title: Methods for identification of CA125 from ovarian cancer ascites by high resolution mass spectrometry.
abstract: CA125 is the most widely used tumour marker in ovarian cancer with unsatisfactory sensitivity and specificity especially at early stage. It is quantified by antibody-based immunoassays; however different molecular weight isoforms have been described in the literature which have never been validated by mass spectrometry, potentially affecting the diagnostic accuracy and clinical reliability of the test. In this study, CA125 was detected by Western blot and its identity confirmed by mass spectrometry. Two-dimensional (2D) gel electrophoresis in combination with mass spectrometry revealed that positive Western blot signals up to 500 kDa are most likely false-positive interactions of M11-like and OC125-like antibodies. Fibronectin, identified as one of these false-positive interaction partners, increased the reading for CA125 in a first generation ELISA significantly (p = 0.02). The existence of low-molecular weight isoforms of CA125 is therefore questionable and is most likely reflecting cross-reactivity of the antibodies with other proteins. This would explain the conflicting reports on the molecular structure of CA125 and also the inconsistency of CA125 levels by different ELISAs. Our results are also the first steps towards a mass spectrometric assay for CA125 quantification, which would improve sensitivity and reliability.
authors.affiliation: Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Florian
authors.initials: F
authors.lastname: Weiland
authors.affiliation: Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Fritz
authors.affiliation: Research Centre for Reproductive Health, Robinson Institute, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Martin K
authors.initials: MK
authors.lastname: Oehler
authors.affiliation: Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Hoffmann
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000097:blood*; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000097:blood* / Q000175:diagnosis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D018394:CA-125 Antigen