Determination of radioligand specific activity using competition binding assays.
Radioligand binding assays are routinely utilized in laboratories throughout the world to study receptors and their related binding sites, carrier proteins, and enzymes. To accurately estimate equilibrium binding parameters, such as the equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax), the investigator must know the correct value of the specific activity of the radioligand. If the specific activity is overestimated the Kd and Bmax values will be underestimated, while underestimation of the specific activity results in an overestimation of the Kd and Bmax. The present communication describes a simple and rapid method for determining the specific activity of a radioligand using homologous competition binding assays. Performing the competition assays at two or more different concentrations of the radioligand allows the specific activity to be determined from the IC50 values without the need of analytical methods to quantify minute amounts of the radioligand. In addition to providing the specific activity, use of this method estimates the Kd for the radioligand. This method was utilized to determine the specific activity and Kd for two blockers of the dopamine uptake carrier, [3H]GBR-12935 and [3H]-CFT, which share a common binding site in the striatum.
Wiener
H L
HL
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, New York 11439.
Reith
M E
ME
D000818:Animals; D001667:Binding, Competitive; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D003342:Corpus Striatum / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008024:Ligands; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014316:Tritium
D008024:Ligands; D010879:Piperazines; D014316:Tritium; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; C044630:1-(2 (diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine; D003042:Cocaine