Automated Time-Resolved Immunofluorometric Assay for Progastrin-Releasing Peptide

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 54; no. 5; pp. 919 - 922
• Main Authors: Nordlund, Marianne S, Warren, David J, Nustad, Kjell, Bjerner, Johan, Paus, Elisabeth
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.05.2008; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Amino acids; Analytical, structural and metabolic biochemistry; Animals; Antibodies, Monoclonal - isolation & purification; Autoanalysis; Biological and medical sciences; Biomarkers, Tumor - blood; Carcinoma, Small Cell - diagnosis; E coli; Enzyme-Linked Immunosorbent Assay; Female; Fluoroimmunoassay; Fundamental and applied biological sciences. Psychology; Gastrin-Releasing Peptide - blood; Gastrin-Releasing Peptide - immunology; Humans; Immunization; Investigative techniques, diagnostic techniques (general aspects); Lung cancer; Lung Neoplasms - diagnosis; Medical sciences; Mice; Mice, Inbred BALB C; Monoclonal antibodies; Peptides; Phosphopyruvate Hydratase - blood; Polyethylene glycol; Protein Precursors - blood; Protein Precursors - immunology; Assay; Time resolution; Peptides; Clinical biology; Biochemistry; Automatic analysis; Molecular biology
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.101436

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. 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). 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 (13-13 500 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. We describe a sensitive and robust mAb-based TR-IFMA for proGRP. The assay is fully automated and displays high quality performance.