“Production of Monoclonal Antibody
Using Hybridoma Technology”
Course Code: PHRM 407
Course Title: Pharmaceutical Biotechnology
Department of Pharmacy
East West University
Taaha Sadman Sakib Hassan
Date of Submission: 06th November, 2018
A method for producing large numbers of identical by using myeloma cell line which is selected for the ability to grow in culture is called hybridoma technology. Two scientists Georges Kohler of West Germany and Cesar Milstein of Argentina become in style for the discovery of hybridoma technology in 1975. They are awarded at 1984 acclamation for physiology and medication. Parasite substances are easily analyzed by monoclonal antibody and permitted that UN agency ought to organized a conference that brought along (those UN agency those that people who) have established and refined the technology and people who are exploitation it, or desiring to use it for the study of organism answerable for a number of the main diseases poignant group. Such monoclonal antibodies, as they’re known, have opened exceptional new approaches to preventing diagnosing, and treating diseases. Global-research-online. net. 2010.
The following steps are allowing in Hybridoma Technology:
Immunization and Isolation of B Cells from the spleen: Laboratory animals (e.g. mice)
Are initial vaccinated by exposing to a specific antigen so that we isolate an antibody against spleen cells of mouse’s are removed out.
Screening of mice for protein production: Immunization Blood sample of mice for the measurement of serum fluid antibodies concentration determined after many weeks passed (Technique: – ELISA/ Flow cytometry). Titre is just too low mice are often boosted until adequate response is achieved. Titre just too low high mice are unremarkably boosted by injecting antigen while not adjuvant present.
Cultivation of malignant neoplasm cells: Malignant neoplasm cells or myeloma cells are immortalized cells that cultured with eight Azaguanine to their sensitivity to HAT medium.
Generation of the hybridoma cells by fusing prime B cells and malignant neoplasm cells: The mammalian spleenocyte are isolated and then the B cells are combined with immortalized myeloma cells using synthetic resin glycol. Synthetic resin glycol (PEG) is employed to extend the cell division.
Adapted from: University of Nevada, Reno School of Medicine (n.d.). Monoclonal antibodies. Online Available at: https://med.unr.edu/ technology/monoclonal-antibodies Accessed 4 Nov. 2018.
Culture in HAT medium: Incubation of combined cells within the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin within the myeloma cells die, this can be as a result of myeloma cells doesn’t synthesize hypoxanthine-guanine phosphoribosyl enzyme (HGPRT) catalyst. They con not manufacture nucleotides by the First State novo or salvage medium blocks the pathway that enables for nucleotide synthesis. Therefore, infused B cells die as they have a brief lifetime. Solely the fused myeloma hybrids survive, since the HGPRT gene coming back from the B cells is useful. These cells manufacture antibodies (a property of B cells) and are immortal (a property of malignant neoplasm cells).
Separation of cell lines and Multiplication: The medium is incubated and then “Limiting Dilution” into multiwall plates to such an extent that every well contains only one cell. Then the supernatant in every well are often checked for desired antibody. Once the characterized specific hybridoma cell then clone-it is increased either in vivo or in vitro.
Cloning by propagation the desired hybridoma: Once a hybridoma colony is established, it’ll regularly grow in culture medium and manufacture for antibody. The following stage may be a speedy primary screening method that identifies and selects solely those hybridomas that manufacture antibodies of acceptable specificity. Jana, S. n.d., researchgate.net (2012).
Applications of Monoclonal Antibodies:
The hybridoma technique is the production of homogeneous antibody against a specific epitope of an antigen. These monoclonal antibodies have wide applications such as:
Immunodiagnostic regents, either for detection of the causative agent’s directly in tissues or body fluids or as a chemical agent employed in indirect diagnosing like serologic detection of antibodies to the borne in upon agent.
For experimental functions starting from molecular dissection of matter epitopes to being anti idiotype protein utilized as an immunogenic to induce protecting immunity.
Immunoprophylaxis or immunotherapeutic applied to infectious, parasitic, microorganism and infective agent communicable disease diagnosing.
For Biotechnological application the Moab’s is employed for purification of macromolecule, antigens, medication treating and sequence biological research & expression.
Moab’s is applied to medical purpose for bioassay, interference ; treatment of diseases, cancer medical aid and Bone marrow ; organ transplantation.
Research tools for the popularity of specific aminoalkanoic acid sequences of polypeptides.
Also applied for neology in vitro and in vivo for identification and localization of disorders. researchgate.net (2012)
Global-research-online.net. (2010). online Available at: http://www.global-research-online.net/volume1issue2/Article%20017.pdf Accessed 4 Nov. 2018.
researchgate.net (2012). online Available at: https://www.researchgate.net/publication/282076158_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_De Accessed 4 Nov. 2018.
Jana, S. (n.d.). HYBRIDOMA TECHNOLOGY ;amp; ITS APPLICATION. online Academia.edu. Available at: https://www.academia.edu/6017218/HYBRIDOMA_TECHNOLOGY_and_ITS_APPLICATION Accessed 4 Nov. 2018.