A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer.
A chemiluminescent derivative of cyclic AMP, aminobutylethylisoluminol succinyl cyclic AMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 X 10(18) luminescent counts X mol-1 at pH 13), pure (greater than 95%) stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cyclic AMP) immunoglobulin G which could detect cyclic AMP at least down to 25fmol. A homogeneous immunoassay for cyclic AMP was established by measuring the shift in wavelength from 460 to 525nm which occurred when ABEI-scAMP was bound to fluorescein-labelled anti-(cyclic AMP) immunoglobulin G. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cyclic AMP over the range 1-1000nM. The homogeneous chemiluminescent energy transfer assay was also able to quantify the association and dissociation of antibody-antigen complexes. Chemiluminescence energy transfer occurred between fluorescein-labelled antibodies and several other ABEI-labelled antigens (Mr values 314-150000) including progesterone, cyclic GMP, complement component C9 and immunoglobulin G. The results provide a homogeneous immunoassay capable of measuring free cyclic AMP under conditions likely to exist inside cells.
D000818:Animals; D000941:Antigens / Q000276:immunology; D010856:Columbidae; D003429:Cross Reactions; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000032:analysis / Q000097:blood / Q000138:chemical synthesis / Q000276:immunology; D004735:Energy Transfer; D004837:Epinephrine / Q000494:pharmacology; D004912:Erythrocytes / Q000032:analysis / Q000187:drug effects; D007118:Immunoassay / Q000379:methods; D008163:Luminescent Measurements; D009712:Nucleotides, Cyclic / Q000032:analysis* / Q000276:immunology
D000941:Antigens; D009712:Nucleotides, Cyclic; C039787:aminobutylethylisoluminol succinyl cyclic AMP; D000242:Cyclic AMP; D004837:Epinephrine