MLP a novel Mucin-like cancer biomarker

Malignant ovarian tumours pose a significant risk to women all over the world. Ovarian cancers are the fifth most common cancers among women in the UK. Mortality due to ovarian malignancies is unacceptably high and the disease is often called a “silent killer” as the early stages of ovarian malignancies present with a little to no symptoms. Ovarian cancer is therefore mostly diagnosed at a very late stage of tumour progression when treatment is hampered by metastastatic spread of the primary tumour. Ovarian tumours express and release a previously undescribed novel mucin-like protein (MLP), which is absent in normal ovarian tissue or benign ovarian cysts (Schwaeble, W and Walker, R unpublished results). This novel mucin-like protein is a glycoprotein composed of a protein backbone, which is highly glycosylated with O-linked oligosaccharides. The high degree of glycosylation accounts for most the molecular weight of the native glycoprotein. The aim of this project was to develop a reliable diagnostic assay for the early and non-invasive detection of ovarian malignancies using specific monoclonal antibodies for MLP detection. The early detection of ovarian malignancies poses a so far unmet clinical necessity. The presently used tumour-associated biomarkers (mainly based on the relatively non-specific tumour marker CA-125) unfortunately all fail to detect ovarian malignancies at an early stage. Recombinant MLP was produced in a bacterial expression system, purified, analysed by Western blotting and used to raise polyclonal antibodies for the development of an MLP- specific sandwich ELISA. Eight different hybridomas producing mouse monoclonal antibodies against recombinant human MLP were also grow up and supernatant tested. Three of the eight monoclonal antibodies were shown to recognise native, glycosylated MLP as well as the recombinant antigen. These three were characterised in detail by ELISA, immunohistochemical staining of tissue sections and Western blot analysis. Preliming experiments were performed using the three monoclonal antibodies and the goat anti-MLP antiserum to investigate the properties of the antibodies and their sutability for establishing a sandwish ELISA able to detect the presence of MLP in serum for the development of a test for the early diagnosis of ovarian cancer.