Switchable Reporter Enzymes Based on Mutually Exclusive Domain Interactions Allow Antibody Detection Directly in Solution

• Published in: ACS chemical biology Vol. 8; no. 10; pp. 2127 - 2132
• Main Authors: Banala, Sambashiva, Aper, Stijn J.A, Schalk, Werner, Merkx, Maarten
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.10.2013
• Subjects: Antibodies - analysis; beta-Lactamase Inhibitors - chemistry; beta-Lactamase Inhibitors - metabolism; beta-Lactamase Inhibitors - pharmacology; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Colorimetry; Humans; Immunoassay - methods; Limit of Detection; Protein Structure, Tertiary; Solutions
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb400406x

Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an intramolecular interaction between TEM1 β-lactamase and its inhibitor protein BLIP. Using the HIV1-p17 antibody as an initial target, the interaction between enzyme and inhibitor was carefully tuned to yield a reporter enzyme whose activity increased 10-fold in the presence of pM antibody concentrations. Reporter enzymes for two other antibodies (HA-tag and Dengue virus type I) were obtained by simply replacing the epitope sequences. This new sensor design represents a modular and generic approach to construct antibody reporter enzymes without the cumbersome optimization required by previous engineering strategies.