The successful production of antibodies that specifically target neo-epitopes depends on peptide design, peptide quality and antibody purification strategy. Done right, synthetic peptides are perfect as antigens for neo-epitope antibody projects. They present a short linear epitope, which is just what is required. In general there is no need to go for a monoclonal antibody if the objective is to obtain a research antibody that specifically targets the neo-epitope.
In addition to some other general design considerations, there are a few things to keep in mind.
The longer the peptide is, the more trivial epitopes are exposed. Thus the peptide should be just long enough so that a decent yield is likely to be obtained. About 7-10 amino acids is typically right, although other issues like solubility and cross reactivity may have to be factored in.
Since we are limiting the number of epitopes that are exposed it is likely that yields are comparatively lower. Every epitope counts and thus - in contrast to most polyclonal antibody projects, where ImmunoGrade peptides work very well - the immunogenic peptide for generating neo-epitope should be of high purity.
A common misconception is that peptide quality should be judged by HPLC purity alone, and that this purity will be the same in any analytical HPLC set-up. This is discussed in detail in our views on producing high quality peptides. Here it suffices to point out that the quality of the peptide may be pivotal for the outcome of the project.
It is true that we guarantee that the host will yield antibodies against the synthetic peptide when we have designed and produced the synthetic peptide. However, there are individual differences in the animals and for these types of projects in particular the differences can be striking. It is recommended that two animals are used.
Careful design, high quality peptides and the use of multiple animals will increase the likelihood for obtaining the desired antibody. Affinity purification should always be done in order to increase the usability of the antibody. But, although we have done everything right, cross reactivity with the non-cleaved protein may be an issue. This is overcome by using a second peptide, a few amino acids longer, spanning over the cleavage site, for absorbing the cross reacting antibodies.
Innovagen was founded in 1992, at a time when anti-peptide antibodies were not a standard research tool in a way that they have become today. Over the years we have produced antibodies against a variety of targets, including more demanding projects such as neo-epitopes that require the dedication to quality that we provide.
Examples of references of customers using our custom neo-epitope specific antibody service include;
Struglics A, Hansson M.
Calpain is involved in C-terminal truncation of human aggrecan.
Biochem J. 2010 Sep 15;430(3):531-8.
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