Weiland Florian, Fritz Katarina, Oehler Martin K, Hoffmann Peter
Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
Research Centre for Reproductive Health, Robinson Institute, University of Adelaide, Adelaide 5005, Australia.
Int J Mol Sci. 2012;13(8):9942-9958. doi: 10.3390/ijms13089942. Epub 2012 Aug 9.
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.
CA125是卵巢癌中使用最广泛的肿瘤标志物，但其敏感性和特异性并不理想，尤其是在疾病早期。它通过基于抗体的免疫测定进行定量；然而，文献中描述了不同分子量的异构体，但从未通过质谱法进行验证，这可能会影响检测的诊断准确性和临床可靠性。在本研究中，通过蛋白质印迹法检测CA125，并通过质谱法确认其身份。二维（2D）凝胶电泳结合质谱分析表明，高达500 kDa的蛋白质印迹阳性信号很可能是M11样抗体和OC125样抗体的假阳性相互作用。纤连蛋白被确定为这些假阳性相互作用伙伴之一，它在第一代酶联免疫吸附测定（ELISA）中显著增加了CA125的读数（p = 0.02）。因此，CA125低分子量异构体的存在值得怀疑，很可能反映了抗体与其他蛋白质的交叉反应。这可以解释关于CA125分子结构的相互矛盾的报道，以及不同ELISA检测CA125水平的不一致性。我们的研究结果也是迈向用于CA125定量的质谱测定法的第一步，这将提高检测的敏感性和可靠性。
