Determination of cAMP in plant cells by a modified enzyme immunoassay method

Presently, there is no doubt about the functioning of the adenylate cyclase signaling system in plants, but the role of this system in various physiological-biochemical processes has been investigated insufficiently. Cyclic adenosine monophosphate (cAMP), the key component produced by adenylate cyclase, whose concentrations in plant cells vary rather widely, is the indicator of functional activity for this signaling way. In the latter case, in the process of determination of concentrations of this messenger, one encounters difficulties related to insufficient sensitivity of the methods most frequently applied. In this connection, the proposed mechanism is a modification of the method of the enzyme immunoassay (EIA), which is based on immediate measurement of cAMP concentrations in the sample with the use of antibodies. This modification allows us to determine the concentrations of cAMP with the precision of 5 pM, which exceeds the sensitivity of other methods by approximately 10 times. The specificity of the assay has been confirmed by other two independent tests--the capillary electrophoresis and the nuclear magnetic resonance (NMR). It has also been compared to the data obtained with the use of the commercial kit from Sigma-Aldrich. The modification has been tested on such plant objects as in vitro potato plants, and suspension cells of potato and Arabidopsis.