Investigation of monoclonal antibodies raised to human ovarian carcinoma cell lines
Blacoe-Masterson, Allan
(1998)
Investigation of monoclonal antibodies raised to human ovarian carcinoma cell lines.
PhD thesis, Dublin City University.
Monoclonal antibodies (MAb) were generated to multidrug resistant (MDR) and sensitive variants of the ovarian carcinoma cell line OAW42 which over-express the M D R associated protem LRP/MVP MAb 3/B6 was raised to the intrinsically resistant variant OAW42-SR Immunofluorescence and immunocytochemical analysis indicated that the antigen detected by MAb 3/B6 was expressed primarily on the external surface of the plasma membrane but was also found to be expressed in the cytoplasm of a series of human M D R cell lmes 3/B6 over-expression was associated primarily with cell lines which expressed the LRP/MVP.
3/B6 expression was studied in paraffin-embedded normal and malignant adult and in foetal tumour tissue. There was heterogeneous expression of the 3/B6 antigen and the LRP/MVP in normal adult and foetal kidney Low-level LRP/MVP expression was observed in 1/10 untreated malignant ovarian tumours while 3/B6 was absent. In two paired pre- and post-chemotherapy breast tumours sections, 3/B6 expression was observed in the post-chemotherapy sections only LRP/MVP expression was also observed in these sections.
A new commercially available immunoprécipitation protocol based on biotm labelling of cellular proteins was extensively modified and improved for this project. The MAb 3/B6 was found to immunoprecipitate a 115 kDa un-glycosylated protein Immunoprécipitation experiments with anti-rat vault polyclonal serum, N2 and purified rat vault protems mdicated that MAb 3/B6 and LRP-56 (the standard MAb used to detect LRP/MVP) did not cross-react Competitive immunocytochemical studies confirmed these results Incubation of OAW42-SR cells with MAb 3/B6 did not have any effect on adnamycm drug accumulation or cellular proliferation.
The antt-OAW42-SR MAb 5/C4 was also characterised by immunofluorescence and immunocytochemistry Results from these studies revealed that this MAb recognised a cytoplasmic antigen which migrated as 2 protein bands at 110 and 85 kDa by Western Blotting Further Western Blotting analysis indicated that MAb 5/C4 did not cross react with purified rat vault particles.
The anti-OAW42-S MAb 3/E3 was partially characterised by immunofluorescence and immunocytochemistry. There did not appear to be any significant difference in expression of this antigen m a panel of multidrug resistant cell lines. It was not possible to determine the molecular weigh of the antigen by Western Blotting or immunoprécipitation. This suggested that the epitope was destroyed during sample preparation.