Abstract:
Ricin is a highly toxic heterodimeric protein derived from castor beans, posing significant risks as a potential toxin due to its enzymatic subunit that inhibits protein synthesis. Given its rapid onset of symptoms and high mortality rate, there is an urgent need for effective point-of-care tests (POCT) to facilitate quick detection and medical intervention [1]. These POCTs rely on high-affinity antibody capture and detection pairs. Our study integrates complementary approaches—synthetic nanobody (Nb) constructs generated via yeast display of synthetic libraries and monoclonal antibodies (mAbs) isolated from immune libraries with phage display—to incorporate as high-affinity constructs in immunoassay development against RCA. By panning against RCA, we screened a synthetic nanobody (Nb) library with a diversity of 10^8 encoded within yeast display platform against the antigen, identifying clones with binding affinities in the nanomolar range, with the most promising clone exhibiting a KD of ~24 nM. We utilized rational engineering to create multimeric constructs, linking complementarily binding Nbs, as informed by epitope binning and computational modeling, finding that these multimeric constructs capitulated on the avidity effect. We compared these constructs to mAbs and evaluated them in flourescence sandwich immunoassays (“D4 assay”) [2] through bioprinting antibody microarrays on POEGMA-coated glass slides, ultimately optimizing antibody pairs for diagnostic assays. The techniques utilized here demonstrate the methodologies and experimental workflow for developing effective POCTs, being broadly applicable across multiple disease targets.
[1] M. L. Y. Diakite et al., “Point-of-care diagnostics for ricin exposure,” Lab on a Chip, vol. 15, no. 10, pp. 2308–2317, 2015, doi: https://doi.org/10.1039/c5lc00178a
[2] D. Y. Joh et al., “Simple and accurate quantification of molecular biomarkers in blood using a digital microfluidic platform,” Proceedings of the National Academy of Sciences of the United States of America, vol. 114, no. 44, pp. E7054–E7062, 2017.
Completed senior thesis work can be requested through email: amaris.huang@duke.edu
