Hybridoma Technology



General description


Hybridoma technology makes it possible to obtain very large amounts of homogeneous antibodies and to replenish the suplly of the exact same antibodies whenever they are needed. The term hybridoma it's a hybrid cell line resulting due to a fusion of following two types of cells: an antibody producing lymphocyte cell (B) and a single myeloma cell (bone marrow tumour cell) which is capable of multiplying indefinitely. So the hybridoma have the antibody producing capability inherited from lymphocytes and have the ability to grow continuously (immortal) like malignant cancer cells.



The hybridoma technology involves the following steps:Montagem.jpg

1. Immunize a rabbit through repeated injection of a specific antigen for the production of a specific antibody, facilitated due to proliferation of the desired B cells;

2. Produce tumours in another mouse or a rabbit;

3. From the two animals, culture separately spleen cells (which are rich in B cells and T cells) that produce the specific antibodies, and myeloma cells that produce tumours. The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves and that they lack the enzyme HGPRT (hypoxanthine guanine phosphoribosyl transferase);

4. Induce fusion of spleen cells to myeloma cells, using PEG (polyethylene glycol) to produce hybridoma. It is performed by making the cell membranes more permeable. Then, the hybrid cells are grown in a selective HAT medium (hypoxanthine aminopterin thymidine). HAT allows selection of hybridoma cells. The medium contains a drug aminopterin, which blocks one pathway for nucleotide synthesis, making the cells dependent on another pathway that needs HGPRT enzyme (absent in myeloma cells). Therefore, only hybridoma will survive, since B cells lack tumorigenic property of immortal growth;

5. Rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. The hybridoma culture supernatant, secondary enzyme labelled conjugate, and chromogenic substrate are then uncubated. The formation of a coloured product indicates a positive hybridoma. Alternatively, immunochytochemical screening can also be used. Only one in several hundred cell hybrids will produce antibodies of the desired specificity;

6. Culture selected hybridoma cells for the production of monoclonal antibodies in large quantity. These hybridoma cells may be frozen for future use and may also be injected in the body of an animal so that antibodies will be produced in the body and can be recovered later for the body fluid.


The use of antibodies produced in animals, often leads to the creation of HAMA - Human Anti-Mouse Antibodies. So,nowadays this thecnology has evolved in order to become "more human and less animal". The new variants are Human HybridomaTechnology, Human Antibody Library, Trangenic Mouse Technology.


History


1975 - Discovery of Hybridoma Technology by Georges Kรถhler (from West Germany) and Cesal Milstein (from Argentina)
1984 - Nobel Prize for Physiology and Medicine, jointly with Neils Jerne (from Denmark)


Products and Companies


Here are some of the companys that produce antibodies from Hybridoma Technology
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References




By: Joana Pereira , 57901

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