Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone

Abstract

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Paper‐Based Antibody Detection Devices Using Bioluminescent BRET‐Switching Sensor Proteins

“…As observed previously, the absolute bioluminescence emission intensities decreased when switching from aqueous solutions to the blood serum matrix, which has been attributed to a lower activity of the NanoLuc enzyme in that matrix 11a. For this reason, the amount of LUMABS deposited onto signal detection areas was increased to 500 fmol for all of the investigated sensor variants.…”

“…Because the response of the system is independent of the applied sample volume over a wide volume range, no complex reagent handling and no quantitative sample metering are required. Although a standard digital camera was used in this study for signal acquisition, future applications of the 3D‐μPADs may use a smartphone camera, allowing integration of signal detection and application‐based analysis without a dedicated measurement instrument 11a. The application of these 3D‐μPADs is also not restricted to LUMABS‐based detection of antibodies but may be combined with any BRET‐based sensor, including recently developed sensors for small‐molecule drugs (LUCIDs)12 and nucleic acids (BRET‐beacons) 13.…”

Engineering BRET-Sensor Proteins

“…Being able to define a personalized dose per patient based on continuous monitoring of therapeutic antibody levels would, therefore, be beneficial both for the individual patient and for the hospital's budget. Our group recently developed a new platform of BRET-sensors for antibody detection named LUMABS (Arts et al, 2016). These NanoLuc-based sensor proteins allow measurement of antibodies directly in blood plasma, eliminating the need for any washing steps.…”

“…(D) Mobile phone photograph showing the color changes of DEN1-LUMABS (LUMABS sensor for detection of antidengue-1; 1 nM sensor) in blood plasma in response to increasing antibody concentrations. Reprinted with permission fromArts, R., den Hartog, I., Zijlema, S. E., Thijssen, V., van der Beelen, S. H. E., & Merkx, M. (2016). Detection of antibodies in blood plasma using bioluminescent sensor proteins and a smartphone.…”

Bioluminescent Antibodies for Point‐of‐Care Diagnostics

“…SNAP-tag is labeled with amolecule composed of aligand for the binding protein and ared-emitting fluorophore suitable for BRET. [5][6][7] Binding of the drug by the sensor displaces the tethered fluorescent competitor from the binding protein, which disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and the fluorophore and thus shifts the color from red to blue in ad rug-concentration-dependent manner, thereby permitting the quantification of drugs by spotting samples onto paper, followed by analysis with adigital camera (Figure 1a). However, up to now,nogeneral strategy exists to identify suitable binding proteins.Here,wedemonstrate how antibodies can be used as the binding proteins in LUCIDs, thereby establishing ag eneral design principle for PoCcompatible biosensors towards ab asically unlimited number of analytes.A ntibodies have three features that make them ideal binding proteins for LUCIDs:1 )all antibodies share Figure 1.…”