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Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays.

ABSTRACT: Here we present LUCOS (Luminescent Competition Sensor), a modular and broadly applicable bioluminescent diagnostic platform enabling the detection of both small molecules and protein biomarkers. The construction of LUCOS sensors entails the covalent and site-specific coupling of a bioluminescent sensor component to an analyte-specific antibody via protein G-mediated photoconjugation. Target detection is accomplished through intramolecular competition with a tethered analyte competitor for antibody binding. We established two variants of LUCOS: an inherent ratiometric LUCOSR variant and an intensiometric LUCOSI version, which can be used for ratiometric detection upon the addition of a split calibrator luciferase. To demonstrate the versatility of the LUCOS platform, sensors were developed for the detection of the small molecule cortisol and the protein biomarker NT-proBNP. Sensors for both targets displayed analyte-dependent changes in the emission ratio and enabled detection in the micromolar concentration range (K_D,app = 16-92 μM). Furthermore, we showed that the response range of the LUCOS sensor can be adjusted by attenuating the affinity of the tethered NT-proBNP competitor, which enabled detection in the nanomolar concentration range (K_D,app = 317 ± 26 nM). Overall, the LUCOS platform offers a highly versatile and easy method to convert commercially available monoclonal antibodies into bioluminescent biosensors that provide a homogeneous alternative for the competitive immunoassay.

SUBMITTER: van Aalen EA

PROVIDER: S-EPMC10964239 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays.

van Aalen Eva A EA Lurvink Joep J J JJJ Vermeulen Leandra L van Gerven Benice B Ni Yan Y Arts Remco R Merkx Maarten M

ACS sensors 20240221 3

Here we present LUCOS (Luminescent Competition Sensor), a modular and broadly applicable bioluminescent diagnostic platform enabling the detection of both small molecules and protein biomarkers. The construction of LUCOS sensors entails the covalent and site-specific coupling of a bioluminescent sensor component to an analyte-specific antibody via protein G-mediated photoconjugation. Target detection is accomplished through intramolecular competition with a tethered analyte competitor for antibo ...[more]

PMID: 38380622

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Project description:Serological assays for antibody detection have contributed significantly to the diagnosis and control of infectious diseases. African swine fever is the most devastating infectious disease of domestic pigs and wild boars, severely threatening the global pig industry in recent years. Here, we developed a rapid, simple, and sensitive immunoassay based on the split-luciferase system to detect IgG antibodies against African swine fever virus (ASFV). In this assay, the p30 protein of ASFV was genetically coupled to the LgBiT and SmBiT subunits of nanoluciferase, which were used as fusion probes for specific antibodies. Target engagement of the probes results in the reconstitution of a functional nanoluciferase, which further catalyzes bioluminescent reactions. Different orientations of the LgBiT and SmBiT-p30 fusion sensors were designed and investigated, and N-LgBiT/p30 and N-SmBiT/p30 were identified as a promising sensor pair for reforming active nanoluciferase in the presence of specific antibodies. After optimization, this split-luciferase complementation assay showed high sensitivity and specificity for the detection of ASFV antibodies. The analytical sensitivity of the assay was 16 times greater than that of the blocking enzyme-linked immunosorbent assay (ELISA) by the detection of serial dilutions of serum, and no cross-reaction was observed with other swine pathogens. As demonstrated in clinical samples, its performance is highly consistent with that of a commercial ELISA kit, with a concordance rate of 98.19%. This assay is simple and easy to perform, providing a more flexible and efficient approach for the measurement of ASFV antibodies in clinical applications.ImportanceThe study is about a homogeneous split-luciferase assay for antibody detection. Split nanoluciferase biosensors for the detection of ASFV antibodies were designed. This sensor platform enables the sensitive and specific detection of antibodies. The split-luciferase assay is simple, rapid, and easy to use.

Dataset Information

Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays.

Publications

Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays.

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