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KMID : 0371319930440040497
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Journal of the Korean Surgical Society
1993 Volume.44 No. 4 p.497 ~ p.507
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Immunofluorescent Monoclonal Antibody(AE1/Ae3) Detection of MCF-7 Human Breast Cancer Cells
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Abstract
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There have been many reports that an occult micrometastasis could be detected in the bone marrow of breast cancer patients by an indirect imunofluorescent assay using a epithelial specific moncolonal antiboies.
Some reports suggest that antikeratin moncolonalantibody may be more useful probe than anti-epithelial membrane antigen in the detection and confirmation of the metastatic breast cancer in the bone marrow by reducing the cross reactivity.
Immunofluorescence method could permit easy identification of tumor cells.
We used the cytokeratin monocolnal antibody(AE₁/AE₃) and FITC-conjugated antimouse antiserum as a primary and secondary antibody.
In order to detect the best combination f the concentration of priamry monclonal antibody and concentration of immunofluoescent dye to alleviated nonspecific staining and to minimize backgroud uptake.
We tested serial dilutions of antibody and fluorescene from 1: 20 to 1 : 150 respectively in this study.
We used an established human metastaic breast cancer cell line (MCF-7) mixed with normal bone marrow cells as the test specimen.
The background staining and cross reactivity were difference according to the concentration of primary and secondry antibody.
All the cytoplasm of the MCF-7 cells were stained but the cytoplasm of the normal bone marrow cells were not stained.
AE₁/AE₃Antibody decorated a network of cytoplamic fibers characteristic of keratins in the MCF-7 cells but not in normal bone marrow cells.
The most appropriate dilution of primary and secondary antibody were 1 : 40-75 and 1 : 75 respectively and the combination of 1 : 20 of primary antibody and 1 : 100-150 secondary antibody also showed a good staining.
The slide should be examined immediately or stored in the dark at 4℃for no more than 12 hours prior to examination with a fluorescence microscope, because the density of staining was weakened progressively as the times goes by.
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