SdAb heterodimer formation using leucine zippers

Ellen R. Goldman; George P. Anderson; P. Audrey Brozozog-Lee; Dan Zabetakis

doi:10.1117/12.2016145

29 May 2013 SdAb heterodimer formation using leucine zippers

Ellen R. Goldman, George P. Anderson, P. Audrey Brozozog-Lee, Dan Zabetakis

Proceedings Volume 8723, Sensing Technologies for Global Health, Military Medicine, and Environmental Monitoring III; 872313 (2013) https://doi.org/10.1117/12.2016145
Event: SPIE Defense, Security, and Sensing, 2013, Baltimore, Maryland, United States

Abstract

Single domain antibodies (sdAb) are variable domains cloned from camel, llama, or shark heavy chain only antibodies, and are among the smallest known naturally derived antigen binding fragments. SdAb derived from immunized llamas are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. We hypothesized that the ability to produce heterodimeric sdAb would enable reagents with the robust characteristics of component sdAb, but with dramatically improved overall affinity through increased avidity. Previously we had constructed multimeric sdAb by genetically linking sdAb that bind non-overlapping epitopes on the toxin, ricin. In this work we explored a more flexible approach; the construction of multivalent binding reagents using multimerization domains. We expressed anti-ricin sdAb that recognize different epitopes on the toxin as fusions with differently charged leucine zippers. When the initially produced homodimers are mixed the leucine zipper domains will pair to produce heterodimers. We used fluorescence resonance energy transfer to confirm heterodimer formation. Surface plasmon resonance, circular dichroism, enzyme linked immunosorbent assays, and fluid array assays were used to characterize the multimer constructs, and evaluate their utility in toxin detection.

Citation Download Citation

Ellen R. Goldman, George P. Anderson, P. Audrey Brozozog-Lee, and Dan Zabetakis "SdAb heterodimer formation using leucine zippers", Proc. SPIE 8723, Sensing Technologies for Global Health, Military Medicine, and Environmental Monitoring III, 872313 (29 May 2013); https://doi.org/10.1117/12.2016145

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