NON-RADIOISOTOPIC HOMOGENEOUS STEROID IMMUNOASSAYS

Homogeneous non-isotopic immunoassays for steroids were developed utilizing as markers steroid-coenzyme conjugates and steroid-fluorescent-dye conjugates. Carboxy derivatives of steroids were coupled to nicotinamide-6-(2-amino-ethylamine)-purine dinucleotide or 7-hydroxy-coumarin to provide the corresponding steroid-cofactor conjugates or steroid-umbelliferone (fluorescent-dye) conjugates. The steroid-coenzyme markers were then used in competitive binding assays monitored by enzymic cyclic reactions. The steroid-umbelliferone labels were utilized in competitive binding assays monitored by enzymatic hydrolysis or by antibody-enhanced hydrolysis of steroid-fluorescent-dye conjugates. These three reaction schemes allowed the measurement of unbound conjugate without separation of free and bound hormone. These assays required simple instrumentation and used short reaction times (45-60 min). Sensitivity comparable to that of radioimmunoassay (2nM or 50 pg steroid/tube) was obtained with the coenzymatic cyclic reactions. © 1979.