Generating antibodies
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 (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 selection
![Example of a protein's hydrophobicity according to 2 different scales. Made with R. Bowen's [Image:Hydrophobicity plot example.png tool].](/wiki/File:Hydrophobicity_plot_example.png)
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
- ExPASy's classic ProtScale tool allows you to check a protein sequence against various amino acid scales including several for hydrophilicity/-phobicity
- Peptide design check by Innovagen
- Hydrophobicity tool by Colorado State Uni - well documented
- 2 hydrophobicity tools from the Institute Pasteur: pepwindow and octanol
- 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.
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
