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We are developing immunodiagnostics for toxins and viruses based on single domain antibodies (sdAbs). Camelids and sharks possess IgG subsets that consist of only a heavy chain. The variable region can be expressed recombinantly as an sdAb. The sdAb recognition elements are both smaller and more stable than conventional antibody fragments. We have constructed large libraries of sdAb derived from nonimmunized llamas and sharks and have mined the libraries for toxin and virus binders. Our laboratory has isolated binders to toxins, such as ricin, while the group at The Southwest Foundation for Biomedical Research (SFBR) has panned the libraries for binders to targets, including Marburg and Ebola viruses, in their maximum containment BSL4 facility. We have isolated binders that are specific and thermally stable, able to be heated to 85°C for an hour without losing their binding ability. These binders can be incorporated into any antibody-based biosensor to ruggedize the system. We continue our efforts to isolate binders to an extended range of toxins and viruses. In addition, we are exploring the mechanisms of sdAb antigen interactions to answer fundamental questions about antibody recognition.
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The Luminex platform, a specialized flow cytometer for performing multiplexed sandwich fluoroimmunoassays on the surface of coded microspheres, can be used to screen individual phage. We examined phage from the third round of a selection for ricin binders. This technique allows us to get a sense of the specificity of the binders before moving them to the expression vector and expressing them as soluble protein. We were able to identify phage through the Luminex that did not get picked up on a colorimetric monoclonal phage ELISA. Positive phage were sequenced. Unique binders were expressed as soluble proteins and characterized in terms of their specificity, thermal stability, and the ability to function as capture elements in a sandwich assay. We continue to use this method, in parallel with ELISA, to identify binders and to get a measure of their specificity. We find it a valuable tool, especially for our most recent toxin binder selections.
See “Multiplexed fluid array screening of phage displayed anti-ricin single domain antibodies for rapid assessment of specificity” on page 806.
