I am working as a senior Research Fellow at the Department of Biophysics, Panjab University, Chandigarh, India. I had an opportunity to use and analyze the Cyclic AMP EIA Kit from Cayman Chemical. As part of my work, I was in search of a convenient and efficient protocol for evaluation of Cyclic AMP as I am working on phytochemicals in the area of cancer research. Cyclic AMP is a second messenger and is utilized by biological systems for intracellular signal transduction and it also regulates calcium ion channels. Further, it has also been linked with carcinogenesis. I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
The Cyclic AMP EIA Kit is a competitive enzyme linked immunosorbent assay which involves competition between free cyclic AMP (cAMP) and a cAMP acetylcholinesterase tracer conjugate (cAMP tracer) for the limited number of binding sites on a cAMP specific rabbit antibody as the concentration of tracer cyclic AMP is held constant and the concentration of free cyclic AMP varies. The antibody-bound forms of both tracer and free cAMP binds further to a monoclonal anti-rabbit IgG antibody which has been previously attached to a well in a multi-well plate. This is followed by addition of Ellmann reagent (which contains the acetylcholinesterase substrate); this results in the production of a yellow color which is read at 412 nm. The intensity of the color is directly proportional to the amount of bound tracer cAMP and inversely proportional to free cAMP.
I found the Cyclic AMP EIA Kit instructions to be clear and concise with well explained, diagrammatic, theoretical details. Secondly, in the same kit, it is very easy to verify the specificity of the substrate with help of a specific inhibitor. Also, the protocol is sensitive and specific enough to allow estimation in a small sample size. The drawback of the Cyclic AMP EIA Kit is its cost and lengthy protocol.
My research work involves the use of tissue homogenates. So, prior to dissection, lung tissues were perfused with Tris Buffer, pH 7.4 to remove any red blood cell contamination. Tissue homogenates were then made and centrifuged at 10.000 rpm for 15 minutes at 4°C. The resultant supernatant was used for the assay. Then, as directed in the kit, different reagents: EIA buffer solution, wash buffer solution, cyclic AMP tracer solution, cyclic AMP EIA antiserum, and cyclic AMP standards, to be used in the assay were prepared. The kit also includes directions for the use of wells on the plate. Accordingly, EIA buffer was pipetted into the standard wells. In the sample wells 100 ul of the EIA buffer was added which was followed by the addition of 50 ul of the sample. Further, this was followed by addition of 50 ul of cAMP tracer. Then 50 ul of EIA antiserum was added to each of the wells. Then the plate was incubated for 18 hrs at 4°C. After incubation, the wells were made empty and rinsed 5 times with wash buffer. This was followed by the addition of Ellman reagent to each well. The plate was then kept on a shaking rocker for 120 minutes for color development. The plate was read at 412 nm using a plate reader.
I recommend the Cyclic AMP EIA Kit for its technology, reproducibility, sensitivity, and specificity. The instructions were clear, precise and accurate. Further, the reference standard curves helps a lot in calculation part of the assay.