A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
A homogeneous single-label quenching resonance energy transfer assay for a delta-opioid receptor-ligand using intact cells
Tekijät: Kopra K, Shweta, Martikkala E, Hanninen P, Petaja-Repo U, Harma H
Kustantaja: ROYAL SOC CHEMISTRY
Julkaisuvuosi: 2013
Journal: Analyst
Tietokannassa oleva lehden nimi: ANALYST
Lehden akronyymi: ANALYST
Numero sarjassa: 17
Vuosikerta: 138
Numero: 17
Aloitussivu: 4907
Lopetussivu: 4914
Sivujen määrä: 8
ISSN: 0003-2654
DOI: https://doi.org/10.1039/c3an00736g
Tiivistelmä
This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.
This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.
Turun yliopiston Tutkimustietojärjestelmän portaali