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Hybridoma technology and application for monoclonal antibodies
- 1. Presented by:- Jagohool Singh Roll no=1237 MSc.Biotechnology 1st MDU ROHTAK
- 2. An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines.
- 3. 1975, Georges Köhler and Cesar Milstein - awarded Nobel Prize in1984
- 4. In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma." The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.
- 5. PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)
- 6. PRODUCTION OF MONOCLONAL ANTIBODY Step 2: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Myeloma Cells HGPRT- (Hypoxanthin Guanine phosphoribosyltransferase)
- 7. PRODUCTION OF MONOCLONAL ANTIBODY Step 3: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLSSPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium 1. Plating of Cells in HAT selective Medium 2. Scanning of Viable Hybridomas
- 11. Selected by using HAT medium (Hypoxanthine- Aminopterin-Thymidine) Myeloma cells are unable to grow B cells are able to survive, but can not live for extended periods
- 13. on of myeloma and B cells (using PEG) ration of cell lines
- 14. ) Screening of suitable cell lines
- 15. in vitro (a) or in vivo (b) multiplication Harvesting
- 17. Monoclonal antibodies are proving to be very useful as diagnostic, imaging, and therapuetic reagents in clinical medicine. Many monoclonal antibody diagnostic reagents now available are products for detecting pregnancy, diagnosing numerous pathogenic microorganisms, measuring blood levels of various drugs, and detecting antigens shed by certain tumors.
- 18. A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine. Monoclonal antibodies to HCG have been produced. These have been attached to enzymes which can later interact with a dye molecule and produce a colour change.
- 19. The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s blood serum.
- 20. a) HIV antigen is attached to the plate. b) Patients serum passed over the plate. Any HIV antibody in the patients serum will attached to the antigen already on the plate. c) A second antibody which is specific to the HIV antibody is passed over the plate. This antibody will attach to the concentrated HIV antibody on the plate. This second antibody has an enzyme attached to its structure. d) Chromagen dye is passed over the complex of concentrated HIV antibody/conjugated antibody. e) The enzyme will turn the chromagen to a more intense colour. The more intense the colour, the greater the HIV antibody level. This would be the a positive result for a HIV test.
- 21. Cancer cells carry specific tumour-associated antigens (TAA) on their plasma membrane. Monoclonal anti-TAA antibodies have been produced. Drugs which kill tumour cells or inhibit key proteins in tumour cells are attached to monoclonal anti-TAA antibodies. Cancer cells are specifically targeted, avoiding damage to healthy host cells.