title: A new immunoenzymometric assay for bioactive N-terminal human parathyroid hormone fragments and its application in pharmacokinetic studies in dogs.
abstract: Advances in the treatment of clinical disorders of mineral in homeostatis 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 immunoenzymometric assay for the quantitative determination of bioactive hPTH-1-37 (human parathyroid hormone) at picomolar concentrations was developed. Monoclonal antibodies (mAB) against hPTH-1-37 were raised by hybridoma cells in serum-free cell culture. Furthermore, sequence-specific polyclonal antibodies were obtained by immunisation of rabbits using multiple antigenic peptides (MAP) representing the conspicuous regions of the primary structure of hPTH-1-37. The polyclonal and monoclonal antibodies were characterised by epitope mapping. The combination of a monoclonal antibody (13C63/5) recognising hPTH fragment 16-24 with a polyclonal antibody (k2) showing a predominant binding sequence at hPTH-1-5 led to a sandwich assay specific for N-terminally intact and therefore 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 concentrations of hPTH-1-37 ranging at 2139 +/- 857 pmol/l were observed 45 min after the injection. Clearance of the peptide calculated from the exponential disappearance curve was 32.0 +/- 9.1 ml/min/kg with a mean t1/2 of 37 +/- 10 min.
authors.affiliation: Pharmazeutisch-Chemisches Institut Universität Heidelberg Germany.
authors.forename: M
authors.initials: M
authors.lastname: Mägerlein
authors.forename: D
authors.initials: D
authors.lastname: Hock
authors.forename: K
authors.initials: K
authors.lastname: Adermann
authors.forename: B
authors.initials: B
authors.lastname: Müller-Beckmann
authors.forename: R
authors.initials: R
authors.lastname: Neidlein
authors.forename: W G
authors.initials: WG
authors.lastname: Forssmann
authors.forename: K
authors.initials: K
authors.lastname: Stein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002138:Calibration; D002682:Chinchilla; D004285:Dogs; D005260:Female; D006207:Half-Life; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000032:analysis* / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D010446:Peptide Fragments / Q000032:analysis / Q000138:chemical synthesis / Q000493:pharmacokinetics; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D010281:Parathyroid Hormone; D010446:Peptide Fragments