A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer

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Abstract
A chemiluminescent derivative of cAMP, aminobutylethylisoluminol succinyl cAMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 .times. 1018 luminescent counts .cntdot. mol-1 at pH 13), pure (> 95%), stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cAMP)IgG which could detect cAMP at least down to 25 fmol. A homogeneous immunoassay for cAMP was established by measuring the shift in wavelength from 460 to 525 nm which occurred when ABEI-scAMP was bound to fluorescein-labeled anti-(cAMP)IgG. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cAMP over the range 1-1000 nM. 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-labeled antibodies and several other ABEI-labeled antigens (MW values 314-150,000) including progesterone, cGMP, [human] complement component C9 and [rabbit] IgG. The results provide a homogeneous immunoassay capable of measuring free cAMP under conditions likely to exist inside cells [pigeon erythrocytes].