Development of a highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34): application to pharmacokinetic study on intranasal parathyroid hormone (1-34) in human.

A highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34) (PTH(1-34)) and its usability for the pharmacokinetic study are described. Plasma samples were incubated simultaneously with 2,4-dinitrophenylated anti-PTH(1-34) IgG and anti-PTH(1-34) Fab'-beta-D-galactosidase conjugate. The immune complex formed of the three components was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the polystyrene balls was assayed by fluorometry. The practical detection limit of PTH(1-34) was 50 fg (12 amol)/0.05 ml of sample and 1 pg/ml as the concentration and practically no interference occurred by PTH(1-84) and PTH-related protein (1-34) up to 300 pg/ml and 10 ng/ml, respectively. The application of this method has enabled us to directly estimate the bioavailability of PTH(1-34) dosed intranasally at the prescribed level (0.090 mg). The pharmacokinetic parameters of the intranasal PTH(1-34) (n = 4) thus estimated were as follows: the area under the plasma concentration-time curve (AUC) = 20,500+/-15,900(SD) pg.min/ml; the mean residence time (MRT) = 194+/-16.3(SD) min; and the maximal concentration (Cmax) = 98+/-51 (SD) pg/ml with the maximal time (Tmax) = 35.0+/-12.2(SD) min.