Improved benzodiazepine radioreceptor assay using the MultiScreen Assay System.
In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time. The filtration in this method was performed by using the MultiScreen Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom, which allows both the incubation and the filtration of the specimen in the same plate. After the filtration, the filters were punched out for quantitation of the bound labeled ligand [3H]flunitrazepam. The results obtained with the MultiScreen Assay System did not differ significantly from the data obtained with the conventional filtration manifold (48S): The Ki's of lorazepam were 2.4 +/- 0.30 and 1.9 +/- 0.15 nM, respectively. In case a radioactive label is replaced by a fluorescent label, the bound labeled-ligand usually cannot be determined in the presence of the receptor material. Here, the bound labeled-ligand has to be dissociated after the filtration step. To dissociate the ligand-receptor complex, Tris- HCl buffer, containing 10 microM flumazenil, was added to the filters and the second filtrates were collected containing the previously bound fractions in the absence of receptor material. This approach showed the same Ki for lorazepam, 2.5 +/- 0.04 nM as without dissociation, when using the radio-labeled benzodiazepine [3H]flunitrazepam.
Janssen
M J
MJ
Department of Analytical Chemistry and Toxicology, Groningen Institute for Drug Studies, University Centre for Pharmacy, The Netherlands.
Ensing
K
K
de Zeeuw
R A
RA
D005374:Filtration / Q000379:methods; D008140:Lorazepam / Q000737:chemistry; D011869:Radioligand Assay / Q000379:methods*; D011963:Receptors, GABA-A / Q000032:analysis* / Q000737:chemistry
D011963:Receptors, GABA-A; D008140:Lorazepam