Department of Pharmacy
Topic: Production of Monoclonal Antibody Using Hybridoma Technology
Course Code: PHRM 407
Course Title: Pharmaceutical Biotechnology
Department of pharmacy
Submission Date: 06.11.2018
A specialized cluster of cells called B-lymphocytes generate protein molecules called antibodies or immunoglobulin’s. The invasive foreign substance specially antigens attack the defense system of our body. Antibodies are part of defense system of our body which protect from antigens. Each substance contain a unique substance determinant (epitopes). The main reason for the antigen specificity is the complimentary determinative regions (CDRs) of antibodies. When an antigen comes in contact with body’s defense system, various antibodies are made from B-lymphocyte. These form of antigens are termed as polyclonal antibody which might react with specific antigen. There are many difficulties for using polyclonal antibodies for therapeutic and diagnostic use.
Monoclonal antibodies are single kind of antibody that is specific for a particular epitope. It had been a dream of scientist to produce monoclonal antibody for various antigens. Within a few years, animals were immunized against a specific antigen and B-lymphocytes had been collected and cultured in vitro for producing MABs. This approach became failed since culturing normal B-lymphocytes were troublesome.
In 1975, George Kohler and Cesar Milstein achieved large scale manufacturing of MAbs. They fused antibody producing B-lymphocytes with myeloma cells to produce a hybridoma cells in vitro.
Immortalized B-lymphocytes will multiply indefinitely and produce MABs. The expansion and multiplying properties of myeloma cell secrete antibody of B lymphocyte. The production of monoclonal antibody by hybridoma cell is referred to as hybridoma technology. To provide desired antibody in large amount hybridoma cells are used.1
Figure: steps involved in production of monoclonal antibody by hybridoma technology Adapted from: Cdn.biologydiscussion.com. (2018). online Available at: http://cdn.biologydiscussion.com/wp-content/uploads/2015/09/clip_image004_thumb17.jpg Accessed 4 Nov. 2018.
Following are several steps involving the production of monoclonal antibodies:
Fusion of cell
Selection of hybridoma cell
Cloning and propagation
Characterization and storage.1
The first step in hybridoma technology is to immunize an animal with a specific antigen. The injection given at multiple sites for many times. This triggers the stimulation of B-lymphocytes which are responding to the antigen. A final dose of antigen in intravenously administered before killing the animal. The production of maximum immune-stimulated cells for synthesis of antibodies have grown through this approach. In the serum of the animal the concentration of specified antibodies is assayed at frequent time interval throughout the course of immunization. The animal is sacrificed when the serum concentration of antibodies is optimum. By mechanical or enzymatic method, spleen of the animal is aseptically removed and disrupted to release the cells. By density gradient centrifugation, the lymphocytes of the spleen are separated from the remainder of the cells.1
Fusion of cell
The lymphocytes were washed and mixed with HGPRT (Hypoxanthine-Guanine Phosphoribosyl Transferase) enzyme. The mixture of cells were exposed to polyethylene glycol (PEG). The exposure should be for a short period because of its toxic property. Then PEG should be removed by washing. After that the cells are kept in a fresh medium. These cells are made from myeloma cells and B-lymphocytes. Thus the cells are called hybridomas (fused cells).1
Selection of Hybridomas
Only the hybridoma cells will grow in the HAT(Hypoxanthine-aminopterin-thymidine) medium. The remainder of the cells will disappear slowly, after 7-10 days of culture. It is very important to select a hybrid cell producing a single antibody. If the hybridomas are isolated and grown separately, it is possible to select only one hybrid cell. After dilution of the suspension of hybridoma cell, the individual aliquots contain one cell each on an average. The required antibody produced if these cells are grown in a regular culture medium.
For the required specificity the hybridoma cell should be screened for antibody secretion. Each culture medium of hybridoma cells are tested for the required specificity periodically. ELISA (Enzyme-linked-immunosorbent-serologic assay) is the most common assay method. To the bottom of 96- well plates antigens are absorbed. So, the specific antigen will be bind to the antibody and the unbound antibody and rest of the medium may be washed off. By screening, the hybridoma cell producing required antibody can be identified. Thus the antibody secreted from the hybrid cell is termed as monoclonal antibody.
Cloning and propagation
After screening, the hybrid cell producing desired antibody is separated and cloned. There are two technique normally used for cloning.
Limiting dilution method:
In this technique, serial dilution of suspension of hybridoma cells are done. In small culture wells, the aliquots of every dilution are placed. Each aliquots in a well contains only one hybrid cell. For this reason dilutions are created. This confirms that the antibody made is monoclonal.
Soft agar method:
In this technique, in soft agar hybridoma cells are cultured. Several cells can be grown in a semi solid medium at the same time to form colonies.1
Characterization and storage
For the required specificity, the antibody is subjected to biochemical and biophysical characterization. To find the MAbs for the sub class or class of immunoglobulin, specificity for an epitope, range of binding site it contain characterization is very important.
The stability of monoclonal antibody is also important. To withstand freezing and thawing, the ability of the cell should be characterized. At many steps of cloning and culture, the required cells are frozen in liquid nitrogen.
Conclusion: For the preparation of monoclonal antibody in vitro hybridoma technology is an appropriate method. For diagnosing, preventing and treating diseases monoclonal antibody have opened remarkable approaches.2
1.Biology Discussion. (2018). Monoclonal Antibodies: Production, Advantages and Limitations. online Available at: http://www.biologydiscussion.com/antibodies/monoclonal-antibodies-production-advantages-and-limitations/10068 Accessed 4 Nov. 2018.
2.Global-research-online.net. (2018). online Available at: http://www.global-research-online.net/volume1issue2/Article%20017.pdf Accessed 4 Nov. 2018.