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Producing monoclonal antibodies

Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs) . [link] compares some of the important characteristics of monoclonal and polyclonal antibodies.

Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive ( [link] ).

Diagram showing production of monoclonal antibodies. Antigen is injected into an animal (such as a mouse) Spleen cells are extracted. Myeloma line cells from a cell culture are added to the spleen cells in a test tube. Then, hybrid cells are selected and grown. Hybrid cells are separated and allowed to proliferate into clones (hybridomas). Each hybrid produces a different antibody and the desired antibody is selected. This hybridoma is then grown to produce large batches of desired mAB.
Monoclonal antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B cells from the mouse’s spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to the antigen. Hybridomas producing the desired mAb are then grown in large numbers on a selective medium that is periodically harvested to obtain the desired mAbs.
Characteristics of Polyclonal and Monoclonal Antibodies
Monoclonal Antibodies Polyclonal Antibodies
Expensive production Inexpensive production
Long production time Rapid production
Large quantities of specific antibodies Large quantities of nonspecific antibodies
Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen
Production is continuous and uniform once the hybridoma is made Different batches vary in composition

Clinical uses of monoclonal antibodies

Since the most common methods for producing monoclonal antibodies use mouse cells, it is necessary to create humanized monoclonal antibodies for human clinical use. Mouse antibodies cannot be injected repeatedly into humans, because the immune system will recognize them as being foreign and will respond to them with neutralizing antibodies. This problem can be minimized by genetically engineering the antibody in the mouse B cell. The variable regions of the mouse light and heavy chain genes are ligated to human constant regions, and the chimeric gene is then transferred into a host cell. This allows production of a mAb that is mostly “human” with only the antigen-binding site being of mouse origin.

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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