Generating antibodies

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[[Image:Antibody schematic.jpg|thumb|right|200px|Drawing of an antibody - heavy chain red, light chain yellow.]]
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[[Image:Antibody schematic.jpg|thumb|right|200px|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.
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.
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=== Type of antibody ===
=== Type of antibody ===
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* polyclonal (faster to generate, less specific)
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{| {{table}} cellpadding=10
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* monoclonal (slower to generate, more specific, after initial setup no animals required)
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|valign=top width=50%| '''polyclonal''' (a mix of antibodies with different specificities)
 +
* faster to generate
 +
* less specific
 +
* cheaper than monoclonals
 +
|valign=top| '''monoclonal''' (many copies of the same antibody)
 +
* 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 ===
=== Source organism ===
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* rabbit
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Common animals for raising antibodies include rabbit, goat, mouse (required for monoclonal antibodies), and even llamas [http://science.howstuffworks.com/bio-llama.htm]. The choice depends on which antibodies will be used in conjunction with antibody to be generated and which type of antibody is desired.
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* goat
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* mouse,..
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Depends on choice of antibody (monoclonal requires mouse) and antibodies to be used in conjunction with antibody to be generated.
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=== Type of antigen to use ===
=== Type of antigen to use ===
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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)
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{| {{table}} cellpadding=10
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* peptide (cheaper, can typically be synthesised without problems, known epitope, possibly low antigenicity)
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|valign=top width=50%| '''full-length protein'''
 +
* likely to produce a ''conformational epitope'' useful for recognition of folded protein
 +
* purchase or purification of full-length protein typically more expensive/time consuming
 +
* full-length protein may not be availalbe and purification may prove difficult
 +
* natural selection of epitope but epitope location initially unknown
 +
* epitope may not be specific to protein
 +
|valign=top| '''peptide'''
 +
* likely to produce a ''linear epitope'' useful for recognition of primary sequence as in immunoblots
 +
* cheaper, less effort (more readily available instruments)
 +
* antigen can typically be synthesised without problems
 +
* known epitope
 +
* but possibly low antigenicity
 +
|}
 +
 
 +
== Peptide selection ==
 +
 
 +
[[Image:Hydrophobicity plot example.png|thumb|right|300px|Example of a protein's hydrophobicity according to 2 different scales. Made with R. Bowen's [http://www.vivo.colostate.edu/molkit/hydropathy/index.html tool].]]
 +
 
 +
If you decide to raise antibodies against a peptide or several peptides from the target protein, you will be faced with the question which region to choose. The gist is, you want to pick a '''15-20 residue peptide''' that is on the surface of the protein, i.e. has a high '''hydrophilicity'''. Another common approach is to pick sequences from the '''ends''' of the protein, since they are typically exposed. '''Avoid cysteines''' if possible, since peptides will often be coupled via Cys residues to carriers and since disulphide bridges alter the shape and thus the recognition by antibodies. Still, peptide selection is trial and error.
 +
 
 +
=== Online tools ===
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* [http://swift.cmbi.kun.nl/swift/future/overview/antigen.html Instructions for peptide prediction (EMBL)]
 +
* [http://bio.dfci.harvard.edu/Tools/antigenic.html#Introduction Background of peptide prediction (Antigenic tool, Harvard)]
 +
 
 +
'''Sequence plots'''
 +
* [http://us.expasy.org/cgi-bin/protscale.pl ExPASy's classic ProtScale tool] allows you to check a protein sequence against various amino acid scales including several for hydrophilicity/-phobicity
 +
* [http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=pepwindow pepwindow] hydrophobicity tool from the Institute Pasteur (default Kyte-Doolittle)
 +
* [http://www.vivo.colostate.edu/molkit/hydropathy/index.html Hydrophobicity tool] by Colorado State Uni - well documented but x scale not sufficiently subdivided
 +
 
