In this article, we discuss methods for generating custom antibodies using peptide antigens including peptide antigen design considerations and options for generating antigenic peptides.
Custom Peptide Synthesis: Peptides for Raising Antibodies
In general, synthetic peptides are too small to elicit an immunogenic response. Peptide antigens can be coupled onto a carrier protein or synthesised onto a larger molecule to ensure that it will be large enough to be detected by the immune system.
1. Multiple Antigenic Peptides (MAPs)
MAPs are octomeric molecules with peptide chains branching out from a central poly-lysine core. The eight peptide chains increase the molecular weight of the compound sufficiently for it to be easily recognised as an antigen. It provides an easy and flexible method for antibody production and is a method best suited for regions away from the C-terminus of a protein.
It is also possible to make chimeric MAPs containing two different peptide sequences, each forming four of the chains of the 8-branched structure. This option could provide a method of presenting 2 peptide antigens in a single inoculation.
Optional purification for MAP peptides is through a 2-3kDa membrane.
In addition to antigen synthesis we can also simultaneously synthesise an affinity column for use in antibody purification.
2. PEG-Peptide Antigens
An alternative to MAPs, antigens made from peptides conjugated to polyethylene glycol (PEG) provide a prolonged residence post inoculation, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity.
3. Peptide–protein conjugates
By conjugating keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) or other suitable proteins via a linker molecule, peptides can be prepared for use in antibody generation. The size of the protein triggers the antibody system, which recognises the attached peptides. The most popular carrier proteins are BSA and KLH, which elicits a strong antibody response and contains a very large number of lysine residues which are used to attach the peptide antigen.
This particular approach can be used to attach the peptide in any orientation, i.e. at either the N or the C terminus. However, it is not suitable for peptides containing cysteine, as that amino acid is added to the sequence to act as the linker to the protein.
Peptide antigen design considerations
We have considerable experience in peptide antigen design and will work with you to achieve the best chance of success. In general, peptides for antibodies will be hydrophilic and flexible coming from the exterior of the parent protein. A hydophilicity plot will indicate which parts of the protein are likely to be on the outside of the structure. The Kyte-Doolittle or the Hopp-Woods algorithms will be very useful here. Structure predictions can be done with Chou-Fasman plots. Peptide epitope regions containing cysteine should be avoided where possible as these in nature would present a very different shape to the immune system compared with the disulphide bridged cystine.
We work with partners to provide full monoclonal and polyclonal antibody generation and purification programmes, please contact us to discuss your requirements.