Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays
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 (KD,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 (KD,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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:9 |
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| Enthalten in: |
ACS sensors - 9(2024), 3 vom: 22. März, Seite 1401-1409 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
van Aalen, Eva A [VerfasserIn] |
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| Links: |
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| Themen: |
Antibodies |
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| Anmerkungen: |
Date Completed 25.03.2024 Date Revised 28.03.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acssensors.3c02478 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM368706486 |
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| 520 | |a 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 (KD,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 (KD,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 | ||
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