Assignment Topic: Production of Monoclonal Antibody using Hybridoma Technology. Course code: PHRM-407 Course Title: Pharmaceutical Biotechnology.
Section: 01. Submitted To: Najneen Ahmed Senior Lecturer Department of Pharmacy East West University Submitted By : Shanzida Hossain Anonna Id. 2016-1-70-069 Submission date: 5-11-2018 1. Introduction: Monoclonal antibodies are laboratory-produced molecules designed to serve as substitute antibodies that can restore, improve or mimic the immune system’s attack on cancer cells.
They are planned to tie to antigens that are generally more various on the surface of cancer cells than healthy cells. (Mayo Clinic, 2018) A hybridoma is a biologically constructed hybrid of a mortal, antibody- producing, lymphoid cell, and a malignant, or immortal myeloma cell. Hybridomas cells are been designed to produce a desired antibody in large amounts, to produce monoclonal antibodies and now this procedure is popularly known as hybridoma technology. (Pandey, 2010) The production of monoclonal antibodies was invented by César Milstein and Georges J.
F. Köhler in 1975. (Wakchaure and Ganguly, 2015) 2. Production of Monoclonal Antibodies: 2.1 Hybridoma technology procedure: There are basically two stages within the production of monoclonal antibodies (mAbs) – a) The acceptance of antibody producing lymphoid cells in vivo and the selection of antibody-producing hybridoma cells in vitro; and b) The in vitro/in vivo proliferation of selected hybridoma clones. (Marx et al., 1997) The generation of mAbs producing cells requires the use of animals, usually mice; and is carried out in the following way: Step 1: Immunization Mice are immunized with an antigen that’s prepared for infusion either by emulsifying the antigen with Freund’s adjuvant or other adjuvant. Intact cells, entire membranes, and microorganisms are sometimes utilized as immunogens.
In nearly all laboratories, mice are used to deliver the specified antibodies. In common, mice are immunized every 2-3 weeks. When an adequate antibody concentration is come to in serum, immunized mice are put to dead and the spleen cut out to use as a source of cells for combination with myeloma cells. (Institute for Laboratory Animal Research National Research Council, 1999) Step 2: Preparation of Myeloma Cells Myeloma cells are immortalized cells that are refined with 8 azaguanine to ensure their sensitivity to the hypoxanthine aminopterin-thymidine (HAT) choice medium utilized after cell combination.
A week before cell fusion, myeloma cells are developed in 8-azaguanine. to testing the growth ability. (Institute for Laboratory Animal Research National Research Council, 1999) Fig 1: Stages of hybridoma production. (Greenfield, 2014) Step 3: Fusion of Myeloma Cells with Immune Spleen Cells The altogether washed lymphocytes (? cells) are mixed with Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) imperfect myeloma cells. The mixture of cells is exposed to polyethylene glycol (PEG) for a short period, since it is harmful.
PEG is expelled by washing and the cells are kept in a new medium. These cells are composed of a blend of hybridomas, free myeloma cells and free lymphocytes. (Jha, 2018) Step 4: Selection of Hybridomas The cells are then cultured in (HAT) the hypoxanthine aminopterin-thymidine medium, only the hybridoma cells develop, whereas the rest will gradually die. (Jha, 2018) Step 5: Screening the Products The culture medium from each hybridoma culture is occasionally tested for the specified antibody specificity. The two procedures specifically ELISA and RIA are commonly used for this purpose. In both the tests, the antibody binds to the particular antigen and the unbound antibody and other components of the medium can be washed off.
Thus, the hybridoma cells creating the specified antibody can be identified by screening. The antibody emitted by the crossover cells is referred to as monoclonal antibody. (Jha, 2018) Step 6: Cloning and Propagation: The single hybrid cells producing the desired antibody are isolated and cloned. (Jha, 2018) Step 7: Characterization and Storage: The monoclonal antibody should be subjected to biochemical and biophysical characterization for the required specificity.
The mAbs must be characterized for their capacity to resist freezing, and defrosting. (Jha, 2018) 3. Application of Monoclonal antibody In cancer treatment monoclonal antibody used as chemotherapy drug. Radioimmunoassay is a alternative cancer therapy, where monoclonal antibody is coupled with radioisotope such that the cancer cell is killed by irradiation.
In organ transplantation monoclonal antibody is used because they help to better cross matching. Humanized monoclonal antibody is used to detect the infant with bronchopulmonary dysplasia. For the detection of bacterial and viral disease monoclonal antibody are applied. Various disease like renal cancer, lymphoma, myeloma cancer, rheumatoid arthritis, Metastatic breast cancer etc.
where monoclonal antibody treatment is approved. (Kumar, et al., 2012) 4. Conclusion The use of monoclonal antibodies is various and incorporates the prevention, determination, and treatment of disease. Also monoclonal antibodies are important for the investigation of parasites antigen.
This hybridoma technology creates great opportunity for treatment and curing the disease by producing monoclonal antibody, which is more specific and more potent drug. So this hybridoma technology brought a revolutionary in life science. 5. Reference Greenfield, E. (2014). Antibodies.
2nd ed. New york: Cold Spring Harbor Laboratory Press, pp.208-209. Institute for Laboratory Animal Research National Research Council (1999). Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. Washington, DC: NATIONAL ACADEMY PRESS, pp.6-8. Jha, N.
(2018). Monoclonal Antibodies: Production, Advantages and Limitations. online Biology Discussion. Available at: http://www.biologydiscussion.com/antibodies/monoclonal-antibodies-production-advantages-and-limitations/10068 Accessed 4 Nov. 2018.
Kumar, A. Singh, M. ; Gupta, SM. (2012) “Hybridoma Technology”. In: Biotechnology in medicine and agriculture: principles and practices.
(eds. Kumar A, Pareek A ; Gupta SM) I. K. International publishing house Pvt. Ltd., New Delhi, India, pp.
338-367. Marx, U., Embleton, M., Fischer, R., Gruber, F., Hansson, U., Heuer, J., de Leeuw, W., Logtenberg, T., Merz, W., Portetelle, D., Romette, J. and Straughan, D. (1997).
Monoclonal Antibody Production. The Report and Recommendations of ECVAM Workshop 231,2. Angera, Italy: The European Centre for the Validation of Alternative Methods, pp.121-137. Marx, U., Embleton, M., Fischer, R., Gruber, F., Hansson, U., Heuer, J., de Leeuw, W., Logtenberg, T., Merz, W., Portetelle, D., Romette, J. and Straughan, D. (1997).
Monoclonal Antibody Production. The Report and Recommendations of ECVAM Workshop 231,2. Angera, Italy: The European Centre for the Validation of Alternative Methods, pp.121-137. Mayo Clinic.
(2018). Monoclonal antibody drugs for cancer: How they work. online Available at: https://www.mayoclinic.org/diseases-conditions/cancer/in-depth/monoclonal-antibody/art-20047808 Accessed 4 Nov. 2018. Pandey, S.
(2010). HYBRIDOMA TECHNOLOGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES. International Journal of Pharmaceutical Sciences Review and Research, 1(2), pp.88-94. Wakchaure, R. and Ganguly, S. (2015).
Importance of Transgenic Fish to Global Aquaculture: A Review. Fisheries and Aquaculture Journal, 06(04).