Protein vs Peptide Antigens
To generate a custom antibody, there are two popular approaches that can be considered. The first strategy is to immunize with the full length protein, which can be soluble or insoluble and can be provided in forms such as recombinant, fusion, gel band, etc. The second strategy is to immunize with a peptide sequence corresponding to a fragment of the full length protein.

If an existing supply of the full-length protein is available, then using this as the antigen may be a convenient and cost effective option. The biggest benefit with using the full-length protein is that antibodies will be generated against multiple epitopes from throughout the sequence. As a result, there is a very high probability that antibodies against at least one of these epitopes will bind with the native protein in the target assay.

The downside with this approach is that because antibodies are being generated against multiple epitopes, there is a higher probability that antibodies against some of these epitopes could recognize other proteins in the sample that contain homologous epitopes. As a result, non-specific cross reactivity might be a problem when assaying with antibodies developed against the full length protein.

Affinity purification of the serum against the protein can help eliminate non-specific, naturally occurring antibody complement that is in the serum. But, this doesn’t help eliminate the problem of cross reactivity against homologous epitopes contained within the full length protein sequence. In addition, affinity purification isn’t possible with all proteins depending on their solubility and ability to be successfully coupled to an affinity column.

One last downside to consider is that recombinant expression of the protein is sometimes costly and that the expressed protein may not completely match all characteristics of the native protein.

As a result, immunizing with a peptide sequence that corresponds to a specific region of the full length protein is a popular strategy for developing custom antibodies. With this approach, it isn’t necessary to have a supply of the full length protein – only the protein’s amino acid sequence is needed. From this, it is possible to analyze the sequence and, with the use of sophisticated algorithms (or 3D structural models), predict regions that are predicted to correspond to exposed regions of the native protein. In addition, homologous regions can be avoided, thereby helping minimize cross reactivity when assaying with the antibody.

Most peptide sequences will be 10-20 amino acids in length, which provides optimal epitope diversity while still offering superior specificity compared to immunizing with the full length protein sequence.

Once a peptide sequence has been chosen, it must be coupled to a carrier protein before immunizations begin (this is because the molecular weight of the peptide by itself isn’t large enough to stimulate an immune response). KLH is most commonly used for this and helps to further enhance the immune response because of its distinct phylogenetic separation from mammalian species.

Antibodies will be generated against the peptide sequence and the carrier protein, but the serum is affinity purified against the peptide sequence in order to isolate only those antibodies in the serum that bind with the peptide sequence. This offers a significant advantage compared to immunizing with a full length protein since the resulting highly specific “polyclonal” antibodies against the peptide sequence will approach a monoclonal antibody in specificity.

In addition, because the resulting purified antibodies may recognize several epitopes on the peptide sequence, antibody affinity will be superior to a monoclonal antibody where only a single epitope can be recognized (see Polyclonal vs Monoclonal Antibodies).

In our opinion, this strategy offers the best combination of specificity and affinity towards the native protein. Our Monospecific Custom Antibody Production package is our most popular service and includes everything needed to pursue this strategy.

The downside with using a peptide sequence is that, like a monoclonal antibody, it corresponds to a very specific region of the native protein and this region may not be accessible in the protein’s native conformation in a particular assay. As a result, it may be necessary to synthesize several peptides from unique regions of the native protein to find one region that is accessible in the target assay (or in multiple assays).

To help minimize the trial and error that can be associated with using a peptide sequence, careful selection of the peptide sequence is critical. Pacific Immunology® offers its innovative and proprietary protein analysis and sequence recommendation service at no additional charge for its customers.

Next: Selecting a Peptide Sequence