Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Dual-Color Bioluminescent Sensor Proteins for Therapeutic Drug Monitoring of Antitumor Antibodies.

ABSTRACT: Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection directly in blood plasma. In this study, we targeted four clinically important therapeutic antibodies, the Her2-receptor targeting trastuzumab, the anti-CD20 antibodies rituximab and obinutuzumab, and the EGFR-blocking cetuximab. A strong correlation was found between the affinity of the antibody binding peptide and sensor performance. LUMABS sensors with physiologically relevant affinities and decent sensor responses were obtained for trastuzumab and cetuximab using mimotope and meditope peptides, respectively, with affinities in the 10-7 M range. The lower affinity of the CD20-derived cyclic peptide employed in the anti-CD20 LUMABS sensor ( Kd = 10-5 M), translated in a LUMABS sensor with a strongly attenuated sensor response. The trastuzumab and cetuximab sensors were further characterized with respect to binding kinetics and their performance in undiluted blood plasma. For both antibodies, LUMABS-based detection directly in plasma compared well to the analytical performance of commercial ELISA kits. Besides identifying important design parameters for the development of new LUMABS sensors, this work demonstrates the potential of the LUMABS platform for point-of-care detection of therapeutic antibodies.

SUBMITTER: van Rosmalen M

PROVIDER: S-EPMC5843950 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

Project description:Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

| S-EPMC5706068 | biostudies-literature

Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity.

Project description:FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

| S-EPMC5485374 | biostudies-literature

Ratiometric Bioluminescent Sensor Proteins Based on Intramolecular Split Luciferase Complementation.

Project description:Bioluminescent sensor proteins provide attractive tools for applications ranging from in vivo imaging to point-of-care testing. Here we introduce a new class of ratiometric bioluminescent sensor proteins that do not rely on direct modulation of BRET efficiency, but are based on competitive intramolecular complementation of split NanoLuc luciferase. Proof of concept for the feasibility of this sensor principle was provided by developing a blue-red light emitting sensor protein for the detection of anti-HIV1-p17 antibodies with a 500% change in emission ratio and a Kd of 10 pM. The new sensor design also improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4-fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma. The modular sensor architecture allows easy and systematic tuning of a sensor's dynamic range and should be generally applicable to allow rational engineering of bioluminescent sensor proteins.

| S-EPMC6350203 | biostudies-literature

Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins.

Project description:This work reports on fully integrated "sample-in-signal-out" microfluidic paper-based analytical devices (?PADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20-30??L) of sample (serum, whole blood) and the acquisition of a photograph 20?min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

| S-EPMC6282528 | biostudies-literature

Bioluminescent Antibodies through Photoconjugation of Protein G-Luciferase Fusion Proteins.

Project description:Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody-luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fc-binding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG's using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.

| S-EPMC7086395 | biostudies-literature

Portable solid-state sensor for therapeutic monitoring of an antineoplastic drug; vinblastine in human plasma.

Project description:During cancer treatment, doses must be carefully administered and monitored to guarantee efficacy and minimize side-effects. A potentiometric sensor was developed for the direct real-time assay of a widely used antineoplastic drug (vinblastine (VB)) in plasma samples. Membrane cocktails were drop-casted over a glassy-carbon electrode coated with a lipophilic conducting polymer (polyaniline). The study investigated five cation exchangers, five plasticizers (of different polarities and dielectric constants), and four ionophores with different physicochemical characters on the sensor performance. The study substantiates a data-driven selection of the optimum membrane recipe. The latter included sodium tetraphenylborate as an ion exchanger, dioctylphthalate as a plasticizer, and hydroxypropyl-β-cyclodextrin as ionophore. The membrane proved a near-Nernstian slope of 37.5 mV per decade, a LOQ of 2.99 × 10-6 M, and a stable fast response. The selectivity study proved poor responses to common physiological ions. The developed sensor was used for the determination of VB in its pure powder form, marketed formulation, and plasma samples. The fast and direct sensor response enables a wide range of applications in quality control laboratories and clinical studies.

| S-EPMC9058239 | biostudies-literature

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

Project description:Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5-13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

| S-EPMC9791688 | biostudies-literature

MARCH Proteins Mediate Responses to Antitumor Antibodies.

Project description:CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

| S-EPMC9382848 | biostudies-literature

A selective dual quenching sensor (EY/BG@CDs) for simultaneous monitoring of gentamicin and ketorolac levels in plasma: a highly efficient platform that caters to the needs of therapeutic drug monitoring.

Project description:This research work introduces a novel sensor that utilizes two fluorophores to enable simultaneous monitoring of gentamicin sulphate (GNT) and ketorolac tromethamine (KET). The innovative sensor is composed of carbon dots (CDs) derived from black grapes (BG) and eosin Y (EY) dye. The interaction between the studied drugs and EY/BG@CDs sensor components allows for their simultaneous detection where GNT quenches the fluorescence of EY at 535 nm without affecting the fluorescence of CDs, while KET quenches the fluorescence of BG@CDs at 385 nm without impacting EY fluorescence. The BG@CDs probe was successfully characterized using various techniques such as absorption spectrophotometry, spectrofluorimetry, TEM imaging, infrared spectroscopic analysis, and XRD analysis. The suggested methodology was observed to be highly sensitive for the simultaneous determination of GNT and KET in their spiked rabbit plasma samples, with wide linear ranges and low limit of detection (LOD) values. The studied drugs were extracted using a highly selective extraction method involving protein precipitation followed by mixed mode solid phase extraction using an Oasis WCX cartridge. The simultaneous determination of GNT and KET is essential due to the potential interactions between the studied drugs. Therefore, this analysis can be used to evaluate the necessity of dose monitoring and the potential adverse effects of co-administration of these drugs.

| S-EPMC10545982 | biostudies-literature

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae.

Project description:Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases.

| S-EPMC4542259 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data