It is mentioned that there should be two screening during the preparation of monoclonal antibody, however, many people are still puzzled about the aims and methods of the two screening. So, how to make it? The summary is as followed.
First screening, researchers should figure out those possible conditions that may occur while (effect) B lymphocytes and myeloma cells are hybridizing.
1.The fusion between (effect) B lymphocytes and (effect) B lymphocytes
2.The fusion between (effect) B lymphocytes and myeloma cells (namely hybridoma cells)
3.The fusion between myeloma cells and myeloma cells (namely tumor cells)
4.Single myeloma cells
5.Single (effect) B lymphocyte
Aim of the screening: obtaining the hybridoma cells
Accomplish the process by selective medium (HAT medium) because it contains hypoxanthine, aminopterin and thymine, wherein aminopterin can block the main approach of DNA synthesis. After the main approach has been blocked, on the basis of a emergency approach, namely under the affection of HGPRT (hypoxanthine-guanine phosphoribosyl transferase) and TK (thymidine kinase), DNA can be synthesized by using thymine and hypoxanthine. The most important is that both the two elements cannot be absent because only in this way can the synthesis be accomplished. Additionally, myeloma cell lines applied in the hybridization are made of those metobolism-deficient cells. No TK or HGPRT exists inside the cells. In this way, a single cell or fused myeloma cells will die in the HAT medium. In fact, although B cells possess HGPRT and TK, they cannot survive for a long term and proliferate under the external typical culture conditions, especially in a single cell environment. Therefore, only the hybridoma cells can grow and reproduce in HAT medium.
Then, according to the methods above, hybridoma cells can be selected. Whereas, although two cells are both hybridoma cells, they can still be produced under different antigenic determinants’ stimulation from the same antigen. Thus, the produced antibody is impure. Without further purification, it is thus obtained a polyclonal antibody. Therefore, a second screening is necessary.
Second screening, in order to obtain a monoclonal antibody, a cell group formed by the fission of a cell must obtained first because such antibody can really be regarded as the undoubted monoclonal antibody.
Aim of the screening: focusing on a particular hybridoma cells where the antigenic determinants can produce antibody, namely obtaining a cell group formed by the fission of a cell and the cell group is capable of producing the required antibody.
1.Separate the single cells and then place them into each foramina of the porous culture plates;
2.Test whether the cells in each foramina produced antibody of the injected antigen. Thus, the screening’ conditions include two connotations. Not all the cells in each foramina can produce antibody so that those cells cannot produce antibody should be eliminated. After that, continue to clone positive cells that can secrete antibody to against a certain antigenic determinants, trying to make sure that the necessary antibodies are in massive production.
3.Apply two ways to clone the selected cells. First way, they can be cultured in vitro. The second way is to extract the required monoclonal antibodies when the proliferation appears.
To be brief, the two screening should be conducted differently, because only in this way can the monoclonal antibody be extracted in a accurate way.