MONOCLONAL ANTIBODY HYBRIDOMA TECHNIQUES AND PRODUCTION.pptx

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1. MONOCLONAL ANTIBODY HYBRIDOMA TECHNIQUES AND PRODUCTION -MERIN K THOMAS MSc MLT-CLIN .BIOCHEM

2. HYBRIDOMA TECHNOLOGY Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies. It was discovered by G.Kohler and C.Milstein in 1975. They were awarded nobel prize for physiology and medicine in 1975.

3. HYBRIDOMA TECHNOLOGY The hybrid cells are produced by fusing B-lymphocyte with myeloma cells (tumour cells). The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth. This is why two cells are used for the production of hybrid cells.

4. PRINCIPLE: The hybrid cell has the capacity of antibody production derived from B-cells(spleen cell). At the same time it can divide continuously by the quality derived from myeloma cells. By combining the desired qualities of both the cells, the technology ensures large, antibody production of single specificity. Specific hybridomas(spleen cell and myeloma cell) obtain monoclonal antibodies in artificial media, this technology is known as HYBRIDOMA TECHNOLOGY

5. MONOCLONAL ANTIBODY: Monoclonal antibodies ( mAb ) are antibodies that are identical because they are produced by one type of immune cell , all clones of a single parent cell. Basically produced by white blood cell which is called as plasma cell. mAb are used as anti-venom and for the treatment of cancerous cells.

6. PROCEDURE: Immunization of specific animal. Isolation of myeloma cells Fusion between spleen cell and myeloma cell. Selection at HAT medium Isolation of hybridoma cell. Screening of hybridoma cell.

7. IMMUNIZATION OF SPECIFIC ANIMAL The first step involves injecting the laboratory animals like rabbits or mice with a selected antigen . A ntibodies 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. Once a sufficient number of antibodies are created in the animal serum , the animal is sacrificed . T he spleen is removed and activated B-cells are isolated.The activated B lymphocytes are then fused with myeloma cells.

8. ISOLATION OF MYELOMA CELLS Myeloma cells are cancerous cells which is isolated from bone marrow. Myeloma cells are generally immortal(no death) in nature and has multiplication property.

9. FUSION OF SPLEEN CELLS AND MYELOMA CELLS(In vitro) It requires PEG(Poly Ethylene Glycone ) medium for fusion. It can be done by electrofusion also. Fusion between spleen cell and myeloma cell produce 5 different types of cells. Fused plasma Fused myeloma Hybridoma(1-2%) Unfused plasma Unfused myeloma

10. SELECTION AT HAT MEDIUM: HAT-hypoxanthine ,aminopterin and thymidine . Before multiplication of antibody, it has to synthesize new copy of DNA and for that it requires synthesis of nucleotide. Nucleotide synthesis (1) denovo and (2)salvage pathway-HGPRT Aminopterin in HAT media blocks the de novo synthesis pathway. Hypoxanthine and deoxythymidine(precursor) allow cells with functional hypoxanthine-guanine phosphoribosyltransferase (HGPRT) genes to survive through salvage pathways. Spleen cells are HGPRT (+ ve ) and Myeloma cells are (HGPRT – ve )

11. SELECTION AND ISOLATION –HYBRIDOMA CELL + FUSED PLASMA & UNFUSED PLASMA-SHORT LIVED SPLEEN CELL/PLASMA CELL HGPRT+ MYELOMA CELL HGPRT- 1)FUSED PLASMA- HGPRT -PRESENT 2)FUSED MYELOMA – HGPRT- ABSENT 3)HYBRIDOMA- HGPRT –PRESENT 4)UNFUSED PLASMA- HGPRT-PRESENT 5)UNFUSED MYELOMA- HGPRT-ABSENT

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14. SCREENING OF HYBRIDOMA CELL ELISA SCREENING method is done by incubating hybridoma culture in which secondary enzyme gets conjugated and formation of colored product + ve hybridoma. Used for multiplying the hybridoma cells in vivo  or  in vitro methods. In vivo-introduction of hybridoma cells into the peritoneal cavity of animals , then from ascetic fluid antibodies are isolated. In vitro-culturing of hybridoma cells in suitable culture media and the Antibodies are isolated and purified.once the hybridoma colony is established , it will continue grow in culture medias like RPMI-1640 and produce Abs. Storage-liquid nitrogen.

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16. REFERRENCE JOURNEL OF GENETIC AND BIOENGINEERING ESSENTIALS OF MEDICAL MICROBIOLOGY-APURVA

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