Angalakurthi, Siva KrishnaVance, David J.Rong, YinghuiNguyen, Chi My ThiRudolph, Michael J.Volkin, DavidMiddaugh, C. RussellWeis, David D.Mantis, Nicholas J.2021-10-052021-10-052018-12-17Angalakurthi, S. K., Vance, D. J., Rong, Y., Nguyen, C., Rudolph, M. J., Volkin, D., … Mantis, N. J. (2018). A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site. Antibodies (Basel, Switzerland), 7(4), 45. doi:10.3390/antib7040045https://hdl.handle.net/1808/31922This work is licensed under a Creative Commons Attribution 4.0 International License.In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim.© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).http://creativecommons.org/licenses/by/4.0/ToxinAntibodyCamelidVaccineBiodefenseHydrogen exchange-mass spectrometryArticle10.3390/antib7040045https://orcid.org/0000-0002-9913-7722https://orcid.org/0000-0003-3032-1211https://orcid.org/0000-0002-5083-8640PMC6374049openAccess