Updated Nov 18, 2015
Using synthetic peptides as antigens is a strategy that may be called for when developing monoclonal antibodies. There are many merits to using synthetic peptides as antibody generators. For a monoclonal antibody project the main advantage is that the antibody production can be directed towards a certain domain or specific site of interest.
When the immunogenic antigen is a synthetic peptide designed and provided by us, we always obtain a polyclonal response when immunised in our BALB/c mice. In fact, we guarantee a positive result in our ELISA; there are antibodies present in the immune sera that recognise the peptide.
Using peptides as antigens is a very successful strategy for raising polyclonal antibodies. For monoclonal antibodies it is a bit more challenging. The main issue here is that there is an increased risk that you may end up with an antibody that specifically targets the peptide with no cross reactivity with the protein of interest. This problem cannot be overcome by peptide design alone.
The principles for the design of a peptide antigen are the same whether the objective is to obtain a polyclonal or a monoclonal antibody. 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;
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 physiochemical properties of a given peptide. Other programs takes a protein sequence as input and 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. In our peptide design service, 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 peptides makes for some interesting reading. Peptide quality does however have a direct impact on the outcome of your monoclonal antibody project, and more so when the peptide is also used for the screening procedure in the hybridoma development step.
Typically a number of mice are immunised for the purpose of producing monoclonal antibodies. Other hosts could be considered, but mice are still the host of choice in most situations. The mice are immunised until the immune response, as evaluated by ELISA, is deemed to be strong enough. One may be tempted to accelerate the immunisation protocol in order to save time, but this may have adverse effects on the end product. The idea is that, over time the immune system will have naturally selected the better antibodies.
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. After fusion with myeloma cells, a selection process starts, where cells that express antibodies that test positive against the antigen are isolated. Typically a selection is made such that the cells that produce the antibodies that perform better in the screening ELISA are retained. This is a crucial moment in the monoclonal antibody development. If a synthetic peptide is used for this screening process there is the possibility that the antibody that is ultimately selected, only targets the peptide and not the native protein. Thus, whenever possible, the screening should be done using the target protein as substrate.
In many cases the obtained monoclonal antibody will work as intended without the need for further manipulation. There are however scenarios where you require a certain isotype, or a chimeric antibody, or even a fully humanised antibody.
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.
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