Mass spectrometric immunoassay for parathyroid hormone-related protein.
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.
Lu
Chuanyi M
CM
Veterans Affairs Medical Center, San Diego, California 92161, USA.
Burton
W Douglas W
WD
Fitzgerald
Robert L
RL
Deftos
Leonard J
LJ
Buchholz
Bruce A
BA
Vogel
John S
JS
Herold
David A
DA
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*
D044162:Parathyroid Hormone-Related Protein; D036361:Peptide Hormones