| Breast cancer is one of the most common cancers, so it is very significance to be diagnosed early. Currently, molecular diagnosis of breast cancer, especially the diagnosis based on monoclonal antibody is attracting extensive attention for its good targeting and high specificity. MUC1 is an important tumor-associated markers, because it is a kind of polymorphic epithelial mucins which can be highly expressed in some cancers as breast cancer. However, currently MUC1 monoclonal antibody has not been reported in China. Therefore, it will lay the foundation for the clinical application of MUC1 to prepare an anti-MUC1 monoclonal antibody with high specificity and compatibility, coupled with Gold Mag nanoparticles, to be used in clinical and basic research on living molecular imaging of breast cancer.ObjectiveThis paper will study the preparation, identification and preliminary application of anti-MUC1 monoclonal antibody and nanoprobes. The whole experiment process can be divided into two parts: 1. To prepare and identify the anti-human MUC1 monoclonal antibody(m Ab) and to detect its application on immunohistochemistry of human breast cancer; 2. To prepare and identify the nanoprobes of targeting MUC1 monoclonal antibody, and to examine the feasibility on preparation of molecular imaging of breast cancer.Method1. Synthetic MUC1 repetitive sequence short peptide coupled with KLH protein was served for antigen to immunize Balb/c mice, cell lines which were able to secreting monoclonal antibodies stably were screened by hybridoma cell fusion technique. The prepared monoclonal antibodies were identified and analyzed by ELISA, Western blot, flow cytometry and immunofluorescence test. The antibodies were applied in the detection of clinical human breast cancer, benign fibro adenoma and normal breast tissue. 2. MUC1 specific MR molecular probe MUC1@Gold Mag was constructed using conjugating anti-human MUC1 m Ab with Gold Mag nano particles, and the coupling efficiency was calculated. The specific binding capability for MUC1@Gold Mag to breast cancer cells was detected using laser confocal microscopy, transmission electron microscope and flow cytometry. Gold Mag-CS nanop articles solution in different concentration was scanned to identify the lowest concentration that can be detected with 3.0T MR system. Breast cancer cel1 line MCF-7 and hepatic cell line Hep G-2 were cultured(the later was control group) and nude mice models were established. The two cell lines were cross-matched with MUC1@Gold Mag and non-related Ig G@Gold Mag.T2 weighted images were obtained and the signal intensity was measured in each group. After conventional scan, T2 weighted images were obtained after the probe was injected into nude mice model through caudal vein 6, 12 and 24 h later respectively. Statistical analyses were performed to assess the statistical differences of tumor signal intensity.Results1. Four hibridoma cell lines secreting specific anti-MUC1 m Abs were obtained, the first antibody of which got the strongest responsiveness. The titers were above 1:107. Antibody subtypes 3 strains of Ig G1, 1 strain of Ig M was identified, and the light chain of which was categorized into chain κ. The m Ab could specifically bind to the MCF-7 cells, which was verified by Western blot, flow cytometry analysis, fluorescence immunoassay and immunohistochemistry test. Significant differences of expression(P<0.05) of the detection of MUC1 monoclonal antibodies were found in breast cancer tissue, fibro adenoma and normal breast tissues. 2. MUC1 specific MR molecular probe MUC1@Gold Mag was successfully constructed. The results of laser confocal microscopy transmission electron micro scope and flow cytometry proved that muc1@Gold Mag could specific bind with MCF-7 cells. T2W1 signal intensity of Gold Mag-CS was significantly lower than that of agarose gel even if the former was diluted by 640 times. MUC1@Gold Mag could specifically decrease T2W1 signal intensity of MCF-7 cells in vitro. MUC1@Gold Mag was injected into nude mice model through caudal vein 6 h, 12 h and 24 h later. T2W1 signal intensity of MCF-7 tumor was significantly lower than that of conventional scan(P<0.05). The signal intensity of control groups did not show significant difference.Conclusion1. The mAb against MUC1 was successfully prepared, which could promote further research on breast cancer early diagnosis and targeted therapy. 2. MUC1 specific MR molecular probe is successfully constructed by conjugating anti-human MUC1 m Ab with Gold Mag nano particles, which can specifically bind with MCF-7 cell and has target-directed enhancement effect in MCF-7 cell line in vitro and in nude mice model grafted with MFC-7 in vivo using a clinical 3.0T MR scanner. |
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