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.

Figure 1

Reaction scheme for synthesis of 2′-O-[4-monosuccinyladenosine 3′:5′-cyclic monophosphate-3-iodotyrosyl methyl ester. Conditions described in Methods (molar excess of precursor) favor the formation of the monoiodinated product (see Figure 2).

Figure 2

Chromatogram of radioiodination. [Bottom tracing] shows injection of cAMP-Sc-TME precursor alone using conditions as described in Methods using 254 nm UV detection. The solvent front emerges at ∼ 2 min, and the precursor elutes at ∼ 6min. The signal in the solvent front and a detectable shoulder on the major peak is consistent with the 95% purity estimated by the supplier. [Top tracing] Actual results from a radioiodination. The monoiodinated product that is immunologically recognized elutes at ∼28 min, and is the fraction to be collected and used for the RIA. This fraction contains from 60-70% of the radioactivity in a typical reaction. The fraction eluting at ∼40 min also contains significant radioactivity (10-20%), and is presumably the diiodinated form. These two peaks account for ∼80% of the total radioactivity injected, with the remainder of the radioactivity largely eluting in the solvent front (representing unreacted iodine or highly polar reaction by-products).

Figure 3

cAMP standard curves generated under varying assay conditions. Standards were incubated for 2 hrs. at room temperature with 50 μL (A) and 10 μL (B) primary antibody and overnight at 4 C [50 μL (C), 10 μL (D)]. Each assay condition yielded a viable standard curve, indicating that the conditions can be tailored according to the user’s needs.

Figure 4

Measurement of D₁ dopamine receptor-mediated cAMP accumulation utilizing [Left panel] secondary antibody-PEG assisted RIA method, and [right panel] our new RIA method (primary antibody conjugated to beads. cAMP production was measured using HEK293 cell membranes transiently expressing human D₁ dopamine receptors. Data are expressed as % maximal cAMP stimulation caused by dopamine. The curves shown represent mean ± SEM for quadruplicate determinations of cAMP accumulation from four separate experiments.

Figure 5

Precision profile demonstrates the Coefficient of Variation as a function of the concentration of cAMP standards.

Figure 6

Schematic flowchart of the described method.