Wang Chien-Wei, Srivastava Anubhuti, Hanson Eliza K, McEntee Caitlin R, Nair Trisha, March Jane C, Whelan Rebecca J
Department of Chemistry, University of Kansas, Lawrence, KS 66047, USA.
Cancers (Basel). 2025 Apr 26;17(9):1458. doi: 10.3390/cancers17091458.
: 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. : 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. : 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). : 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. : 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.
卵巢癌生物标志物CA125是一种肽表位，存在于粘蛋白MUC16的多个串联重复结构域中。尽管已经努力表征CA125与其临床使用抗体之间的相互作用，但CA125表位的分子性质仍不明确。MUC16分子模型的最新修订为全面表征CA125特异性抗体与MUC16串联重复区域之间的结合提供了机会。
本研究的目的是表征CA125抗体与MUC16表达的串联重复蛋白之间的结合，这是一项长期努力的一部分，旨在以氨基酸水平的精度鉴定CA125表位。
表达并纯化了16种MUC16串联重复蛋白。用高分辨率质谱确认了蛋白质表达。使用间接酶联免疫吸附测定（ELISA）和局部表面等离子体共振（SPR）测量了每种串联重复蛋白与四种CA125抗体（临床测试中使用的两种（OC125和M11）以及定义为OC125样和M11样的两种克隆）的结合相互作用。
虽然通过ELISA发现M11与所有16种测试的串联重复蛋白结合，但OC125不与16种重复中的5种结合。ELISA和SPR之间抗体的识别模式在很大程度上是一致的，在ELISA中观察到结合但在SPR中未观察到结合的情况可归因于SPR分析中接触时间不足。
可以推断，M11表位存在于所有测试的串联重复中，而OC125表位存在于较少的串联重复中。
