Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.
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.
Brown
Justin T
JT
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, United States.
Kant
Andrew
A
Mailman
Richard B
RB
D000262:Adenylyl Cyclases / Q000032:analysis* / Q000378:metabolism; D000906:Antibodies / Q000737:chemistry; D001331:Automation / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D019411:Clinical Laboratory Techniques; D003365:Costs and Cost Analysis; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D008280:Magnetics / Q000379:methods; D008863:Microspheres; D011863:Radioimmunoassay / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D015290:Second Messenger Systems / Q000502:physiology; D013997:Time Factors
D000906:Antibodies; D007457:Iodine Radioisotopes; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D006152:Cyclic GMP