title: Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides.
abstract: Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP, a strong cross-reactivity with the corresponding cAMP and cGMP, respectively, immunoassays was observed. The determined EIA-binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10-30% of the extracellular applied concentration, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment.
authors.affiliation: Institute for Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Grombuehlstrasse 12, Wuerzburg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Werner
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Hans-Gottfried
authors.initials: HG
authors.lastname: Genieser
authors.forename: Jörg
authors.initials: J
authors.lastname: Geiger
authors.forename: Elke
authors.initials: E
authors.lastname: Butt
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D011863:Radioimmunoassay
chemical_list: D000242:Cyclic AMP; D006152:Cyclic GMP