New Monoclonal Antibodies for a Selective Detection of Membrane-Associated and Soluble Forms of Carbonic Anhydrase IX in Human Cell Lines and Biological Samples

Monoclonal antibodies (MAbs) selectively targeting tumor-associated antigens such as carbonic anhydrase IX (CA IX) can significantly contribute to research, diagnostics, and treatment of CA IX-related cancers. CA IX is overexpressed in numerous hypoxic cancers where it promotes tumor progression. Therefore, it is considered as a promising tumor biomarker. A novel collection of MAbs against recombinant CA IX was developed and evaluated in different immunoassays for studying CA IX expression. The reactivity of MAbs with native cell surface protein was confirmed by flow cytometry and the presence of hypoxia-inducible CA IX was investigated in several human cancer cell lines. In addition, the applicability of MAbs for visualization of CA IX-positive tumor cells by immunofluorescence microscopy was demonstrated. MAb H7 was identified as the most promising MAb for different immunoassays. It recognized a linear epitope covering CA IX sequence of 12 amino acid residues 55-GEDDPLGEEDLP-66 within the proteoglycan domain. The MAb H7 was the only one of the collection to immunoprecipitate CA IX protein from cell lysates and detect the denatured CA IX with near-infrared fluorescence Western blot. It was also employed in sandwich enzyme-linked immunosorbent assay to detect a soluble form of CA IX in growth medium of tumor cells and blood plasma samples. The diagnostic potential of the MAb H7 was confirmed on formalin-fixed and paraffin-embedded tissue specimen of cervical carcinoma in situ by immunohistochemistry. The generated MAbs, in particularly clone H7, have great potential in diagnostics and research of CA IX-related cancers.