title: A quantitative immunoassay for lung cancer biomarker CIZ1b in patient plasma.
abstract: OBJECTIVE
  Non-invasive tests for early detection of lung cancer are an important unmet clinical need. CIZ1b plasma biomarker can discriminate stage 1 lung cancer from within high-risk groups with clinically useful accuracy, with ROC AUCs in excess of 0.9 for two independent retrospective cohorts, and could therefore meet this need. Our aim was to characterise the native state of the biomarker and develop a quantitative immunoassay.
  
  
  METHODS
  Selective denaturation, preparative electrophoresis and mass spectrometry of human plasma were used to characterise the biomarker and interaction partners. A sandwich ELISA was generated, and specificity for CIZ1b biomarker tested on lung cancer patient plasma.
  
  
  RESULTS
  CIZ1b biomarker is a denaturation-resistant complex between a C-terminal fragment of CIZ1 bearing the CIZ1b epitope specified by alternative splicing of exon14, and fibrinogen alpha chain. Reconstitution of the biomarker epitope with purified fibrinogen and CIZ1b, but not CIZ1a (non-alternatively spliced exon 14) confirmed the specificity of the results. The endogenous complex is highly stable in lung cancer plasma and can be quantified by pairing of a CIZ1b exon-junction specific antibody with detection of fibrinogen. Application of this sandwich ELISA to a prospectively collected development set of plasmas reveals the same level of accuracy as the western blot used to validate the discriminatory capability of the biomarker.
  
  
  CONCLUSIONS
  Unexpected and unusual molecular structure of CIZ1b in native plasma has complicated immunoassay design, and delayed translation of this promising biomarker. However, CIZ1b can now be measured using a high-throughput, hospital-friendly sandwich ELISA format, overcoming an important barrier to further clinical development and application of this blood test for early stage lung cancer.
authors.affiliation: Cizzle Biotech, University of York, YO10 5DD, UK; Department of Biology, University of York, YO10 5YW, UK. Electronic address: dc17@york.ac.uk.
authors.forename: Dawn
authors.initials: D
authors.lastname: Coverley
authors.affiliation: Cizzle Biotech, University of York, YO10 5DD, UK; Department of Biology, University of York, YO10 5YW, UK.
authors.forename: Gillian
authors.initials: G
authors.lastname: Higgins
authors.affiliation: Cizzle Biotech, University of York, YO10 5DD, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: West
authors.affiliation: Department of Biology, University of York, YO10 5YW, UK; Hull-York Medical School, University of Hull, HU6 7RX, UK.
authors.forename: Oliver T
authors.initials: OT
authors.lastname: Jackson
authors.affiliation: Department of Biology, University of York, YO10 5YW, UK.
authors.forename: Adam
authors.initials: A
authors.lastname: Dowle
authors.affiliation: Department of Biology, University of York, YO10 5YW, UK.
authors.forename: Aidan
authors.initials: A
authors.lastname: Haslam
authors.affiliation: Cizzle Biotech, University of York, YO10 5DD, UK; Department of Biology, University of York, YO10 5YW, UK.
authors.forename: Eve
authors.initials: E
authors.lastname: Ainscough
authors.affiliation: Cizzle Biotech, University of York, YO10 5DD, UK; Department of Biology, University of York, YO10 5YW, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Chalkley
authors.affiliation: Department of Respiratory Medicine, York Teaching Hospital NHS Foundation Trust, YO31 8HE, UK.
authors.forename: John
authors.initials: J
authors.lastname: White
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D019540:Area Under Curve; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D015153:Blotting, Western; D016022:Case-Control Studies; D002853:Chromatography, Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008175:Lung Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D009687:Nuclear Proteins / Q000097:blood* / Q000235:genetics; D011446:Prospective Studies; D012189:Retrospective Studies; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; C400423:Ciz1 protein, human; D009687:Nuclear Proteins
keywords: Biomarker; Blood test; CIZ1b; Immunoassay; Lung cancer