Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16

Background: The ovarian cancer biomarker CA125 is a peptide epitope found in multiple tandem repeat domains of the mucin MUC16. Although efforts have been undertaken to characterize the interaction between CA125 and its clinically used antibodies, the molecular nature of the CA125 epitope(s) remains undefined. A recent revision of the molecular model of MUC16 provides an opportunity to fully characterize the binding between CA125-specific antibodies and the tandem repeat region of MUC16. Objectives: The objective of this study was to characterize the binding between CA125 antibodies and expressed tandem repeat proteins from MUC16 as part of a longer-term effort to identify the CA125 epitopes with amino-acid-level precision. Methods: Sixteen MUC16 tandem repeat proteins were expressed and purified. Protein expression was confirmed with high-resolution mass spectrometry. The binding interaction of each tandem repeat protein with four CA125-antibodies-the two used in the clinical test (OC125 and M11) and two clones defined as OC125-like and M11-like-was measured using indirect enzyme-linked immunosorbent assay (ELISA) and localized surface plasmon resonance (SPR). Results: Whereas M11 was found by ELISA to bind to all 16 tandem repeat proteins tested, OC125 does not bind to 5 of the 16 repeats. The recognition pattern of the antibodies was largely in agreement between ELISA and SPR, and cases in which binding is observed in ELISA but not in SPR can be attributed to insufficient contact time in SPR analysis. Conclusions: It can be inferred that the M11 epitope is present on all tandem repeats tested, whereas the OC125 epitope is present on fewer tandem repeats.