Production, Establishment and Characterisation of Monoclonal Antibody Against Breast Cancer Cell Line (Mcf-7)
Ever since hybridoma technology was introduced by Kohler and Milstein in the 70's, numerous efforts have been undertaken to produce monoclonal antibodies (MAbs ) against mammary cancer cells. However, even to this day, all the MAbs produced still possess cross-reactivities toward other type...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English English |
| Published: |
1995
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8364/1/FSMB_1995_2_A.pdf |
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| Summary: | Ever since hybridoma technology was introduced by Kohler
and Milstein in the 70's, numerous efforts have been undertaken
to produce monoclonal antibodies (MAbs ) against mammary cancer
cells. However, even to this day, all the MAbs produced still
possess cross-reactivities toward other types of cancerous cells
and also normal mammary cells.
In this study, the breast cancer eel} line MCF-7 was used as
an immunogen to raise MAb against mammary cancer cells. Fusion
between lymphocytes sensitised with MCF-7 cell line and myeloma
cells, SP2, was performed using 50% of polyethylene glycol (PEG).
The hybridoma secreting MAb against MCF-7 cell line was selected
using cell-ELISA technique. Limiting dilution of five times was
performed to yield a stable hybridoma clone secreting the MAb.The selected clone, C2E7, which secreted MAb of the Igt-1 class and lamda light chains was chosen for further studies.
MAb secreted by C2E7 was found to react with an antigenic
determinant located in the cytoplasm of the MCF-7 cell line.
Immunocytochemical studies showed that apart from the MCF-7 cell
line, the anti genic determinant was also present in mammary
cancer cell line of T47-D. Weak cross-reactivities were also
observed against cell lines Panc-l and Ova-3. Immunohistochemical
studies using the immunoperoxidase technique showed that staining
occurred in the cytoplasmic region of all mammary lobular
carcinoma and 90% of mammary ductal carcinoma examined. Staining
was also found in 50% of mammary fibroadenoma cases studied. On
the contrary, no staining of tissues was found in uterine
leiomyoma, stomach showing intestinal metaplasia, cervical
carcinoma, tonsillitis, neurofibroma, ductal papilloma of the breast
and normal mammary tissues. Biochemical studies showed that the
antigenic determinant on the MCF-7 cell line with reactivity
towards MAb C2E7 was composed of endopeptide chain having
arginine and lysine as the side chains, and possessed a specific
conformational order which was disrupted when the determinant
was electrophoresed on SDS-PAGE. Consequently, characterisation
of the determinant using Western Blotting technique could not
be performed. The hybridoma clone C2E7 was able to grow and proliferate
in serum-free medium of EDRF supplemented with ITES.
Purification technique using a combination of ammonium sulphate
precipitation and gel filtration on Sepharose 6B enabled the
separation of IgM from MAb secretion of C2E7 hybridomas cultured
in serum-free medium. |
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