A NONCOMPETITIVE ENZYME-IMMUNOASSAY (HETERO-2-SITE ENZYME-IMMUNOASSAY) FOR SALMON-CALCITONIN - DETERMINATION OF THE BIOAVAILABILITY OF SUBCUTANEOUS SALMON-CALCITONIN AND ITS CORRELATION WITH THE HYPOCALCEMIC ACTIVITY IN RATS

A noncompetitive enzyme immunoassay method (hetero-two-site enzyme imm unoassay) for salmon calcitonin (SCT) and its usability for the pharma cokinetic study are described. The method in brief proceeds as follows : centrifugal filtration through a polysaccharide membrane to remove p lasma proteins, biotinylation, trapping onto an anti-SCT IgG-coated po lystyrene ball, acid elution, coupling with affinity-purified anti-SCT Fab'-peroxidase conjugate, final trapping onto streptavidin-coated po lystyrene balls, and measurement of peroxidase activity bound to the b alls by fluorometry. The practical detection limit of SCT was 0.1 pg ( 30 amol)/assay and 2 pg/ml as the assay sample's concentration, which was at least fivefold lower than those previously reported by competit ive radioimmunoassays. The application of this method has enabled us t o 1) directly estimate the bioavailability of SCT dosed subcutaneously at the therapeutic levels (1.2 and 4.7 mu g/kg) for its antiosteoporo tic effect as compared to an intravenous dose (1.2 mu g/kg) and 2) sea rch for the relationship between blood level and the hypocalcemic acti vity of SCT The pharmacokinetic parameters of subcutaneous SCT (1.2 an d 4.7 mu g/kg) thus estimated were as follows: the area under the bloo d concentration-time curve (AUC) = 89 and 550 pg . hr/ml, and mean res idence time (MRT) = 44 and 65 minutes, respectively, when the AUC for an intravenous SCT(1.2 mu g/kg)= 160 pg . hr/ml and the MRT = 10 minut es. (C) 1996 Wiley-Liss, Inc.