Downloaded 27 times
Hybridoma technology
The document discusses the concept and production of monoclonal antibodies using hybridoma technology, which involves the fusion of B-lymphocyte cells with myeloma cells to create a hybrid cell that can proliferate endlessly while producing specific antibodies. It details the historical context of this technology, the procedural steps involved in creating and selecting hybridoma cells, and various applications of monoclonal antibodies in diagnostics and treatments. Key highlights include the methods used for selecting hybridoma cells, such as the use of HAT medium, and the purification processes for monoclonal antibodies.
More Related Content
Similar to Hybridoma technology
More from Dr. Harisingh Gour Vishwavidyalaya (A Central Universuty), Sagar, MP
Hybridoma technology
- 1. 1 PRESENTED BY: PRADDUMKUMAR NAMDEV BSc (Hons) ZOOLOGY Enrolment no: 17140007 IGNTU Amarkantak MP
- 2. INTRODUCTION HISTORY WHAT IS MONOCLONAL ANTIBODY? HYBRIDOMA TECHNOLOGY PRNCIPLE PROCEDURE APPLICATIONS OF HYBRIDOMA TECHNOLOGY REFERENCES 2 SYNOPSIS
- 3. Antibodies producedordinarily by infection or immunization are polyclonal because natural antigens have multiple epitopes, each of which generates clones of lymphocytes. This results in antisera containing antibodies from different clones of lymphocytes with specificities against different epitopes of the antigens. On the other hand, monoclonal antibodies (mAB) are monospecific antibodies. 3 Introduction
- 4. The productionof monoclonal antibodies was invented by Cesar milstein and Georges J. kohler in 1975. And in 1984 they shared a noble prize for this discovery. They make a hybrid cell that will make a numbers of monoclonal antibodies against antigen. 4 History
- 5. • Monoclonal antibodiesare produced by hybridoma technology. • The term hybridoma is used to fused cells resulting due to fusion of following two types of cells-a lymphocytes and tumor cell. • An antibody producing B- lymphocytes ( eg. Spleen cell of mouse immunized with RBCs from sheep) • A single myeloma cell (e.g. Bone marrow tumor cell) that can adopted to grow for infinite time in culture • The fused product derived the ability of two different types of cells. i.e. Ability to produce large amount of pure antibodies as lymphocytes and ability to grow or multiply indefinitely like tumor cell. 5 What is monoclonal antibody?
- 6. PRINCIPLE: Thehybrid cell has the capacity of antibody production derived from B-cells. 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. 6 HYBRIDOMA TECHNOLOGY
- 7. 7 Rabbit or labrat is immunized through repeated injection (intravenously) of specific antigen. Where in spleen it activate B-cell which produce plasma cell. Plasma cell to produce monoclonal antibodies Isolation of plasma cell from spleen of animal
- 8. Myeloma cellsare cancerous cells which is isolated from bone-marrow. Myeloma cells are generally Immortal in nature (that which never dies) and has multiplication property. 8 Step 2. Isolation of myeloma cells
- 9. The extractedB-lymphocytes is added to a culture of myeloma cell from bone marrow. The intended result is the formation of hybridoma cells formed by fusion of B-cell and myeloma cell. The fusion is done by using Polyethylene glycol (PEG) or by electrophoration or by using phages. This fusion will produce five types of cells: 1. Fused plasma cell 2. Fused myeloma 3. Hybridoma 4. Unfused plasma 5. Unfused myeloma Step 3. Fusion of myeloma cells with B-lymphocytes 9
- 10. Step 4. Selectionof HAT medium 10 (hypoxanthine, Aminopterin, Thymine) Before multiplication of antibody it has to synthesise new copy of DNA and for that is require synthesis of nucleotide. For synthesis of nucleotide mainly two pathways are there: 1. Salvage pathway 2. De-Novo pathway In salvage pathway it requires degraded part of old nucleotide to produce new nucleotide. In de-Novo pathway it synthesized completely new nucleotide by small molecules (sugar, amino acids)
- 11. So inHAT medium, cells not synthesized by De- novo synthesis due to presence of Aminopterin in HAT medium which blocks Di-hydro folate enzyme which is necessary for these synthesis. For synthesis in salvage pathway it must require HGPRT enzyme (Hypoxanthine Guanine phospho- Ribosyl Transferase) Where hypoxanthine and thymidine are used as precursers. 11 Contd…
- 12. The nextstep is selection of hybridoma cells. 12 Step 5. Selection of hybridoms cells B-cells have HGPRT enzyme Myeloma cells doesn’t have HGPRT enzyme HGPRT Fused plasma……………………………...(+) Fused myeloma……….. ……………….....(-) Hybridoma………………………………..(+) Unfused plasma………………………….(+) Unfused myeloma……………………......(-)
- 13. The myelomacell fused with another myeloma cell or those do not fused at all die in HAT medium since they do not utilize Hypoxanthine. Similarly, B- cell that fuse with another B- cell or those do not fuse at all die eventually because they do not have capacity to divide indefinitely, So, only hybridoma cell ie. fused cell between myeloma and B-cell can survive and divide in HAT medium. Screening is done to select hybridoma cells which are the desired cell for monoclonal antibodies production. Contd… 13
- 14. The selectedhybridoma cells are cultured in suitable culture medium, often supplemented with insulin, transferon, ethanol, amine and other additional hormones. Some commonly used culture media for hybridoma cell for production of monoclonal antibodies are: DMEM (Dulbecco’s modified eagle medium) IMDM (Iscove’s Modified Dulbecco’s Medium) Ham’s F12 RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium) Step 6. Culture of hybridoma cell 14
- 15. These hybridomacells are then injected into lab animal so that they starts to produce monoclonal antibodies. These hybridoma cells may be frozen and store for future use. Step 7. Inoculation of hybridoma cell into suitable host 15
- 16. Monoclonal antibodiesfrom host animal is extracted and purified by one of the following methods; Ion exchange chromatography Antigen affinity chromatography Radial immunoassay Immune precipitation Step 6. extraction & purification of monoclonal antibodies 16
- 17.
- 18.
- 19.
- 20. SEROLOGICAL Identificationof ABO blood group DIAGNOSIS Detection of pregnancy by assaying of hormones with monoclonal Separation of one substance from a mixture of very similar molecules IMMUNOPURICATION Purification of individual interferon using monoclonal antibodies. Inactivation of T-lymphocytes responsible for rejection of organ transplants Removal of tumor cells from bone marrow. Treatment of acute renal failure Treatment of malignant leukemic cells Applications of hybridoma technology 20
- 21. LIFE SCIENCES FUNDAMENTALS AND PRACTICES-1 BY: PRANAV KUMAR Pathfinder publications, New Delhi ,India www.onlinebiologynotes.com www.biologydiscussion.com www.slideshare.com 21
- 22.