Development of thermally stable nanobodies for detection and neutralization of Staphylococcal enterotoxin B

Authors: HUGHES, ANNA - Former ARS Employee; KIRKLAND, MARINA - Former ARS Employee; Du, Wen-Xian; Rasooly, Reuven; Hernlem, Bradley - Brad; Tam, Christina; Zhang, Yuzhu; He, Xiaohua

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/14/2023
Publication Date: 6/16/2023
Citation: Hughes, A.C., Kirkland, M., Du, W.N., Rasooly, R., Hernlem, B.J., Tam, C.C., Zhang, Y., He, X. 2023. Development of thermally stable nanobodies for detection and neutralization of Staphylococcal enterotoxin B. Toxins. 15(6). Article 400. https://doi.org/10.3390/toxins15060400.
DOI: https://doi.org/10.3390/toxins15060400

Interpretive Summary: Due to the classification of staphylococcal enterotoxin B (SEB) as a bioterrorist agent and propensity to cause food poisoning there is a need for continued improvement of diagnostic and surveillance strategies. Conventional monoclonal (mAb) and polyclonal antibodies (pAb) have been shown to be effective at neutralizing the immune response to SEB, however, these antibodies can be laborious to screen or lack of specificity. As an alternative, single domain antibodies or nanobodies (nbs) have beneficial characteristics such as stability in harsh conditions, small size, low production cost and batch-to-batch reproducibility. In this study, we developed 16 unique SEB reactive nbs (10 monovalent and 6 bivalent), and analyzed their physical and biochemical properties, including their binding affinity and specificity to SEB, thermo-stability, shelf life and capacity to neutralize immune response to SEB.

Technical Abstract: In this study, 16 unique staphylococcal enterotoxin B (SEB) reactive nanobodies (nbs), including 10 monovalent and 6 bivalent, were developed. All nbs characterized were highly SEB-specific and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb), the lowest LOD reached 50 pg/mL in PBS. When applied an ELISA to detect SEB spiked in milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the 16 nbs with a subset of nbs, SEB-5, SEB-9, and SEB-62, retaining activity even after exposure to 95°C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat labile properties. Several nbs demonstrated a long shelf life, with one nb (SEB-9) retaining 93% activity after 2 weeks storage at room temperature. In addition to detection, 11 out of 15 nbs were capable of neutralizing SEB’s superantigenic activity demonstrated by their ability in inhibiting IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs identified here are relatively small, thermally stable, and easy to produce, making them useful recognition el-ements in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products.