Sandwich immunoassays are commonly used for in vitro diagnostics of infectious diseases. In this format, one binding protein is immobilized to some surface and captures the target biomarker, while a second binding protein also binds to the biomarker to produce a signal for detection. This format requires the simultaneous binding of affinity reagents to non-overlapping target epitopes. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Furthermore, antibodies are commonly used as the affinity reagents in diagnostic immunoassays; however, due to the limitations of antibodies for in vitro applications—including variable stability and issues with cross-reactivity—alternate binding proteins have been investigated to replace antibodies in in vitro diagnostics. In order to directly engineer pairs of binding proteins against our target biomarkers, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process. This method uses in vitro libraries with directed evolution in order to enable the efficient identification of affinity reagents that function together as complementary pairs. For this study, we used the reduced-charged Sso7d protein (rcSso7d) as an alternative binding protein due to its inherent thermal stability, ease of genetic modification, and low-cost production in bacteria. We used RAPIDS to develop highly-specific affinity pairs against biomarkers of Zika virus, and sepsis. We also incorporated negative selections to ensure specificity and low cross-reactivity of the identified clones. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats including bead-based assays, well-plate ELISAs, and paper-based assays. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or non-targets. With the RAPIDS method, we demonstrate efficiency, versatility, and increased control over the development of functional affinity pairs for use in diagnostic assays.

Engineering complementary pairs of binding proteins via directed in vitro selection

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.