Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: Application in GPCR-mediated adenylate cyclase assays

Abstract

Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high-throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C

“…Stably transfected CHO cells and transiently transfected HEK-293 cells were used for radioimmunoassay, and GloSensor ™ cAMP system used transiently transfected HEK-293 cells. The procedures of radioimmunoassay have been previously published (Brown et al , 2009; Harper and Brooker, 1975). Briefly, the transfected cells were seeded at a density of 10 5 cells/well in a 48-well plate and grown overnight.…”

Receptor Conformations Involved in Dopamine D2LReceptor Functional Selectivity Induced by Selected Transmembrane-5 Serine Mutations

“…I for cAMP assays was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA), and processed as described by Brown et al (2009). cAMP primary antibody was obtained from Dr. Gary Brooker (George Washington University, Washington DC); secondary antibody, rabbit anti-goat IgG, was purchased from Advanced Magnetics (Cambridge, MA).…”

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

“…A reasonable alternative to HPLC separation is to apply either competitive binding assays to cAMP-binding proteins or cAMP-directed antibodies. Typically measurements are performed either with [ 3 H]-or [ 125 I]-cAMP as competitors [7,9,13]. Both approaches have been successfully and frequently applied in the last decades.…”