Development of a Surface Programmable Activation Receptor system (SPAR): A living cell biosensor for rapid pathogen detection

Abstract

8Efficient pathogen detection is essential for the successful treatment and prevention of infectious 9 disease; however, current methods are often too time intensive to be clinically relevant in cases 10 requiring immediate intervention. We have developed a Surface Programmable Activation 11 Receptor (SPAR) diagnostic platform comprised of universal biosensor cells engineered for use 12 in combination with custom or commercial antibodies to achieve rapid and sensitive pathogen 13 detection. SPAR cells are stably transfected Jurkat T cells designed to constitutively express a 14 modified T cell mouse FcγRI receptor on the cell surface and a high level of the luminescent 15 reporter protein aequorin in the cytoplasm. The modified mFcγRI-CD3ζ receptor protein binds 16 with high affinity to the Fc region of any full-length mouse IgG2a and some IgG2 antibodies: 17 this allows customized target detection via the selection of specific antibodies. T-cell receptor 18 aggregation in response to target antigen binding results in signal transduction which, when 19 amplified via the endogenous T cell signal cascade, triggers the rapid intracellular release of 20 calcium. Increased Ca 2+ concentrations activate the expressed reporter protein aequorin resulting 21 in the immediate emission of detectable light. Testing demonstrates the accurate and specific 22 2detection of numerous targets including P. aeruginosa, E. coli O111, and E. coli O157. We 23 report that the SPAR biosensor cell platform is a reliable pathogen detection method that enables 24 the rapid identification of bacterial causative agents using standard laboratory instrumentation. 25The technology lends itself to the development of efficient point-of-care testing and may aid in 26 the implementation of effective and pathogen-specific clinical therapies. 27 Introduction 28The rapid and accurate identification of causative agents is critical to the prompt application of 29 directed, pathogen-specific antibiotic therapies. Effective and timely clinical intervention is 30 essential for the control of infectious disease as well as in the successful treatment of bacterial 31 infections. The observed increases in the frequency and severity of nosocomial infections (1) 32 and the increasing prevalence of antibiotic resistance induced by non-specific antibiotic use (2) 33 further highlight the need for informed antibiotic selection based upon precise and efficient 34 pathogen detection. 35 Current bacterial identification methods include both classic procedures and novel molecular 36 techniques. Traditional culture-based methods, while sensitive and reliable, are also labor-37 intensive and time-consuming and therefore often cannot provide definitive diagnostics within a 38 clinically relevant timeframe (3). Molecular diagnostic methods include immunological assays 39 such as ELISA (4), microarray immunoblot (5), or serological assays (6); nucleic acid-based 40 techniques including PCR (7), DNA sequencing (8), hybridization techniques (9), or DNA/RNA 41 microarrays (10);...