A NEW IMMUNOENZYMOMETRIC ASSAY FOR BIOACTIVE N-TERMINAL HUMAN PARATHYROID-HORMONE FRAGMENTS AND ITS APPLICATION IN PHARMACOKINETIC STUDIES IN DOGS

Advances in the treatment of clinical disorders of mineral in homeosta tis and metabolic bone disease with intact parathyroid hormone 1-84 or one of the biologically active N-terminal fragments require a precise and sensitive measurement in serum. Therefore, a two-site immunoenzym ometric assay for the quantitative determination of bioactive hPTH-1-3 7 (human parathyroid hormone) at picomolar concentrations was develope d. Monoclonal antibodies (mAb) against hPTH-1-37 were raised by hybrid oma cells in serum-free cell culture. Furthermore, sequence-specific p olyclonal antibodies were obtained by immunisation of rabbits using mu ltiple antigenic peptides (MAP) representing the conspicuous regions o f the primary structure of hPTH-1-37. The polyclonal and monoclonal an tibodies were characterised by epitope mapping. The combination of a m onoclonal antibody (13C63/5) recognising hPTH fragment 16-24 with a po lyclonal antibody (K-2) showing a predominant binding sequence at hPTH -1-5 led to a sandwich assay specific for N-terminally intact and ther efore biologically active hPTH. The validated assay ranging from 4 to 1000 pmol/l was applied to pharmacokinetic studies of hPTH-1-37. After s.c. administration of 30 mu g/kg in 5 beagles, the maximum serum con centrations of hPTH-1-37 ranging at 2139 +/- 857 pmol/l were observed 45 min after the injection. Clearance of the peptide calculated from t he exponential disappearance curve was 32.0 +/- 9.1 ml/min/kg with a m ean t(1/2) of 37 +/- 10 min.