PMID- 32749852
OWN - NLM
STAT- MEDLINE
DCOM- 20210921
LR  - 20210921
IS  - 1557-8127 (Electronic)
IS  - 1540-658X (Linking)
VI  - 18
IP  - 7
DP  - 2020 Oct
TI  - Development and Evaluation of a Versatile Receptor-Ligand Binding Assay Using 
      Cell Membrane Preparations Embedded in an Agarose Gel Matrix and Evaluation with 
      the Human Adenosine A(1) Receptor.
PG  - 328-340
LID - 10.1089/adt.2020.991 [doi]
AB  - Guanosine-5'-triphosphate (GTP)-binding protein-coupled receptors are the target 
      of up to 40% of prescribed medications worldwide. To evaluate the suitability of 
      novel receptor ligands, frequently elaborate, time-consuming, and expensive 
      receptor-ligand interaction studies have to be carried out. This work describes 
      the development and proof of principle of a rapid, sensitive, and reliable 
      receptor-ligand binding assay. CHO cells were stably transfected with a construct 
      encoding the human A(1) adenosine receptor (hA(1)AR). For ligand binding assays, 
      membranes from these cells were prepared and embedded in low melting point 
      agarose. These "immobilized" samples were incubated with tritiated 
      8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX), a well-established receptor 
      antagonist. The K(D) and B(max) values as well as kinetic parameters (k(on) and 
      k(off)) of receptor-ligand interaction were determined. Unspecific binding of 
      various radiotracers to either the carrier material or the agarose gel matrix was 
      negligible. The dissociation constant (K(D)) for [(3)H]DPCPX at the hA(1)AR was 
      determined by saturation, competition binding, and kinetic experiments. These 
      studies resulted in K(D) values of  approximately 3 nM, which is in good accordance with 
      previously published data obtained from conventional receptor-ligand binding 
      assays. The procedure described in this study simplifies classical binding 
      studies to a kit-like assay. The receptors retained their binding properties even 
      when preparations were dried completely. Transport and delivery of the material 
      are conceivable without loss of biological activity. Therefore, other 
      laboratories can perform binding studies without special equipment or the 
      necessity to run a cell culture laboratory and/or to dissect tissue on their own.
FAU - Bier, Dirk
AU  - Bier D
AD  - Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), 
      Forschungszentrum Julich GmbH, Julich, Germany.
FAU - Schulze, Annette
AU  - Schulze A
AD  - Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), 
      Forschungszentrum Julich GmbH, Julich, Germany.
FAU - Holschbach, Marcus
AU  - Holschbach M
AD  - Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), 
      Forschungszentrum Julich GmbH, Julich, Germany.
FAU - Neumaier, Bernd
AU  - Neumaier B
AD  - Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), 
      Forschungszentrum Julich GmbH, Julich, Germany.
AD  - Institute of Radiochemistry and Experimental Molecular Imaging, University of 
      Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
FAU - Baumann, Arnd
AU  - Baumann A
AD  - Institute of Biological Information Processing, Molecular and Cell Physiology 
      (IBI-1), Forschungszentrum Julich GmbH, Julich, Germany.
LA  - eng
PT  - Journal Article
DEP - 20200729
PL  - United States
TA  - Assay Drug Dev Technol
JT  - Assay and drug development technologies
JID - 101151468
RN  - 0 (Gels)
RN  - 0 (Ligands)
RN  - 0 (Receptor, Adenosine A1)
RN  - 0 (Xanthines)
RN  - 9012-36-6 (Sepharose)
RN  - 9PTP4FOI9E (1,3-dipropyl-8-cyclopentylxanthine)
SB  - IM
MH  - Animals
MH  - Cells, Cultured
MH  - Cricetulus
MH  - Gels/chemistry
MH  - Ligands
MH  - Male
MH  - Rats
MH  - Rats, Wistar
MH  - Receptor, Adenosine A1/*metabolism
MH  - Sepharose/*chemistry
MH  - Xanthines/chemistry/*pharmacology
OTO - NOTNLM
OT  - DPCPX
OT  - adenosine A1 receptor
OT  - low melting point agarose
OT  - radioligand
OT  - receptor kinetics
OT  - stably transfected cell lines
EDAT- 2020/08/05 06:00
MHDA- 2021/09/22 06:00
CRDT- 2020/08/05 06:00
PHST- 2020/08/05 06:00 [pubmed]
PHST- 2021/09/22 06:00 [medline]
PHST- 2020/08/05 06:00 [entrez]
AID - 10.1089/adt.2020.991 [doi]
PST - ppublish
SO  - Assay Drug Dev Technol. 2020 Oct;18(7):328-340. doi: 10.1089/adt.2020.991. Epub 
      2020 Jul 29.