(109c) Engineering Thermostable Binding Protein rcSso7d Against Zika Virus for Paper-Based Diagnostic Tests

Zika virus is a flavivirus transmitted by mosquitoes and has gained widespread attention in recent years. Although the virus typically only causes mild symptoms in adults, Zika virus has recently been linked to severe birth defects such as microcephaly in infants and other neurological disorders such as Guillain-Barré Syndrome in adults. Therefore, rapid and accurate diagnostics are important to track, control, and prevent spread of Zika virus. As symptoms and diagnostic biomarkers for Zika virus can be similar to other flaviviruses like Dengue virus, it is important that the diagnostic test is specific to Zika virus to minimize cross-reactivity and inaccurate results.

Antibody pairs are widely used as affinity agents in rapid diagnostic tests (RDTs). These tests are frequently designed as a sandwich assay with a pair of antibodies that bind to separate epitopes of the target antigen. One binding protein is immobilized to capture the target disease biomarker, while another binding protein is labeled to produce a signal for detection. Unfortunately, antibodies have some limitations for in vitro applications, including difficulty in developing antibodies with adequate stability and minimal cross-reactivity. Therefore, alternate binding proteins have been investigated to replace antibodies in RDTs.

We used the reduced-charged Sso7d protein (rcSso7d) as our protein scaffold due to its intrinsic stability, high yield expression in bacteria, and ease of genetic modification. We previously incorporated rcSso7d binders in paper assays as either the capture binding protein or the reporter binding protein; however, we had yet to demonstrate the use of rcSso7d as both the capture and reporter reagents simultaneously. In this study, we developed a pair of dual-epitope binding rcSso7d binders against a Zika virus antigen, non-structural 1 (ZNS1) protein. We used directed evolution techniques via yeast surface display to engineer clones with binding affinity to ZNS1. Using negative selections, we ensured that the binders would not cross-react with another similar flavivirus NS1 protein, Dengue virus NS1. We incorporated these binders into soluble protein formats as capture and reporter reagents. When the performance of the rcSso7d sandwich was compared to an antibody sandwich in a paper-based format, we found that the rcSso7d sandwich had a lower limit of detection than the antibody sandwich. By demonstrating the use of rcSso7d as both capture and reporter reagents in a diagnostic test format, we validate that rcSso7d is a strong potential antibody alternative in rapid diagnostic tests.