Hybridoma Technology: Introduction, Procedure and applications (BP605T).pptx

Hybridoma technology is a groundbreaking method for producing monoclonal antibodies, identical molecules that target a specific antigen with precision. Pioneered by Georges Köhler and César Milstein in 1975, this innovative technique involves fusing an antibody-producing B-cell with a myeloma (cancer) cell. The fusion creates a hybridoma, a super cell that combines the B-cell's ability to produce a specific antibody with the myeloma cell's capacity for indefinite division.

The process begins by immunizing an animal, typically a mouse, with the target antigen to trigger an immune response. B-cells from the animal's spleen are then harvested and fused with myeloma cells using a fusogen like polyethylene glycol. The resulting hybridomas are cultured in a selective medium that ensures only the successful fusions survive. These hybridomas are then meticulously screened to identify those producing the desired antibody.

Once identified, the chosen hybridomas are cloned to produce a uniform, high-purity supply of monoclonal antibodies. These antibodies are invaluable in a myriad of applications, from cutting-edge diagnostics and groundbreaking therapeutics to advanced scientific research. Hybridoma technology has revolutionized biomedical research, leading to significant breakthroughs in disease diagnosis and treatment, particularly in cancer and autoimmune disorders, marking a new era in medical science.

1. Presentation by: Jaskiran Kaur Assistant Professor, SCOP, Barabanki HYBRIDOMA T ECHNOLOGY

2. Introduction Monoclonal antibodies were discovered in 1975. In 1975, Kohler and Milstein discovered a technique called hybridoma technology for the production of monoclonal antibodies at Oxford University . Kohler and Milstein developed a system in which antibody-producing B cells ( Plasma B cells) were fused with immortal cancerous cell lines such as myeloma cells , creating an immortal hybrid cell line that produces antibodies limitlessly. Hybridoma technology produces monoclonal antibodies ( mAbs ) specific to antigens.  Monoclonal antibodies produced by this method are highly specific antibodies, which are derived from a single parental B cell clone.

3. Activation of B cells and production of antibodies

4. Antibodies are divided into two types based on their origin from lymphocytes monoclonal antibodies polyclonal antibodies . 

5. Preparation of monoclonal antibodies using H ybridoma technology Immunization-  The first step involves injecting the laboratory animals like rabbits or mice with a selected antigen against which the antibodies are raised through a series of injections over a period of several weeks to stimulate B cell differentiation into plasma B cells and memory B cells.  2. Isolation of B lymphocytes  – After animal sacrifice, the spleen is removed to isolate the activated B-cells. This procedure is performed using centrifugation. The serum contains the activated B lymphocytes (that produce antibodies). The activated B lymphocytes are then fused with myeloma cells.

6. 3. Preparation of myeloma cell lines – Few weeks before the cell fusion, metastatic tumor cells are incubated in 8-azaguanine to get non-functional hypoxanthine-guanine phosphoribosyltransferase (HGPRT) genes in the myeloma cells. Non-functional HGPRT can stop the assembly of nucleotides (formation of DNA) from the salvage pathway and makes the metastatic tumor cells sensitive to HAT media as the preferred method in hybridoma technology. 4. Cell fusion – Cell fusion is the process in which the activated B lymphocytes are fused with HAT-sensitive myeloma cells . Polyethylene glycol (PEG) is used in this procedure . PEG helps in the fusion of cells by promoting the fusion of the plasma membrane of the myeloma cells with the plasma membrane of the antibody-producing cells, thus giving rise to a cell with more than one nucleus, forming heterokaryon . Another method used for fusion is electrofusion, in which cells are fused under the effect of an electric field.

7. Possible fusion products

8. 5. Hybridoma selection-   After fusing cells, you need to separate the actual fused cells ( hybridomas ) from the unfused parental cells. HAT medium is a special selection media containing: Aminopterin : This blocks the cells' ability to synthesize nucleotides through the de novo pathway (the normal way cells make nucleotides using Dihydro Folate Reductase). Hypoxanthine and Thymidine: These are salvage pathway precursors. Unfused B cells: These cells have the HGPRT gene and .They have a limited lifespan and die within days. Unfused Myeloma cells (malignant): These typically lack the HGPRT gene . With aminopterin blocking de novo synthesis, they cannot make nucleotides and die. Fused cells ( Hybridomas ): They inherit the functional HGPRT gene from the B lymphocytes. This allows them to utilize the salvage pathway with hypoxanthine and thymidine, and they can survive and grow in HAT media. After incubation in HAT media, only the hybridomas remain viable and can be continuously cultured

9. 6. Screening of hybridoma cells – HAT-selection hybridoma cells are transferred to ELISA plates, where each well houses a single hybridoma cell.  8. Cloning and propagation of hybridoma cell – Hybridomas producing desired antibodies are selected and are then transferred into large culture vessels or flasks; the hybridoma cell lines are cultured using in vivo  or  in vitro methods. 

10. Flowchart of the process

11. Applications of hybridoma technology Diagnostic testing: It is used to check the presence of any foreign antigen such as toxins, drugs, hormones, or internal and surface proteins of bacteria or viruses. Testing of pregnancy: Monoclonal antibodies are used to identify the presence of human chorionic gonadotropin [ hCG ] as a mark for recognition of pregnancy. Identification of different strains of pathogens : Monoclonal antibodies can be used to differentiate between different strains of a single pathogen, for example,  Neisseria gonorrhoeae   Cancer treatments through drugs : Monoclonal antibodies are also used in targeted chemotherapy. A drug named rituximab, sold under the brand name of Rituxan , was approved by FDA for commercial use, for the targeted treatment of cancers mainly lymphomas  Viral disease treatment : Monoclonal antibodies are also being tested in treatments of previously incurable diseases such as AIDS.

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