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.

Analysis of CA125 antigen in normal human seminal plasma highlightsthe molecular heterogeneity of underlying glycosylated species

“…Although the occurrence of several low molecular mass CA125-immunoreactive species was reported for different sources, their origin is still not elucidated, in contrast to the high molecular mass species. They are supposed to result from auto/proteolytic activity and muc16 gene polymorphism, but there is also a suggestion that some of them are actually cross-reactive molecules (Weiland et al, 2012;McLemore et al, 2016).…”

Deciphering the Molecular Nature of Ovarian Cancer Biomarker CA125

“…Interestingly, one identified structure was identical to an unusual glycostructure expressed by uromodulin [7,45]. In light of our recent results these contradicting reports may arise from cross-reactivity of the M11-like and OC125-like antibodies with other proteins [41]. This may have lead to the characterization of proteins that were wrongly assigned as CA125.…”

Selection of small plasma peptides for the auxiliary diagnosis and prognosis of epithelial ovarian cancer by using UPLC/MS‐based nontargeted and targeted analyses

“…Clinical endpoints were determined according to the date of death from the disease or from the date of the last follow‐up. The levels of cancer antigen 125 ( CA125) in the plasma samples of both EOC and BOT patients were measured with an enzyme linked immunosorbent assay (ELISA) kit after admission . The recruitment and usage of all samples were approved by the ethical committee at the Affiliated Tumor Hospital of Harbin Medical University.…”

“…The levels of cancer antigen 125 (CA125) in the plasma samples of both EOC and BOT patients were measured with an enzyme linked immunosorbent assay (ELISA) kit after admission. 14 The recruitment and usage of all samples were approved by the ethical committee at the Affiliated Tumor Hospital of Harbin Medical University. Meanwhile, informed consent was obtained from all enrolled participants prior to this study.…”