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

Abstract

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.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells

“…The third column in Fig. 3 shows cell staining with the monoclonal CAIX antibody H7 31 . Staining by GZ19-32 perfectly co-localized with the staining by the antibody (fourth column).…”

“…Cells were fixed by submerging glass coverslips into 20 ℃ methanol for at least 5 min. To visualize CAIX, live cells were rehydrated and stained using H7 antibody 31 diluted 1:200 in PBS, supplemented with 0.05% Tween-20 (PBS-Tw) in 37 ℃ incubator for 30 min. After washing 3 × 10 min with PBS-Tw at room temperature (RT), cells were incubated with secondary Alexa Fluor 488-conjugated anti-mouse antibodies (Cat # A-11034, ThermoFisher), dilution 1:200 in PBS-Tw, in 37 ℃ incubator for 30 min.…”

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies

“…ELISA is suitable for large-scale clinical epidemiological investigations [ 31 ]. Antibodies are considered one of the most effective reagents for developing sensitive and specific ELISA [ 32 , 33 ]. Based on the advantage of easy genetic manipulation of nanobody, K205R protein-specific Nb-HRP fusion protein was used as a probe to develop immunoassays for ASFV antibody detection in this research.…”

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

“…Antibodies are considered one of the most effective biomolecules to be used for detection methodologies and applications [42][43][44]. Due to their particular characteristics, such as high affinity and specificity, a large variety of immunoassay formats implementing antibodies as reagents have been developed for disease diagnoses.…”