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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 half breed of a mortal, antibody- producing, lymphocyts, and a malignant or undying myeloma cell. Hybridomas are programmed to produce wanted antibody in huge amount to manufacture monoclonal antibodies and now this process 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 (mAbs) Monoclonal Antibodies:
2.1 Hybridoma technology procedure: There are basically two stages within the production of monoclonal antibodies (mAbs) –
a) Introduction of antibody producing lymphocytes in vivo and the selection hybridoma cells in vitro which produce antibody, and
b) The in vitro/in vivo proliferation of selected hybridoma clones. (Marx et al., 1997)
The initiation of mAbs producing cells needs to use the animals, usually mice; it 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 appear in serum, then immunized mice is place to dead, then the spleen cut out and utilized as a originator of cells for fusion with myeloma cells. (Institute for Laboratory Animal Research National Research Council, 1999)
Step 2: Formaation of Myeloma Cells
Myeloma cells are refined with 8 azaguanine to testing the susceptibility to the hypoxanthine aminopterin-thymidine (HAT) medium utilized after cell combination. A week before cells fusion, they are developed in 8-azaguanine, for the growth ability detection. (Institute for Laboratory Animal Research National Research Council, 1999)

Fig 1: Step of hybridoma production. (Greenfield, 2014)
Step 3: Fusion of myeloma with immune spleen Cells
The ? lymphocytes are mixed with Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) defected myeloma cells. The cells mixture is uncovered to polyethylene glycol (PEG) for a short period, since it is harmful. PEG is removed from the cells by washing and then cells are kept in an unused medium. These unused medium cells are composed of a mix of free myeloma cells and free lymphocytes and hybridomas. (Jha, 2018)
Step 4: Collection of Hybridomas
The cells are then cultivated in the hypoxanthine aminopterin-thymidine (HAT) medium, only the hybridoma cells develop, the rest will gradually die.

(Jha, 2018)
Step 5: Screening the Products
The culture medium from each hybridoma culture is sometimes tested for the required antibody specificity. Specifically ELISA and RIA are two procedures of testing. In those tests, the antibody ties to the specific antigen and the unbound antibody and other components of the medium can be washed off. Hence the hybridoma cells making the required antibody can be distinguished by screening. the antibody radiated by the hybrid cells is alluded to as monoclonal. (Jha, 2018)
Step 6: Cloning and Propagation:
The single half breed cells creating the required antibody are separated and cloned. (Jha, 2018)
Step 7: Identification and Storage:
The monoclonal antibody ought to be undergoes to biochemical and biophysical identification for the required specificity.. The mAbs should be indentified for their holding ability to stand up to freezing, and defrosting. (Jha, 2018)
3. Application of Monoclonal antibody
Monoclonal antibodies are used as chemotherapy drug in cancer treatment.

Radioimmunoassay is another cancer therapy, where monoclonal antibody is coupled with radioisotope so that the cancer cell is destroyed by irradiation.
In organ transplantation monoclonal antibody is used because they aid to better cross matching.

Humanized monoclonal antibody is used to identify the infant with bronchopulmonary dysplasia.

For the encounter of viral and bacterial 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 benefit of monoclonal antibodies is numerous and associate the prevention, identification, and treatment of disease. Also monoclonal antibodies are important for the inspection 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: 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: 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).

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