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 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.
Mägerlein
M
M
Pharmazeutisch-Chemisches Institut Universität Heidelberg Germany.
Hock
D
D
Adermann
K
K
Müller-Beckmann
B
B
Neidlein
R
R
Forssmann
W G
WG
Stein
K
K
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
D000911:Antibodies, Monoclonal; D010281:Parathyroid Hormone; D010446:Peptide Fragments