Poster Presentations
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ELISA in solution: enzyme-inhibitor fusion proteins as switchable reporter enzymes for homogenous antibody detection
Sambashiva Banala, Postdoc, Howard Hughes Medical Institute
Detection of antibodies is essential for the diagnosis of many diseases including infectious diseases, autoimmune diseases and allergies. Current heterogeneous assays such as ELISA (enzyme-linked immune sorbent assay) require immobilization of antigens and involve multiple time-consuming binding and washing steps, which limits their application in low cost point-of-care diagnostics and high-throughput screening. Our goal is to develop a new approach that allows one-step detection of antibodies directly in solution. Towards this goal, we have developed switchable reporter enzyme (also called as sensor) strategy. In this sensor design, the enzyme TEM1-ß-lactamase is fused to its natural inhibitor protein (BLIP) via a long, semi-flexible peptide linker. Bivalent binding of antibody to two epitope sequences introduced at the ends of the linker disrupts the enzyme-inhibitor complex, resulting in an increase in enzyme activity that can be monitored using simple colorimetric or fluorescent read outs. Using this strategy three reporter enzymes were obtained by simply replacing the epitope sequences for detection of anti-HIV1-p17, anti-Dengue-1 and HA-tag antibodies. Our approach provides a generic, modular framework for the rational design of antibody sensors for homogenous immunoassays.
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