Bioluminescent Antibodies for Point-of-Care Diagnostics

Abstract

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR_max >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.

Keywords: antibodies; bioluminescence; point-of-care monitoring; sensors; therapeutic drug monitoring.

Figure 1

The design of LUCIDs for PoC diagnostics. a) Schematic representation of the paper‐based device. The LUCID is a fusion protein of SNAP‐tag, NanoLuc luciferase (NLuc), and a binding protein (BP). SNAP‐tag is labeled with a molecule containing a fluorophore (red star) and a ligand (green ball) that binds to BP. The filter paper was printed with wax circles and the signal was collected by a digital camera. b) The variable fragment of the methotrexate antibody (PDB ID: 4OCX) bound to methotrexate (yellow). The N‐termini of both chains are indicated in green. The three CDRs (H1‐3, blue) on the heavy chain (light blue) and three CDRs (L1‐3, red) on the light chain (pink) are involved in antigen binding. c) In Fab‐based LUCIDs, the binding protein is an antibody Fab fragment. SNAP‐tag and NanoLuc are attached to the light chain.

Figure 2

Antibody‐binding of methotrexate (a), theophylline (b), and quinine (c). The chemical structures of the three drugs are shown on the left. The crystal structures of antibodies with the methotrexate (PDB ID: 4OCX), theophylline derivative (PDB ID: 5BMF), and quinine (PDB ID: 4UIN) were superimposed, showing the interactions of the residues with the ligands (carbon atoms in green). Extensive hydrogen bonding (dotted orange lines) links the three antigens to the antibodies, either directly or via water molecules (red balls).

Figure 3

Molecules used for semisynthesis of the sensor proteins. The benzylguanine group serves as the reactive moiety for SNAP‐tag labeling (blue). The fluorophore Cy3 is colored in red. The ligands for different analytes are shown as R groups, which conjugate to the polyethylene glycol linker through amide bonds (green).

Figure 4

LUCIDs for methotrexate, theophylline, and quinine. Emission spectra of 5 nm MTX₁‐Lucid (a), Theo₁‐Lucid (b), and Quin₂‐Lucid (c) in human serum spiked with known concentrations of the corresponding analyte. Emission ratios of methotrexate LUCIDs (d), theophylline LUCIDs (e), and quinine LUCIDs (f) as a function of analyte concentration. The measurements were performed on paper spotted with 5 μL sample (50 mm HEPES, 50 mm NaCl, pH 7.45, 50 % (v/v) serum, 1/100 furimazine). The data (mean±SD) are fitted to a single‐site binding isotherm (dashed line). Insets: pictures of the paper‐based devices in the detection of serum samples with the corresponding analytes (Top: LUCIDs with low c₅₀ values; Bottom: LUCIDs with high c₅₀ values).

Figure 5

Correlation of the results obtained for the spiked samples (black: theophylline, red: methotrexate, and blue: quinine) using a traditional UV/Vis spectrophotometric method and LUCID. The LUCID measurements were performed on paper by spotting 5 μL sample/serum mixture (50 mm HEPES, 50 mm NaCl, pH 7.45, 50 % (v/v) serum, 1/100 furimazine), and the UV/Vis measurements in HEPES buffer (50 mm HEPES, 50 mm NaCl, pH 7.45). Each LUCID measurement is given as the mean±SD of three independent measurements. Regression analysis yielded a Pearson correlation coefficient R=0.994.