MONOCLONAL ANTIBODY PRODUCTION BY HYBRIDOMA TECHNOLOGY

A hybridoma, is produced by the injection of a specific antigen into a mouse, procuring the antigen-specific plasma cells (antibody-producing cell) from the mouse and the subsequent fusion of this cell with a cancerous immune cell called a myeloma cell. 
The hybrid cell, which is thus produced, can be cloned to produce many identical daughter clones. These daughter clones then secrete the immune cell product. Since these antibodies come from only one type of cell (the hybridoma cell) they are called monoclonal antibodies. The advantage of this process is that it can combine the qualities of the two different types of cells; the ability to grow continually, and to produce large amounts of pure antibody. 
HAT medium (Hypoxanthine Aminopetrin Thymidine) is used for preparation of monoclonal antibodies. 
Laboratory animals (eg. mice) are first exposed to an antigen to which we are interested in isolating an antibody against. Once isolated from the mammal, the B cells are fused with immortalized myeloma cells - which lack the HGPRT(hypoxanthine-guanine phosphoribosyltransferase) gene - using polyethylene glycol or the Sendai virus. 
Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin in the myeloma cells die, as they cannot produce nucleotides by the de novo or salvage medium blocks the pathway that allows for nucleotide synthesis.

Note-Because of their high specificity, monoclonal antibodies are useful in diagnosing microbial diseases.

MCQs.
1.  In hybridoma technology ,the myeloma cells used lack 
a.HGPRTase
b.the ability to prduce Ig
c.both HGPRT ase and ability to produce Ig
d.thymidine kinase 

ans. c
2.Monoclonal antibodies are -
a.heterogenous antibodies
b.Homogenos antibodies produced from single close of plasma cells
c.Both a and b 
d.None of these

ans.b

3.Hybridoma technology was developed by -
a.Beadle and tatum
b.Francis crick
c.Jacob and monad
d.kohler and milstein

Ans.d

4.

In monoclonal antibody technology, tumor cells that can replicate endlessly are fused with mammalian cells that produce an antibody. The result of this cell fusion is a

A. hybridoma B. myeloma C. natural killer cell D. lymphoblast Ans. A 5.Hybridomas are produced by combining with- a.B cell and Myeloma cells  b.B cell with particular epitopes c.Both a and b  d.None of the above ans.a 6. All of the following are needed to produce monoclonal antibodies except- a. Animal to inject b. Polyvalent antigen c.PEG d.None of the above Ans.b (Polyvalent antigen is not used. A single specific antigen with one epitope is used.) 7.Monoclonal antibodies (mAb) can be potentially used as therapeutic agents. The major advantage is that they can specifically target aberrant cells. However, there is a practical difficulty. Monoclonals are raised in mouse and therefore it is expected that an immune reaction will develop if these are injected into humans. It is therefore necessary to 'humanize' monoclonal antibody by 1. expressing the genes for the mAb in cultured human cells and isolating the mAb from these cells 2. replacing the Fv region of a mAb with one derived from a human IgG 3. replacing CL and CH regions of the Mab with that obtained from a human IgG. 4. taking a human IgG and replacing the CDRs by those derived from the mouse mAb  (csir june 2011)

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