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

The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with cAMP- and cGMP-immunoassays from Cayman, IBL (both IBL International, Hamburg, Germany) and ENZO Life Sciences (Loerrach, Germany).

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 ENZO cAMP- respectively cGMP-EIA and the IBL cGMP-RIA was observed (Table 1). As a consequence we tested these derivatives with the Cayman cGMP-EIA. This assay is less sensitive to cGMP (1.0 pmol/ml) than the ENZO cGMP-EIA (0.01 pmol/ml), however the specificity concerning cGMP-analogs is superior and therefore advantageous when measuring cGMP in the presence of 8-Br-cGMP or 8-pCPT-cGMP.

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 after 20 min (Table 1).

Affiliations

1. Institute for Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Germany
   • Katharina Werner
   • , Jörg Geiger
   •  & Elke Butt
2. Biolog Life Science Institute, Flughafendamm 9a, D-28199, Bremen, Germany
   • Frank Schwede
   •  & Hans-Gottfried Genieser

Corresponding authors

Correspondence to Katharina Werner or Elke Butt.

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