Hybridoma technology

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Hybridoma technology.and mAb production..

• Prsented by..
• Abhilasha tiwari
• Ankita Dwivedi
• Arijit Biswas
• Jasmine Kaur

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HYBRIDOMA TECHNOLOGY

• A hybridoma is a hybrid cell obtained by fusion
  of B lymphocyte with usually a tumor cell of
  antibody forming system or b lymphocyte,(these
  are called myelomas).
• The hybrid cell thus formed posses ability to
  produce antibodies due to B lymphocyte genome and
  the capability for the indefinte growth in vitro
  due to tumor(myeloma)cell involved in the fusion.
• Therefore ,specific hybridomas are either
  cultured in vitro or passed through mouse
  peritoneal cavity to obtain monoclonal
  antibodies,this is called as hybridoma technology.

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PRINCIPLE

• Antibodies are produced by B lymphocyte,each B
  lymphocyte being pre programmed to respond to a
  single antigenic determinant.
• Antigenic determinant denotes the
  region of antigen molecule which reacts with an
  antibody that is specific to it.
• When an antigen reacts to the
  cell surface reactor of B lymphocyte,it
  proliferates rapidly to yield a population of B
  cells ,all of which produces antibodies of same
  specificity,this is called as clonal selection.

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• Thus a B lymphocyte produces antibodies of
  only one specificity. In addition , an antibody
  producing B lymphocyte cells called plasma cell
  is fully differentiated and does not divide when
  cultured in vitro,these features are critical to
  hybridoma technology.
• B lymphocyte are isolated from spleen of an
  animal eg mouse wich had been immunized with
  antigen against which monoclonal antibodies
  are to be raised.
• Immunization is achived by
  injecting the antigen along with the suitable
  adjuvant either subcutaneously or in peritoneal
  cavity followed by the booster doses of antigen.

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• Immunization enhances the population of B
  lymphocytes producing antibodies specific to
  antigen used(clonal selection),which greatly
  increases the chance of obtaining desired
  hybridoma clone.
• A large no. of these b
  lymphocytes are mixed with cells of selected
  myeloma and induce to fuse to form hybrid cell.
• Myeloma cells are selected for mainly 2
  features
• 1These cells must not produce antibodies
  themselves.
• 2They must contain a genetic marker egHGPRT-
  trait,which permits an easy selection of
  resulting hybrid cells.

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Improvements in hybridoma technology

• A continuous cell line was used as a fusion
  partner for the antibody producing B cell
• Feeder layers consisting of extra cells to feed
  newly formed hybridomas were used for optimal
  growth and hybridoma production.
• The most common feeder layers consisted of the
  following
• Murine peritoneal cells
• Macrophages derived from mouse,rat,or guinea pigs
  ..
• Human fibroblast peripheral blood monocytes or
  thymus cells

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Steps in production of monoclonal antibodies
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Immunisation.
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Fusion.
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Culture in HAT.
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Screening.

• ELISA
• RIA
• WESTERN BLOT