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Duygu Yazicion 11 March 2013
Transcript of Hybridoma Technology
Conjugated Monoclonal Antibody Therapy
Investigational and Analytical Application
Cancer Treatment Advantages
Disadvantages The method of generating a hybridoma cell that avoids the use of animals has not been found.
May be rejected by the human immune system.
All advantages are about preventing, diagnosing and treating disease. Introduction Hybridoma Technology Duygu YAZICI / 500312009 Hybridoma Technology The Nobel Prize in Physiology or Medicine 1984 was awarded jointly to Niels K. Jerne, Georges J.F. Köhler and César Milstein "for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies". Myeloma cells + Antibody Producing B cell Hybridoma As a result monoclonal antibodies are produced because are all of a single specificity. César Milstein Georges J.F. Köhler Niels K. Jerne The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Köhler in 1975. Cloning by propagating the desire hybridomas. Selection and the screening of resulting clones. Generation of B cell hybridomas by fusing B cells and myeloma cells. Fusion= polyethylene glycol (PEG)
HAT medium (Hypoxanthine Aminopetrin Thymidine) is used for preparation of monoclonal antibodies.
In the presence of aminopterin the cells are unable to use the de novo pathway.
In this medium only fused cells will grow. Unfused myeloma cell does not have ability to grow in this HAT medium because they lack HGPRT, and thus the cell are not able to produce the DNA The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody.
The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity.
The hybridoma culture supernatant, secondary enzyme labelled conjugate, and chromogenic substrate, is then incubated, and the formation of a colored product indicates a positive hybridoma. References Shanu Tyagi, P. K. Sharma, Nitin Kumar, Sharad Visht, Hybridoma Technique in Pharmaceutical Science, 2011.
Shivanand Pandey, HYBRIDOMA TECHNOLOGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES, 2010. The use of myelomas that do not secrete their own antibodies and that therefore do not interfere with the production of the required antibody.
Feeder layers consisting of extra cells to feed newly formed hybridomas were used for optimal growth and hybridoma production.
marcrophages derived from mouse, rat or guinea pig
extra non immunized spleen cells
human fibroblasts, human peripheral blood monocytes or thymus cells