 +
'''Peptide prediction'''
 +
 
 +
Tools attempting prediction of immunogenic peptides given the protein sequence. Both are based on ''Kolaskar and Tongaonkar "A semi-empirical method for prediction of antigenic determinants on protein antigens." FEBS Letters 1990'' (PMID 1702393). The estimated accuracy of prediction is 3 in 4 peptides or 75%.
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* [http://bio.dfci.harvard.edu/Tools/antigenic.pl Harvard Antigenic tool] (fast but no scoring of peptides)
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* [http://liv.bmc.uu.se/cgi-bin/emboss/antigenic EMBOSS antigenic tool] (slow, peptides ordered by score)
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 +
'''Other'''
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* [http://www.innovagen.se/peptide-design-tools.asp Peptide design check] by Innovagen (no explanation of reasoning)
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* [http://www.ebi.ac.uk/cgi-bin/proteincol/ProteinColourer.pl Protein Colourer tool] from the EBI may be helpful to inspect the sequence by eye
 +
 
 +
=== Further reading ===
 +
* Rapberger 2007 "Identification of discontinuous antigenic determinants on proteins based on shape complementarities." PMID 17421048
 +
* Söllner 2006 "Machine learning approaches for prediction of linear B-cell epitopes on proteins." PMID 16598694
 +
* Batori 2006 "An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context." PMID 16193533
 +
* Kulkarni-Kale 2005 "CEP: a conformational epitope prediction server." PMID 15980448, http://bioinfo.ernet.in/cep.htm
 +
 
 +
== Boosting the immune reaction ==
 +
 
 +
[[Image:Polyclonal ab overview.png|thumb|right|350px|Approximate overview of polyclonal antibody generation. Total duration around 2 months.]]
 +
 
 +
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. Multimers can be made by linking several copies of the peptide as branches to a carrier peptide chain.
 +
* '''Adjuvants''' are mixtures co-injected with the antigen to stimulate 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]] and [[wikipedia:Titermax|Titermax]] 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.
 +
 
 +
== Pitfalls ==
 +
* antigen is a poor immunogen; consider coupling to a carrier if not already done; consider polymersiation of antigen with glutaraldehyde; choose a different peptide
 +
* too little antigen was used; this will be an issue if your antigen is hard to produce and therefore scarce
 +
* protein or peptide preparation is not pure; contaminant is more immunogenic than desired epitope; resulting antibody does not recognised the right target
 +
* adjuvant might not be suitable; try a different one and a different antigen/adjuvant ratio
 +
* oil-in-water emulsion might not be good enough (a drop of a good emulsion will float on cold water)
 +
* if using outbred animals, individual variation of immune reaction will be significant; make sure a large enough group is immunised
== See also ==
== See also ==
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* [[Griffin:Antibody_Basics]]
* [[Protein purification tags]]
* [[Protein purification tags]]
* [[Western blot]]
* [[Western blot]]
== External links ==
== External links ==
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* [http://www.acuc.berkeley.edu/assets/guidelines/antibody.pdf Guidelines for polyclonal antibody production] by  Animal Care and Use Committee, UC Berkeley
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* [[Image:3stars.png]] [http://www.acuc.berkeley.edu/assets/guidelines/antibody.pdf Guidelines for polyclonal antibody production] by  Animal Care and Use Committee, UC Berkeley - full of up-to-date, technical advice viewed from animal health point of view
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* [http://www.hopkins-arthritis.org/physician-corner/education/mono_anti.html Introduction to monoclonal antibody generation] by Mark Soloski, Johns Hopkins
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* [[Image:2stars.png]] [http://www.anaspec.com/resources/antibody.asp Antibody production tips] from AnaSpec, Inc. (detailed information on several pages, little advertisement)
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* [http://www.altabioscience.bham.ac.uk/services/peptide/antibodies.shtml FAQ on antibody generation] by Alta Bioscience, UK
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* [[Image:2stars.png]] [http://www.altabioscience.com/products-and-services/antibodies FAQ on antibody generation] by Alta Bioscience Ltd, UK (useful technical tips and a little advertisement)
 +
* [[Image:2stars.png]] [http://www.hopkins-arthritis.org/physician-corner/education/mono_anti.html Backgrounder on history, generation, and application of monoclonal antibodies] by Mark Soloski, Johns Hopkins
* [http://mabs.unc.edu/services.htm Hybridoma and in vitro generation of antibodies], Antibody Facility, UNC Chapel Hill
* [http://mabs.unc.edu/services.htm Hybridoma and in vitro generation of antibodies], Antibody Facility, UNC Chapel Hill
* [http://books.google.de/books?id=-aikPhH9MrcC&hl=en Monoclonal Antibodies: A Practical Approach] by Philip Shepherd, Christopher Dean on Google Book Search
* [http://books.google.de/books?id=-aikPhH9MrcC&hl=en Monoclonal Antibodies: A Practical Approach] by Philip Shepherd, Christopher Dean on Google Book Search

Current revision

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 (a mix of antibodies with different specificities)
  • faster to generate
  • less specific
  • cheaper than monoclonals
monoclonal (many copies of the same antibody)
  • 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

Common animals for raising antibodies include rabbit, goat, mouse (required for monoclonal antibodies), and even llamas [1]. The choice depends on which antibodies will be used in conjunction with antibody to be generated and which type of antibody is desired.

