Conjugates were diluted with PBS-T buffer in a series from 1:100 to 1 1:12,800 by the dilution factor 2, adding 100 L of each dilution per well and incubated for 1 h at RT
Conjugates were diluted with PBS-T buffer in a series from 1:100 to 1 1:12,800 by the dilution factor 2, adding 100 L of each dilution per well and incubated for 1 h at RT. 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 PCI-34051 research of CA IX-related cancers. Keywords: monoclonal antibodies, human carbonic anhydrase IX, immunoassays, cancer biomarkers 1. Introduction Cancer encompasses serious diseases which are characterized by changes in the cell condition emerged from the alterations in expression of various genes and their coding proteins. Relatively unlimited and uncontrolled proliferation along with improved survival potential of cancer cells disturbs normal homeostasis of various tissues and organs leading to their failure, which deteriorates the life quality of cancer patients and, in many cases, leads to death [1,2,3]. Cancer-specific proteins that are affected by or contribute to malignant transformation are monitored to provide insights for early Rabbit Polyclonal to Histone H2A (phospho-Thr121) cancer detection, differentiation of cancer stages, patient prognosis determination, prediction of response to therapy, and treatment selection [4,5,6]. The growing demand for the advanced technologies and novel biological tools for screening and treatment of cancer patients caused intensive development in fields of immunodiagnostic and immunotherapy [7,8]. Monoclonal antibodies (MAbs) developed to recognize tumor-specific antigens are considered as core agents in many diagnostic methods such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), or positron-emission tomography to analyze serum, saliva, sputum, urine and tissues both ex vivo and in vivo [9,10,11]. Antibody-based immunotherapy use antibodies or their derivatives as mediators between the cancer antigen and the toxic substance, such as chemotherapy drugs or radioactive particles, or components of the hosts immune system capable of destroying the cancerous cell [12]. The major difficulty of antibody-based cancer diagnostics or therapy relates to the identification and validation of reliable tumor antigens. Studies are focusing on proteins PCI-34051 expressed entirely by tumor cells with no or minimal expression in normal tissues to overcome ambiguous or insignificant results when considering identification of pathology as well as emergence of off-target toxicities in case of immunotherapy [7,8]. Two dimeric transmembrane isozymes of carbonic anhydrase (CA) family carbonic anhydrase IX (CA IX) and carbonic anhydrase XII (CA XII) are involved in carcinogenesis by contributing to extracellular pH regulation under hypoxia conditions thus promoting tumor cell growth and survival. Active centers of both cancer-associated CAs are PCI-34051 directed to the outside of the cell which is the most important feature to apply antibody-based detection or therapy [13,14,15]. The expression pattern of CA XII is less appropriate in the context of immunotherapy, as this enzyme is found in various normal tissues [16]. In contrast, CA IX is present only in normal small intestine, pancreas, and gastric and biliary mucosa, while it is overexpressed in many tumors like renal, bladder, colon, breast, cervix, ovary, head and neck, or lung [17,18,19]. The significance of CA IX in malignant processes, its diverse expression in normal and cancerous tissues and external localization in cells makes CA IX an attractive tumor antigen, which diagnostic and therapeutic relevance is under investigation [4,14,20]. First MAbs against CA IX were generated using tumor cells as an immunogen without identifying the target. Mice were immunized with cell homogenates of either primary renal cell carcinoma lesions, which resulted in the MAb G250 [21], or cervical adenocarcinoma cell line (HeLa) cells which resulted in the MAb M75 [22]. It was not until 1996 that the antigen recognized by the MAb M75 was demonstrated to be CA IX [23,24]. Four.