title: Automated time-resolved immunofluorometric assay for progastrin-releasing peptide.
abstract: 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 (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.
  
  
  CONCLUSIONS
  We describe a sensitive and robust mAb-based TR-IFMA for proGRP. The assay is fully automated and displays high quality performance.
authors.affiliation: Department of Medical Biochemistry at Radiumhospitalet, Rikshospitalet Medical Centre, Oslo, Norway. marisno@ulrik.uio.no
authors.forename: Marianne S
authors.initials: MS
authors.lastname: Nordlund
authors.forename: David J
authors.initials: DJ
authors.lastname: Warren
authors.forename: Kjell
authors.initials: K
authors.lastname: Nustad
authors.forename: Johan
authors.initials: J
authors.lastname: Bjerner
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Paus
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001322:Autoanalysis; D014408:Biomarkers, Tumor / Q000097:blood; D018288:Carcinoma, Small Cell / Q000175:diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015200:Fluoroimmunoassay; D019886:Gastrin-Releasing Peptide / Q000097:blood* / Q000276:immunology; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis; D051379:Mice; D008807:Mice, Inbred BALB C; D010751:Phosphopyruvate Hydratase / Q000097:blood; D011498:Protein Precursors / Q000097:blood* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D011498:Protein Precursors; D019886:Gastrin-Releasing Peptide; D010751:Phosphopyruvate Hydratase