Generating antibodies

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* full-length protein (natural selection of epitope, epitope location initially unknown, may not be availalbe, epitope may not be specific to protein)
* full-length protein (natural selection of epitope, epitope location initially unknown, may not be availalbe, epitope may not be specific to protein)
* peptide (cheaper, can typically be synthesised without problems, known epitope, possibly low antigenicity)
* peptide (cheaper, can typically be synthesised without problems, known epitope, possibly low antigenicity)
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== Boosting the immune reaction ==
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A small peptide is often not enough to elicit sufficient antibody production. Several methods, also in combination, can be used to stimulated the immune system.
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* '''Multimers''' of the peptide often increase antibody synthesis. For example, several copies of the peptide linked as branches to a carrier peptide chain
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* '''Adjuvants''' are mixtures co-injected with the antigen to stimulated the immune response. Complete [[wikipedia:Freund's adjuvant|Freund's adjuvant]] (CFA), typically made from inactivated mycobacteria, is a common choice. However, newer adjuvants like [[wikipedia:Ribi's adjuvent|Ribi's adjuvent]] are safer for both scientist and animal. [http://www.acuc.berkeley.edu/assets/guidelines/antibody.pdf] See also [[wikipedia:immunologic adjuvant|adjuvant]] in the wikipedia.
== See also ==
== See also ==

Revision as of 09:40, 23 April 2008

Drawing of an antibody - heavy chains red, light chains yellow.
Drawing of an antibody - heavy chains red, light chains yellow.

You are hot in pursuit of an interesting protein but there are no commercial antibodies or they are bad. It's time to think about raising your own antibodies against the protein. It will be a time and money consuming undertaking but once you developed a specific antibody or set of antibodies, you will be in a unique position to generate interesting data.

Contents

Early decisions

Type of antibody

polyclonal

  • faster to generate
  • less specific
  • cheaper than monoclonals

monoclonal

  • slower to generate
  • more specific
  • after initial setup no animals required; unlimited production from immortal cell line
  • antigen can be impure; subsequent selection of hybridoma clones
  • more expensive than polyclonals

Source organism

  • rabbit
  • goat
  • mouse,..

Depends on choice of antibody (monoclonal requires mouse) and antibodies to be used in conjunction with antibody to be generated.

Type of antigen to use

  • full-length protein (natural selection of epitope, epitope location initially unknown, may not be availalbe, epitope may not be specific to protein)
  • peptide (cheaper, can typically be synthesised without problems, known epitope, possibly low antigenicity)

Boosting the immune reaction

A small peptide is often not enough to elicit sufficient antibody production. Several methods, also in combination, can be used to stimulated the immune system.

  • Multimers of the peptide often increase antibody synthesis. For example, several copies of the peptide linked as branches to a carrier peptide chain
  • Adjuvants are mixtures co-injected with the antigen to stimulated the immune response. Complete Freund's adjuvant (CFA), typically made from inactivated mycobacteria, is a common choice. However, newer adjuvants like Ribi's adjuvent are safer for both scientist and animal. [1] See also adjuvant in the wikipedia.

See also

External links

Protocol Online (methods forum)

Wikipedia

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