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Published on: February 2, 2009
A new monoclonal antibody for specific immunocytochemical staining of nucleoli
C J Vissers1, C C Flohil, A A de Jong
1Department of Pathology, Erasmus University Rotterdam, The Netherlands.
Insights
Researchers identified a novel monoclonal antibody (1E10) that specifically targets a unique RNA epitope within the nucleoli of various cell types. This antibody shows promise for nucleolar research, particularly in cancer cells with enlarged nucleoli.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- The nucleolus is a critical cellular organelle involved in ribosome biogenesis and other nuclear functions.
- Specific markers for nucleolar components are essential for studying nucleolar structure and function, especially in disease states like cancer.
Purpose of the Study:
- To isolate and characterize a monoclonal antibody that specifically recognizes nucleoli.
- To determine the nature and localization of the target epitope recognized by the antibody.
Main Methods:
- Hybridoma technology to generate monoclonal antibody (clone 1E10).
- Immunofluorescence microscopy on cryostat sections of unfixed tissues.
- Immunoelectron microscopy for ultrastructural localization.
- Enzymatic and chemical treatments (DNase, pronase, trypsin, acid hydrolysis, RNase) to characterize the epitope.
- Western blotting and spot blotting to assess epitope nature.
Main Results:
- Monoclonal antibody 1E10 (IgM, k) specifically binds to nucleoli in various cell types, with enhanced reactivity in neoplastic cells.
- The epitope is sensitive to RNase digestion and acid hydrolysis but resistant to proteases and DNase, indicating it is RNA-based.
- Immunoelectron microscopy localized the epitope to the granular component of the nucleolus.
- Western blot analysis confirmed the epitope is not proteinaceous.
Conclusions:
- Clone 1E10 antibody recognizes a conformational RNA epitope exclusively found in the nucleolus.
- This antibody is a valuable tool for studying nucleolar components and may have diagnostic potential, especially for cancers.
- The epitope's resistance to standard tissue processing (paraffin embedding) and its specific nucleolar localization make it a unique marker.
Abstract:
We have isolated a hybridoma cell line (clone 1E10) producing a monoclonal antibody which specifically recognizes nucleoli. The antibody (IgM, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sections of unfixed tissues. Paraffin embedding destroyed the epitope. Tissue specificity or species specificity was not observed. Nucleoli in neoplastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectron-microscopy (using a pre-embedding as well as a post-embedding technique) confirmed the specific nucleolar localization of the immunoreactivity. Immunoreactivity was confined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNase, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific signal was obtained, which supports the non-protein nature of the epitope. Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin orange confirmed the RNA nature of the epitope. In spot blots, immunoreactivity was not found with tRNA or mRNA. These observations indicate that 1E10 recognizes a conformational RNA epitope which occurs only in the nucleolus.
