HYBRIDOMA TECHNOLOGY.pptx

Monoclonal antibodies are been developed an produced from some identical parental immune cells. they can be developed to target and identify specific cells and antigens and to work as antibodies in tandem with the human immune system against them. Hybridoma technology was developed by Georges J.F. Kohler and Cesar Milstein. they made a hybrid cell that will make number of monoclonal antibodies against them.

1. PRODUCTION OF MONOCLONAL ANTIBOBY BY HYBRIDOMA TECHNOLOGY MADHUMA K

2. MONOCLONAL ANTIBODY Monoclonal antibodies are antibodies that been developed and produced from some identical parent immune cell. They can be developed by scientists to target and identify specific cells and antigens and to work as antibodies in tandem with the human immune system against them.

3. APPLICATIONS DIAGANOSTIC TESTING PREGNENCY TESTING RADIO IMMUNO DETECTION OF CANCER TREATMENT OF CANCER THROUGH DRUGS VIRAL DISEASE TREATMENT IDENTIFING PATHOGENS. TRACING SPECIFIC CELLS AND THEIR FUNCTION

4. HYBRIDOMA TECHNOLOGY

5. HYBRIDOMA TECHNOLOGY This technology was developed by Georges J.F. Kohler and Cesar Milstein. in 1984 they shared a Nobel prize for this discovery. They make a hybrid cell that will make a number of monoclonal antibodies against antigen.

6. PROCEDURE 1. Immunization of specific animal which generate hybridoma cell with spleen cell. 2. Isolation of myeloma cells 3. Fusion between spleen cell and myeloma cell 4. Selection of HAT medium 5. Isolation of hybridoma cell 6. Screening of hybridoma cells.

7. 1.IMMUNIATION OF SPECIFIC ANIMAL An antigen immunized to an animal like mice via intravenously ( directly to blood ) by injection. Where in spleen it activates b-cell which produce plasma cell (spleen cells). spleen cell is isolated from the spleen of the animal.

8. 2.ISOLATION OF MYELOMA CELLS. Myeloma cells are cancerous cells which is isolated from bone marrow. Myeloma cells are generally immortal in nature ( that which never dies) and has multiplication property.

9. 3. FUSION OF SPLEEN CELL AND MYELOMA CELLS It requires PEG ( poly ethylene glycone) medium for fusion. It can also done by electro fusion Fusion between spleen cell and myeloma cells produce 5 different types of cells. Fused plasma Fused myeloma Hybridoma Unfused plasma Unfused myeloma

10. 4.SELECTION OF HAT MEDIUM (Hypoxanthine, Aminopterin, Thymidine) the hybridoma cells or fused cells are selected by incubated in the selective media called HAT medium. It contain hypoxanthine , aminopterin and thymidine. The unfused b cell have a very short life span in the medium. The myeloma cells can synthesis DNA nucleotide using two pathway : Denovo pathway and Salvage pathway. In HAT medium myeloma cells cannot replicate because denovo pathway is blocked by the aminopterin in the medium.

11. SELECTION IN HAT MEDIUM when denovo pathway is blocked cell will utilize alternate pathway called salvage pathway. But it cannot take place due to the lack of HGPRT ( hypoxanthine guanine phosphoribosyl transferase). so it is contributed by B cells which is rich in HGPRT+ But the salvage pathway is also inhibited due to the mutation of enzyme thymidine kinase ( TK) an enzyme that catalyse the phosphorylation reaction.

12. 5. ISOLATION OF HYBRIDOMA CELLS Fused myeloma and unfused myeloma cannot survive in HAT medium because of the absence of HGPRT enzyme. Fused plasma and unfused plasma cells have HGPRT enzyme but they have only very short life span. But hybrid cells has HGPRT enzyme from the spleen or plasma cells as well as they have the ability to multiply repeatedly as the myeloma cells. So isolation of hybrid cell is possible because they are the only cell that survive in the HAT medium.

13. 6. SCREENING OF HYBRIDOMA CELLS ELISA screening method is used for the screening of the hybridoma cells Hybridoma cells are multiplied using in vivo or in vitro method. In in-vivo procedure hybridoma cell is introduce in to the peritoneal cavity of animal. In-vitro method involves culturing of hybridoma cells in suitable cultural media an then antibodies are isolated and purified.

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