Identification of a novel monoclonal antibody recognizing CD133
Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455, USA.Journal of immunological methods (Impact Factor: 1.82). 09/2010; 361(1-2):110-5. DOI: 10.1016/j.jim.2010.07.007
Human CD133 (prominin-1), a cell surface glycoprotein, is used as a marker of hematopoietic and neural stem cells. Antibodies that recognize a glycosylation-dependent CD133 epitope have been extensively used for enrichment of tumor initiating cells in a variety of cancers. These currently available antibodies are restricted for use in only a subset of biological assays. We have generated a novel anti-human CD133 monoclonal antibody, using a recombinant protein consisting of highly immunogenic amino acid residues selected from the native CD133 protein as an immunogen. The antibody (identified as clone 7) specifically recognizes the CD133 protein in a variety of immunological applications including Western blot, immunofluorescence, flow cytometry and immunohistochemistry. Further, clone 7 specifically recognizes an unmodified CD133 extracellular domain, and not its glycosylated epitope. In conclusion, the specificity and usefulness in a wide range of applications suggest that clone 7 could be a valuable tool to identify CD133 positive cells as well as to target them for therapy.
- "Subcellular Distribution of HC7 and AC133 Epitopes on Polarized and Mitotic Human HSPCs Recently, a novel anti-CD133 antibody has been described whose epitopes, unlike those of classical AC133 and AC141 anti-CD133 antibodies, were claimed to be insensitive to fixation (Swaminathan et al., 2010). Because of our interest in analyzing the subcellular distribution of CD133 on polarized and mitotic HSPCs, we decided to test for the usability of the HC7 antibody in human hematopoietic stem cell research. "
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- "Here we show that the cytofluorimetrical analysis with anti CD133 antibodies identifies, in the bulk of the cell population, a low basal CD133 expression, and in a small percentage of cells (2-3%), a much higher expression level, making this cell line useful to compare TNBC cells with different levels of CD133 expression. By using antibodies directed against different CD133 epitopes [33,34] and Tunicamycin we ruled out the potential bias arising from variable glycosylation levels and from glycosylation-dependent epitopes in the extracellular portion of CD133 that it was reported to be potentially lost upon differentiation of tumor cells . We also extended the analysis to intracellular CD133 that allowed to definitely confirm the existence, in MDA-MB-231 cells, of a small but stable subpopulation expressing high levels of CD133 in both membrane and cytoplasm compartments. "
ABSTRACT: Beyond its possible correlation with stemness of tumor cells, CD133/prominin1 is considered an important marker in breast cancer, since it correlates with tumor size, metastasis and clinical stage of triple-negative breast cancers (TNBC), to date the highest risk breast neoplasia. To study the correlation between the levels of CD133 expression and the biology of breast-derived cells, CD133low and CD133high cell subpopulations isolated from triple negative MDA-MB-231 cells were compared in terms of malignant properties and protein expression. High expression of CD133 characterizes cells with larger adhesion area, lower proliferation rate and reduced migration speed, indicative of a less undifferentiated phenotype. Conversely, when compared with CD133low cells, CD133high cells show higher invasive capability and increased expression of proteins involved in metastasis and drug-resistance of breast tumors. Among the signalling proteins examined, PLC-beta2 expression inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion, suggesting that, in TNBC cells, the de-regulation of this PLC isoform is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133. Since CD133 plays a role in determining the invasiveness of CD133high cells, it may constitute an attractive target to reduce the metastatic potential of TNBC. In addition, our data showing that the forced up-regulation of PLC-beta2 counteracts the invasiveness of CD133-positive MDA-MB-231 cells might contribute to identify unexplored key steps responsible for the TNBC high malignancy, to be considered for potential therapeutic strategies.
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ABSTRACT: CD133, also known as Prominin-1, is expressed on stem cells present in many tissues and tumors. In this work, we have identified and characterized a single-chain variable fragment (scFv) for the efficient and specific recognition of CD133. Phage display was used to develop the scFv from a previously reported anti-CD133 hybridoma clone 7, which was capable of recognizing both glycosylated and non-glycosylated forms of human CD133. The scFv immunostained CD133+ Caco-2 cells, but not CD133−/low U87 cells. Significantly, it immunostained CD133− cells transiently transfected with the mouse CD133 gene as well as CD133+ mouse cells. Co-immunostaining studies in mouse bone marrow cells, using anti-CD133 scFv-FITC and anti-mouse CD133-PE (clone 13A4) commercial antibody, indicated that the epitopes recognized by these reagents partially overlap. Taken together, these results suggest that the scFv can recognize mouse CD133 protein in addition to recognizing human CD133. This new scFv is expected to be valuable both as a molecular diagnostic reagent for identifying CD133+ cells and as a ligand for targeting therapeutics to CD133+ tumor-initiating cells.
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