Generating antibodies: Difference between revisions
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=== Type of antibody === | === Type of antibody === | ||
{| {{table}} cellpadding=10 | {| {{table}} cellpadding=10 | ||
|valign=top| '''polyclonal''' (a mix of antibodies with different specificities) | |valign=top width=50%| '''polyclonal''' (a mix of antibodies with different specificities) | ||
* faster to generate | * faster to generate | ||
* less specific | * less specific | ||
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=== Type of antigen to use === | === Type of antigen to use === | ||
* full-length protein | {| {{table}} cellpadding=10 | ||
* | |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 == | == Peptide selection == |
Revision as of 05:35, 24 April 2008
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.
Early decisions
Type of antibody
polyclonal (a mix of antibodies with different specificities)
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monoclonal (many copies of the same antibody)
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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
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peptide
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Peptide selection
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
Other
- Peptide design check by Innovagen (no explanation of reasoning)
- Protein Colourer tool from the EBI may be helpful to inspect the sequence by eye
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. 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
- protein or peptide preparation is not pure; contaminant is more immunogenic than desired epitope; resulting antibody does not recognised the right target
See also
External links
- 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
- FAQ on antibody generation by Alta Bioscience, UK (2 pages of useful technical tips and a little advertisement)
- Backgrounder on history, generation, and application of monoclonal antibodies by Mark Soloski, Johns Hopkins
- Hybridoma and in vitro generation of antibodies, Antibody Facility, UNC Chapel Hill
- Monoclonal Antibodies: A Practical Approach by Philip Shepherd, Christopher Dean on Google Book Search
- Protocol for raising polyclonal antibodies in rabbits, Chew lab, Uni of Singapore
Protocol Online (methods forum)
- Protocol-online.org archive, keyword antibody and discussions indexed under antibody
- Discussion on affinity purification
- Discussion on antigen amount
Wikipedia