Assignment Topic

Assignment
Topic: Production of Monoclonal Antibody using Hybridoma Technology.

Course code: PHRM-407
Course Title: Pharmaceutical Biotechnology.

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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, lymphoid cell, and a malignant or immortal myeloma cell. Hybridoma cells are programmed to produce a a wanted antibody in huge sums to create 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 Monoclonal Antibodies:
2.1 Hybridoma technology procedure: There are basically two stages within the production of monoclonal antibodies (mAbs) –
a) the acknowledgment 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 initiation 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, then immunized mice is place to dead and the spleen cut out and use 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 deathless cells that are refined with 8 azaguanine to ensure their susceptibility to the hypoxanthine aminopterin-thymidine (HAT) medium utilized after cell combination. A week before fusion of cells, they are developed in 8-azaguanine, to analysis the growth ability. (Institute for Laboratory Animal Research National Research Council, 1999)

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