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Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides

Please always quote using this URN: urn:nbn:de:bvb:20-opus-141828
  • 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 theImmunoassays 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.show moreshow less

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Metadaten
Author: Katharina Werner, Frank Schwede, Hans-Gottfried Genieser, Jörg Geiger, Elke Butt
URN:urn:nbn:de:bvb:20-opus-141828
Document Type:Journal article
Faculties:Medizinische Fakultät / Institut für Klinische Biochemie und Pathobiochemie
Language:English
Parent Title (English):Naunyn-Schmiedeberg's Archives of Pharmacology
Year of Completion:2011
Volume:384
Issue:2
Pagenumber:169-176
Source:Naunyn-Schmiedeberg's Arch Pharmacol (2011) 384:169–176
DOI:https://doi.org/10.1007/s00210-011-0662-6
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Tag:Cell permeability; Cyclic nucleotides; Enzyme immunoassay; Lipophilicity
Release Date:2019/01/21
Licence (German):License LogoCC BY-NC: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell