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.
Highlights
Cancer antigen 125 (CA125), known as mucin-16 (MUC16), is the most widely used tumour marker in ovarian cancer, and considered the “gold standard”
The reliability of CA125 detection by probing with antibodies is challenged as we identified several antigens interacting with the M11-like and OC125-like antibody resulting in false-positive signals
The results of this study suggest that experimental findings involving the identification and quantification of CA125 should be viewed with caution when they are based on antibody probing alone and have not been verified by detailed mass spectrometric analysis
Summary
Cancer antigen 125 (CA125), known as mucin-16 (MUC16), is the most widely used tumour marker in ovarian cancer, and considered the “gold standard”. Its role for screening and early detection of ovarian cancer is limited due to a low sensitivity and specificity. It is used for diagnostic purposes in combination with other methods such as transvaginal ultrasonography [1]. CA125 was first detected by Bast et al in 1981 using a murine monoclonal antibody purified following immunization of mice with a human ovarian cancer cell line [2]. This antibody reacted with the majority of ovarian carcinoma cells and appeared to be nonreactive with non-malignant tissues
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