Mass spectrometric immunoassay for parathyroid hormone-related protein

This paper describes a novel two-site peptide immunoassay using the isotope C-14 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 C-14 by growing the hybridoma cells in a miniPERM bioreactor in the presence of [U-C-14]L-leucine and [U-C-14]D-glucose. The antibody was purified from the culture media using protein G affinity chromatography. The purified C-14-labeled antibody (C-14-1A5) fractions showed excellent correlation between the levels of radioactivity and binding activity for PTHrP. Using C-14-1A5 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 similar to17 pCi/well (or 1.6 nCi/96-well microtiter plate) C-14-1A5 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.