( Sensitive nonradioactive cyclic-AMP ...)Enzyme Immunoassay for Studying Intracellular Levels of cAMP

Sensitive, nonradioactive cyclic-AMP detection kit and adenylate cyclase (AC) toxin

Danny Q. Hoang
Stratagene Cloning Systems, Inc.

Xinping Yang and Timothy G. Kingan, Ph.D.
American Qualex

Stratagenes EIA cAMP Kit is a highly sensitive and specific antibody-based assay kit for nonradioactive, quantitative measurement of intracellular levels of cAMP. The assay is based on the competition between cAMP present in the sample and a fixed quantity of a cAMP-horseradish peroxidase conjugate for a limited number of binding sites on cAMP-specific antibodies. When the amounts of antibody and peroxidase-labeled cAMP are fixed, the amount of peroxidase-labeled cAMP that binds to the antibody is inversely proportional to the concentration of added, unlabeled cAMP in the sample. In this enzyme immunoassay (EIA) format, each microtiter plate is precoated with a secondary antibody, goat anti-rabbit IgG, which binds to a fixed amount of rabbit anti-cAMP. The amount of peroxidase-labeled cAMP bound to the antibody is determined by the addition of a colorimetric substrate, and the resulting color is measured by reading the spectrophotometric absorbance at 450 nm. The sensitivity of the EIA cAMP Kit is in the nanomolar range, and sensitivity is increased to the picomolar range with acetylated cAMP.

Cyclic adenosine monophosphate (cAMP) plays a key role as an intracellular second messenger for transduction events that follow a number of extracellular signals. Typically, the binding of a hormone or neuromodulator to its receptor is followed by the activation of a G protein, which, in turn, activates the effector adenylyl cyclase evoking the production of cAMP. The activation of a protein kinase by cAMP results in the phosphorylation of substrate proteins, which include enzymes, ion channels and transcription regulators. Because cAMP can activate a cascade of reactions, the involvement of cAMP greatly amplifies the response to a hormonal stimulus.

Nonradioactive Detection of cAMP

figure 1

Since changes in cAMP levels can result in the activation of proteins known to play key roles in signal transduction cascades, quantitative measurment of cAMP can serve to identify signaling events. Popular methods for determining cAMP levels include radioimmunoassays (RIA). Although these assays are highly sensitive, they require radioisotopes and time-consuming protocols. In contrast, Stratagenes nonradioactive EIA cAMP Kit uses a competitive EIA (figure 1) assay for simple and sensitive measurement of intracellualar cAMP levels. The assay is based on the competition between cAMP present in the sample and a fixed quantity of a cAMP-peroxidase conjugate that binds to rabbit anti-cAMP-specific antibodies.

figure 2

The assay is performed in a microtiter plate, with each well precoated with goat anti-rabbit IgG. Rabbit anti-cAMP antibodies are combined with the sample and cAMP-horseradish peroxidase conjugate (tracer) and applied to the microtiter well. Samples containing cAMP compete with a fixed quantity of tracer for the rabbit anti-cAMP-specific antibodies. All unbound reagents are removed by a wash step. The reaction is completed by the addition of the colorimetric substrate, 3,3,5,5-tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂). The addition of TMB/H₂O₂ to each well results in a blue-green product background, and the reaction is stopped by the addition of 1 M phosphoric acid. A bright yellow color results, and absorbance of peroxidase-labeled cAMP that is bound to the cAMP-specific antibody is determined using a spectrophoto- metric plate reader at 450 nm. Since the amount of cAMP-horseradish peroxidase bound to rabbit anti-cAMP antibody is inversely proportional to cAMP in the sample (or standard), a high absorbance value corresponds to low levels of cAMP present in the sample. The EIA cAMP Kit includes cAMP standards for calculating standard curves. As an option for increased sensitivity (figure 2), standards and prepared samples can be acetylated by treatment with acetic anhydride in the presence of triethylamine.¹ Picomolar detection is possible for acetylated cAMP, whereas nonacetylated cAMP levels can generally be assayed in the nanomolar range. The EIA cAMP Kit includes all the re- agents and protocols for quantifying cAMP levels in both nonacetylated and acetylated preparations.

AC Toxin for Increased Intracellular cAMP Levels

Stratagene is offering adenylate cyclase (AC) toxin, a useful reagent for increasing intracellular cAMP levels in cultured cells. AC toxin can be used to study cAMP-dependent protein kinase activation, the key regulatory event in signal transduction pathways involving cAMP. Modulators currently available for examining the effects of cAMP on intracellular signaling include dibutyryl cAMP² or 8-bromo-cAMP, cholera toxin (CT),² 3-isobutyl-1-methylxanthine (IBMX)³ and forskolin.⁴ AC toxin has been shown to elevate intracellular cAMP in cultured cells⁵ and can be used as an alternative to agonists that increase cAMP levels. AC toxin has an apparent molecular weight of 200 to 216 kDa by SDS-PAGE.⁶ Stratagenes AC toxin has been purified from E. Coli ⁷ and is supplied as lyophilized powder sealed under vacuum. When reconstituted in 50 l distilled water, each vial contains 50 g of AC toxin in 20 mM HEPES, pH 7.5 and 8 M urea.

AC toxin activity was determined by quantitation of intracellular cAMP accumulation in Jurkat⁸ cells using Stratagenes EIA cAMP Kit. Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 units/ml penicillin and 50 mg/ml streptomycin in a humidified, 37⁰C, 5% CO₂ incubator. Various concentrations of AC toxin were added to the cells (1 x 10⁶/ml), and intracellular cAMP levels were quantified.

figure 3

AC toxin elevates intracellular cAMP levels in Jurkat cells (figure 3). cAMP levels were elevated by as little as 0.5 g/ml of AC toxin, although 2.0 g/ml seems to be the optimal concentration for elevating values of cAMP. A 5-fold increase in the AC toxin concentration to 10 g/ml resulted in decreased intracellular cAMP levels, suggesting that cAMP production levels off at high concentrations of AC toxin.⁹ Stratagene recommends that researchers vary levels of AC toxin within this range (0.5 to 10 g/ml) to ensure maximal stimulation in the cell line of interest. In addition to Jurkat cells, AC toxin has also been shown to elevate cAMP in monocyte macrophages,¹⁰ polymorphonuclear leukocytes and alveolar macrophages,¹¹ natural killer cells¹² and HL-60 promyelocytic leukemia cells.¹³

Conclusions

The EIA cAMP Kit is a nonradioactive EIA system for measuring intracellular cAMP levels. The kit is simple, convenient and highly sensitive and allows determination of cAMP levels from cell culture or tissue preparations. Intracellular cAMP levels can be detected in the nanomolar or picomolar range. Stratagene also offers AC toxin, which significantly increases intracellular cAMP levels as an alternative to currently available cAMP agonists.

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