Background: Before 1979 monoclonal antibodies were produced using heteroantiserums that randomly discovered a correct matching antibody to the specific antigen. This was seen through rosetting, a process in which antibodies form a flower pattern around a matching antigen present on a erythrocyte. Unfortunately, even when a correct antibody was found, only low titers could be produced, making them clinically irreverent. In this paper, a new technique is introduced that greatly improves the production of monoclonal antibodies, specifically looking at monoclonal antibodies for human T-cell surface antigens.
Methods:
- The hybridoma technique
- Immunofluorescence and Flow Cytometry
Results
- In order to create monoclonal antibodies, myeloma is fused with mouse spleen cells that contain the antigen of interest. At this time antibody producing plasma cells will bind with cancer plasma cells, and the rest of the plasma and cancer cells will dying. The remaining fusion cells are then cultured and capable of producing high titers of the specific monoclonal antibody of interest.
- To distinguish which antibodies produced bound to the correct T-cell of interest, fluorescent flow cytometry was utilized. Multiple T-cell types were introduced to the antibodies of interest with the assumption the antibody would bind to one T-cell type. This culture was then introduced to fluorescence that attached only to bound antibodies. Following, the culture was run through a flow cytometer and the fluorescent cells were characterized to evaluate antibody specificity to human T-cells. After this was done three different monoclonal antibodies were recognized to have specific binding to unique human T-cells.
~JI
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