title: Mass spectrometric immunoassay for parathyroid hormone-related protein.
abstract: This paper describes a novel two-site peptide immunoassay using the isotope 14C as the label and accelerator mass spectrometry as the detection system. A mouse monoclonal antibody (1A5) against the amino terminal region of human parathyroid hormone-related protein (PTHrP) was labeled with 14C by growing the hybridoma cells in a miniPERM bioreactor in the presence of [U-14C]L-leucine and [U-14C]D-glucose. The antibody was purified from the culture media using protein G affinity chromatography. The purified 14C-labeled antibody (14C-1A5) fractions showed excellent correlation between the levels of radioactivity and binding activity for PTHrP. Using 14C-1A5 as the detection antibody in a two-site immunoassay format for PTHrP1-141, a 16-kDa polypeptide, an analytic sensitivity of 10 pmol/L was achieved with a linear measurement range up to 1.3 nmol/L. Only approximately 17 pCi/ well (or 1.6 nCi/96-well microtiter plate) 14C-1A5 was used, which is far below the limit (50 nCi/g) for disposal as nonradioactive waste. This study may serve as a model for the development of sensitive and "nonradioactive" immunoassays for peptides, including polypeptide tumor markers.
authors.affiliation: Veterans Affairs Medical Center, San Diego, California 92161, USA.
authors.forename: Chuanyi M
authors.initials: CM
authors.lastname: Lu
authors.forename: W Douglas W
authors.initials: WD
authors.lastname: Burton
authors.forename: Robert L
authors.initials: RL
authors.lastname: Fitzgerald
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Deftos
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Buchholz
authors.forename: John S
authors.initials: JS
authors.lastname: Vogel
authors.forename: David A
authors.initials: DA
authors.lastname: Herold
mesh_terms: D000818:Animals; D002138:Calibration; D002846:Chromatography, Affinity; D007118:Immunoassay / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D044162:Parathyroid Hormone-Related Protein; D036361:Peptide Hormones / Q000032:analysis*
chemical_list: D044162:Parathyroid Hormone-Related Protein; D036361:Peptide Hormones