Antibodies raised against synthetic peptides
Using synthetic peptides as antigens is a common strategy for developing polyclonal antibodies. There are many merits to using synthetic peptides as antibody generators. Where a protein might be time-consuming, difficult or expensive to produce, a peptide is comparatively inexpensive and easy to make. Furthermore, a protein may not be suitable for immunisations due to such things as being toxic or too similar to proteins native to the host. Using shorter peptides solves these particular problems. When certain domains or sites need to be targeted, as is the case with a post translational modification such as phosphorylation, using a peptide the antibody production can be directed towards the area of interest. When the polyclonal antibodies are purified by antigen affinity purification, they are said to be monospecific antibodies.
This document relates to the production of polyclonal antibodies, and although much applies to mAbs as well, you may also want to have a look at Monoclonal antibodies raised against synthetic peptides, which describes the additional considerations for those types of projects.
When the immunogenic antigen is a synthetic peptide designed and provided by us, we always obtain an antibody when immunised in our hosts, e.g. rabbit, chicken, goat, mouse. In fact, we guarantee a positive result in our ELISA; in the immune sera or purified sample there are antibodies present that recognise the peptide. If the peptide mimics the protein well enough, chances are good that the antibodies will react with the intended protein too. How well they will perform in a certain application with the target protein is however unknown beforehand.
Producing an antibody that reacts with the immunogen (the peptide) is one thing but what matters is that the resulting antibody works for you, in your application, and with your protein. What can be done is to create a basis that increases the likelihood for a successful outcome as much as possible. As more effort is put into the individual steps of the antibody production, the odds for success improve. These steps can be grouped into topics, briefly described on this page but in more detail on other pages;
The task of picking out a suitable sequence might be daunting, but since it can be defining for the success of the project it is well worth the effort. A few of the considerations when designing synthetic peptides as antigens for antibody production are;
- Is the sequence in a region or domain of interest?
- Does the peptide mimic the target protein well enough?
- Is it soluble?
- Is it suitable for immunisation, conjugation, and synthesis?
- Is it made up of a low complexity sequence, common patterns or motifs, or are there other homology issues to consider?
There are a lot of freely available pieces of software that will provide various predictions, which can be very useful. As an example we have our own peptide property calculator PepCalc.com, which will provide estimations of physiochemical properties of a given peptide. Some programs present you with one or more peptide sequences. All these programs are potentially useful as aids in your peptide design work, but no more. The predictions are not very accurate, and not all important aspects that apply to your particular project may have been considered. We use our own proprietary software, PeptideCAD™, and although it takes all relevant parameters into account, it is still treated as just an aid – albeit a very useful one - for our experienced personnel in selecting suitable sequences.
That said; Typically the best results are achieved when we can work together and combine your knowledge of the target protein with the PeptideCAD™ software and our experience from thousands of anti-peptide antibody projects.
Designing a good peptide for immunisations is of course crucial, but the actual quality of the peptide is also important. There is much more to this than the reported purity of one QC set-up and the topic of the production of high quality antibody generating peptides makes for some interesting reading, if perhaps a bit lengthy. It does however have an impact on the final product and may certainly be pivotal for the usefulness of your antibody, and thus the whole project. Some projects will be more demanding than others. For example making phospho specific anti-peptide antibodies, or other PTMs, neo-epitopes, protein fusion sites, or exon-bridges.
Should one, two or more animals be used, and will a short immunisation protocol be fine?
Since we guarantee that a host that we immunise with our peptides will produce anti-peptide antibodies, one animal suffices. Certainly, a second animal might produce better antibodies, but do you really need this? The main concern however is that although the animal produces antibodies that target the peptide, this does not necessarily mean that the antibodies will work in your application with your protein. The careful design and high quality production of the peptide are key to success, but there are potentially large individual differences in the animals. We have seen examples where both animals produce excellent antibodies that react with the peptide, but only one works in the end user’s application. For each additional animal that is immunised the odds for success is improved.
Some rare projects call for short immunisation protocols. These include projects where IgM is favoured over IgG, or where the antigen is such that the immune response drops rapidly over time - such antigens are typically not peptides. When producing anti-peptide antibodies, however, where IgG is the desired antibody class, it is better to have a longer schedule. Taking rabbits as an example, this means a test bleed after 5-7 weeks after primary injection in order to verify that the immunisation has been successful. Although sera containing useful IgG may be collected earlier, the likelihood for obtaining excellent antibodies typically increases with time, towards a plateau, during the first 3-4 months. Depending on the individual rabbit and the antigen, titre levels can then be maintained for several years with booster injections made at intervals.
Over the years we have developed standard immunisation protocols for rabbit anti-peptide antibody production, chicken, goat, mouse, guinea pig and rat. These protocols have proven to work very well. As mentioned before; we will guarantee that the immune serum will contain antibodies targeting the antigen – provided that we have been involved in the design and production of the immunogenic peptide.
With careful selection of the peptide sequence, high quality peptide synthesis and the use of a suitable immunisation strategy, you have increased the likelihood for obtaining a great antibody. In many cases the crude antiserum will work well without the need for any sort of purification, although certain applications, such as immune-precipitation, nearly always require antigen specific affinity purified antibodies. If the antibodies are to be labelled, then they should at least be purified by Protein-G or similar so that the labelling is performed on the total IgG fraction. Other projects may require purification in order to achieve the desired specificity. As an example; an antibody that is to discriminate between a phosphorylated and non-phosphorylated protein.
Innovagen was founded in 1992. Since then we have been providing peptide designs, syntheses, and their resulting antibodies for thousands of projects. You and your research will also benefit from this experience when you turn to us for your custom anti-peptide antibody requirements.
Examples of references of customers using our custom anti-peptide antibody services include;
Parhamifar L, Sime W, Yudina Y, Vilhardt F, Mörgelin M, et al. 2010
Ligand-Induced Tyrosine Phosphorylation of Cysteinyl Leukotriene Receptor 1 Triggers Internalization and Signaling in Intestinal Epithelial Cells.
PLoS ONE 5(12): e14439. doi:10.1371/journal.pone.0014439
Steinfeldt T, Könen-Waisman S, Tong L, Pawlowski N, Lamkemeyer T, et al. 2010
Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii.
PLoS Biol 8(12): e1000576. doi:10.1371/journal.pbio.1000576
M W Hornef, K Putsep, J Karlsson, E Refai, M Andersson. 2004
Increased diversity of intestinal antimicrobial peptides by covalent dimer formation
Nature Immunology 5, 836-843 doi:10.1038/ni1094
Holmlund, U., Cebers, G., Dahlfors, A. R., Sandstedt, B., Bremme, K., EkstrÖm, E. S. and Scheynius, A. (2002)
Expression and regulation of the pattern recognition receptors Toll-like receptor-2 and Toll-like receptor-4 in the human placenta.
Immunology, 107: 145–151. doi: 10.1046/j.1365-2567.2002.01491.x
P-J Jakobsson, S Thorén, R Morgenstern, and B Samuelsson. 1999
Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.
PNAS 1999 96 (13) 7220-7225; doi:10.1073/pnas.96.13.7220