading causes of food poisoning, staphylococcal enterotoxins (SEs) secreted by Staphylococcus aureus pose a serious threat to human health. The immunoassay has become the dominant tool used for the rapid detection of harmful bacteria and toxins as a result of its excellent specificity. However, with regard to SEs, staphylococcal protein A (SpA) is likely to bind with the fragment crystallizable (Fc) terminal of the traditional antibody and result in a false positive, limiting the practical application of this method. Therefore, to eliminate the bottleneck problem, the sandwich immunoassay was development by replacing the traditional antibody with a nanobody (Nb) that lacked a Fc terminal. Using 0.5 × 10⁷ colony-forming units, the Nb library was constructed using Bactrian camels immunized with staphylococcal enterotoxin B (SEB) to obtain a paired Nb against SEB with good affinity. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using one Nb as the capture antibody and a phage-displayed Nb with signal-amplifying properties as the detection antibody. In optimal conditions, the current immunoassay displayed a broad quantitative range from 1 to 512 ng/mL and a 0.3 ng/mL limit of detection. The recovery of spiked milk, milk powder, cheese, and beef ranged from 87.66 to 114.2%. The Nbs-ELISA was not influenced by SpA during the detection of SEB in S. aureus food poisoning. Therefore, the Nb developed here presented the perfect candidates for immunoassay application during SE determination as a result of the complete absence of SpA interference.