Paper-based medical diagnostic tests have appealingly low cost of goods and can be very simple to operate. However, their sensitivity is often found lacking and new tests typically take months to a year or more to develop, limiting adoption and driving up costs. One of the slow and expensive steps in developing diagnostic immunoassays is identification of pairs, or sets in the case of multiplexed assays, of affinity reagents that simultaneously bind non-overlapping target epitopes and also do not cross-react with one another or complex matrix components.
Engineered binding molecules derived from a thermophilic organism will be presented as alternatives to antibodies, human or camelid, along with a method for selecting pairs or sets of these reagents for diagnostic immunoassays. Analysis of reaction rates and fluid flow within paper-based tests suggested further protein engineering strategies to improve sensitivity. Generalized assay design principles for integrating these engineered proteins into antigen and serology tests will be discussed, along with applications to covid-19.