A Genetically Encoded Bioluminescent System for Fast and Highly Sensitive Detection of Antibodies with a Bright Green Fluorescent Protein

Abstract

A method for fast and highly sensitive detection of antibodies in serum would greatly facilitate the early diagnosis of disease and infection and dose optimization of therapeutic antibody. Bioluminescence detection with LUMABS (renamed mNeonG-LUMABS, where mNeonG is short for mNeonGreen) sensors based on bioluminescence resonance energy transfer (BRET) between blue-emitting luciferase Nluc and green fluorescent protein (FP) mNeonGreen has been demonstrated to enable fast detection of antibodies directly in serum with reasonable sensitivity. However, some mNeonG-LUMABS sensors exhibit low sensitivity, and thus, sensitivity improvement remains imperative. Here, we report a bright green FP, Clover4, obtained by structure-guided mutagenesis of green FP Clover. Despite similar brightness and fluorescence spectra of Clover and mNeonGreen, Clover4-LUMABS sensors exhibit a largely increased dynamic range (maximum 20-fold) and much lower limit of detection (LOD) (maximum 5.6-fold), most likely because Clover4 is positioned in a more parallel orientation to Nluc in LUMABS. Due to modular design, Clover4-LUMABS offers a general BRET system for fast and highly sensitive antibody detection in serum.

Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors

“…Tuning the relative affinities of the competing DarkBiT and SmBiT components yielded an optimized sensor for anti-HIV1-p17 with a 48 ± 8-fold increase in luminescent intensity. This dynamic range is 10-fold better than the previously reported sensor based on NanoBiT (NB-LUMABS) and 6-fold better than the most optimized BRET-based LUMABS sensor (Clover4-LUMABS-HIV) . This comparison is not completely fair, as the latter represent ratiometric sensor systems, whereas dark-LUMABS-HIV #3 by itself is an intensiometric sensor.…”

“…This dynamic range is 10-fold better than the previously reported sensor based on NanoBiT (NB-LUMABS) and 6-fold better than the most optimized BRET-based LUMABS sensor (Clover4-LUMABS-HIV). 26 This comparison is not completely fair, as the latter represent ratiometric sensor systems, whereas dark-LUMABS-HIV #3 by itself is an intensiometric sensor. However, we recently showed that intensiometric assays can be rendered ratiometric by the addition of green enhanced Nanolantern (GeNL), a tight fusion protein between NanoLuc and mNeongreen.…”

Fluorescence, ultrasonic and photoacoustic imaging for analysis and diagnosis of diseases

“…16 Numerous kinds of fluorescent probes (e.g., fluorescent proteins, fluorescent dyes and fluorescent nanomaterial-based probes) have been employed to label and trace different molecules, proteins, cells, and tissues and their dynamic progress. [49][50][51][52][53][54][55][56][57][58] In particular, fluorescent proteins are suitable for labeling various cells, viruses, genes and so forth; fluorescent dyes are appropriate for imaging and analyzing antibodies, peptides, small molecule drugs and so on. With significant advances in nanotechnology, nanomaterials (e.g., II-VI quantum dots (QDs), carbon dots (CDs), upconverting nanoparticles (UCNPs) and silicon-based nanoparticles (SiNPs)) featuring attractive optical properties, have been exploited as novel high-performance fluorescent nanoprobes for biomedical imaging applications.…”

“…, fluorescent proteins, fluorescent dyes and fluorescent nanomaterial-based probes) have been employed to label and trace different molecules, proteins, cells, and tissues and their dynamic progress. 49–58 In particular, fluorescent proteins are suitable for labeling various cells, viruses, genes and so forth; fluorescent dyes are appropriate for imaging and analyzing antibodies, peptides, small molecule drugs and so on. With significant advances in nanotechnology, nanomaterials ( e.g.…”

A Reaction-Based Ratiometric Bioluminescent Platform for Point-of-Care and Quantitative Detection Using a Smartphone

“…The ratiometric signal can remain constant, regardless of the decay of the bioluminescence intensity, which makes the detection result more accurate. Proximity-based BRET probes have been proved to be powerful tools for monitoring protein–protein interactions, − protein–ligand interactions, − and molecular proximity. − However, current BRET-based probes mainly employs specific proteins or peptides as recognition moiety, which allows the detection of only a limited number of targets. The recognition of the probes mainly relies on intermolecular interactions, which is difficult to transform the binding of targets into a change in BRET signal .…”