473766 Development of Thermostable Affinity Reagents for Low-Cost, Point-of-Care Diagnostics

Thursday, November 17, 2016: 1:42 PM
Continental 7 (Hilton San Francisco Union Square)
Eric A. Miller1, Hadley D. Sikes1, Michael Traxlmayr1 and Jacqueline Shen2, (1)Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, (2)Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

The timely diagnosis and treatment of disease in resource-constrained settings requires the development of robust point-of-care (POC) diagnostics, which provide accurate results and can be employed by users with minimal medical training and limited access to basic infrastructure. One of the most common POC diagnostic formats is the immunochromatographic rapid diagnostic test (RDT), which traditionally uses nitrocellulose-immobilized IgG antibodies as affinity reagents for the capture of disease-relevant biomarkers from patient samples. However, these antibodies are prone to thermal denaturation and non-specific binding events that can render test results unreliable, and in infrastructure-poor contexts where continuous cold chain storage may be infeasible, this silent test invalidation can occur with regularity. This serves to diminish device performance, resulting in adverse patient outcomes and eroding patient trust in the diagnostic format.

We here report the development of thermostable affinity reagents based on a DNA-binding protein isolated from the hyperthermophilic bacteria S. solfataricus. This scaffold was used to develop a model binding protein, which has been assessed for its surface-bound activity, its utility within the dilute antigen regime, and its activity retention under thermal challenge. The demonstrated kinetic stability of this species is nearly three orders of magnitude greater than that of standard polyclonal antibodies, and the species can be facilely produced in high molar yields via bacterial expression and purified in a single chromatographic step. Using yeast surface display and flow cytometric analysis, rationally-designed mutagenesis libraries have also been screened for potential binders to four TB biomarkers identified in the urine of infected patients via mass spectrometry. These studies have confirmed the scaffold’s potential applicability to a broad suite of disease biomarkers, and have validated the suitability of this scaffold as a replacement for antibodies in in vitro point-of-care diagnostic devices.

Extended Abstract: File Not Uploaded