12-31-2013, 03:02 AM
Specialized methods for monoclonal antibody production: (Decoy Method)
To study the embryonic stem cell and their differentiation pattern, it is very impotent to understand the proteins which are involve in the differentiation and to know that what are the different proteins between differentiated and non differentiated embryonic cells.
There is a method called “Decoy method” of producing the monoclonal antibody. This method will produce the antibody for specific protein which is unique in differentiated and non differentiated embryonic cells. For this method, you need group of naive Balb/c mouse (5-6 numbers) which will be immunized with differentiated embryonic cells in one hind foot-pad (Left) and with Non-differentiated embryonic cells in other side (Right) of hind foot-pad.
Differential immunization of these two types of embryonic cells will neutralize all the protein candidates which are common in these two type of cells and antibody response will be only for unique proteins.
Immunization need to be done at every 3 days with 10000 cells (In Phosphate buffer) of each type and for 0-24 days (9 immunizations). After last immunization, Fusion of lymph node cells (Popliteal) need to be done with sp2/0 (Myeloma) cells as routine monoclonal antibody production protocol.
Note: As immunization is done in foot-pad it is important to have very less volume for immunization. While fusion with myeloma cells, left and right lymph cells need to be fused separately.
Obtained monoclonal antibodies need to be screened on differentiated and non differentiated embryonic cells. This method gives best way to discover embryonic marker and production of antibody for them simultaneously.
This is highly advance method to produce antibodies for embryonic markers and can also be applied to search for the cancer markers. Because the fusion efficiency are low with lymph node cells this techniques in not very popular as a routine method for production of monoclonal antibodies, though it is less time consuming in comparison to routine methods.
To study the embryonic stem cell and their differentiation pattern, it is very impotent to understand the proteins which are involve in the differentiation and to know that what are the different proteins between differentiated and non differentiated embryonic cells.
There is a method called “Decoy method” of producing the monoclonal antibody. This method will produce the antibody for specific protein which is unique in differentiated and non differentiated embryonic cells. For this method, you need group of naive Balb/c mouse (5-6 numbers) which will be immunized with differentiated embryonic cells in one hind foot-pad (Left) and with Non-differentiated embryonic cells in other side (Right) of hind foot-pad.
Differential immunization of these two types of embryonic cells will neutralize all the protein candidates which are common in these two type of cells and antibody response will be only for unique proteins.
Immunization need to be done at every 3 days with 10000 cells (In Phosphate buffer) of each type and for 0-24 days (9 immunizations). After last immunization, Fusion of lymph node cells (Popliteal) need to be done with sp2/0 (Myeloma) cells as routine monoclonal antibody production protocol.
Note: As immunization is done in foot-pad it is important to have very less volume for immunization. While fusion with myeloma cells, left and right lymph cells need to be fused separately.
Obtained monoclonal antibodies need to be screened on differentiated and non differentiated embryonic cells. This method gives best way to discover embryonic marker and production of antibody for them simultaneously.
This is highly advance method to produce antibodies for embryonic markers and can also be applied to search for the cancer markers. Because the fusion efficiency are low with lymph node cells this techniques in not very popular as a routine method for production of monoclonal antibodies, though it is less time consuming in comparison to routine methods.
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