Automated time-resolved immunofluorometric assay for progastrin-releasing peptide

BACKGROUND: Small cell lung cancer accounts for approximately 20% of new cases of lung cancer, and advanced disease is prevalent at the time of diagnosis. Neuron-specific enolase (NSE) has been the primary tumor marker in small cell lung cancer but it has relatively low sensitivity in early-stage disease. Progastrin-releasing peptide (proGRP) is a promising alternative or complementary marker for NSE. We have previously described a time-resolved immunofluorometric assay (TR-IFMA) for proGRP that lacked the necessary sensitivity and robustness for use in the routine clinical laboratory. Herein we describe the development of an improved assay using a novel monoclonal antibody pair. METHODS: Mice were immunized with different conjugated proGRP peptides, including residues 31-98, 1-98, and preproGRP(-23-125). Pair combinations of the resulting monoclonal antibodies (mAb) were tested. The improved TR-IFMA was compared with the only other available proGRP assay, the proGRP ELISA (IBL). RESULTS: A panel of 12 high-affinity mAbs was produced. The best assay combination was between our original E146 mAb as solid-phase antibody and the new mAb M16 as tracer. The new TR-IFMA had a linear dose-response curve, a wide dynamic range (1313 500 ng/L), and a limit of detection of 2.8 ng/L. Total CV was <5.6% over the whole measuring range. Bland-Altman difference analysis indicated a significant positive bias between the IFMA and the ELISA. CONCLUSIONS: We describe a sensitive and robust mAb-based TR-IFMA for proGRP. The assay is fully automated and displays high quality performance.