Type of antigen to use

full-length protein
  • likely to produce a conformational epitope useful for recognition of folded protein
  • purchase or purification of full-length protein typically more expensive/time consuming
  • full-length protein may not be availalbe and purification may prove difficult
  • natural selection of epitope but epitope location initially unknown
  • epitope may not be specific to protein
peptide
  • likely to produce a linear epitope useful for recognition of primary sequence as in immunoblots
  • cheaper, less effort (more readily available instruments)
  • antigen can typically be synthesised without problems
  • known epitope
  • but possibly low antigenicity

Peptide selection

Example of a protein's hydrophobicity according to 2 different scales. Made with R. Bowen's tool.
Example of a protein's hydrophobicity according to 2 different scales. Made with R. Bowen's tool.

If you decide to raise antibodies against a peptide or several peptides from the target protein, you will be faced with the question which region to choose. The gist is, you want to pick a 15-20 residue peptide that is on the surface of the protein, i.e. has a high hydrophilicity. Another common approach is to pick sequences from the ends of the protein, since they are typically exposed. Avoid cysteines if possible, since peptides will often be coupled via Cys residues to carriers and since disulphide bridges alter the shape and thus the recognition by antibodies. Still, peptide selection is trial and error.

Online tools

Sequence plots

  • ExPASy's classic ProtScale tool allows you to check a protein sequence against various amino acid scales including several for hydrophilicity/-phobicity
  • pepwindow hydrophobicity tool from the Institute Pasteur (default Kyte-Doolittle)
  • Hydrophobicity tool by Colorado State Uni - well documented but x scale not sufficiently subdivided

Peptide prediction

Tools attempting prediction of immunogenic peptides given the protein sequence. Both are based on Kolaskar and Tongaonkar "A semi-empirical method for prediction of antigenic determinants on protein antigens." FEBS Letters 1990 (PMID 1702393). The estimated accuracy of prediction is 3 in 4 peptides or 75%.

Other

Further reading

  • Rapberger 2007 "Identification of discontinuous antigenic determinants on proteins based on shape complementarities." PMID 17421048
  • Söllner 2006 "Machine learning approaches for prediction of linear B-cell epitopes on proteins." PMID 16598694
  • Batori 2006 "An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context." PMID 16193533
  • Kulkarni-Kale 2005 "CEP: a conformational epitope prediction server." PMID 15980448, http://bioinfo.ernet.in/cep.htm

Boosting the immune reaction

Approximate overview of polyclonal antibody generation. Total duration around 2 months.
Approximate overview of polyclonal antibody generation. Total duration around 2 months.

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. Multimers can be made by linking several copies of the peptide as branches to a carrier peptide chain.
  • Adjuvants are mixtures co-injected with the antigen to stimulate the immune response. Complete Freund's adjuvant (CFA), typically made from inactivated mycobacteria, is a common choice. However, newer adjuvants like Ribi's adjuvent and Titermax are safer for both scientist and animal. [2] See also adjuvant in the wikipedia.

Pitfalls

  • antigen is a poor immunogen; consider coupling to a carrier if not already done; consider polymersiation of antigen with glutaraldehyde; choose a different peptide
  • too little antigen was used; this will be an issue if your antigen is hard to produce and therefore scarce
  • protein or peptide preparation is not pure; contaminant is more immunogenic than desired epitope; resulting antibody does not recognised the right target
  • adjuvant might not be suitable; try a different one and a different antigen/adjuvant ratio
  • oil-in-water emulsion might not be good enough (a drop of a good emulsion will float on cold water)
  • if using outbred animals, individual variation of immune reaction will be significant; make sure a large enough group is immunised

See also

External links

Protocol Online (methods forum)

Wikipedia

Personal tools