Antigen Design Assistance
Overview:
To maximize the probability that antibodies against a synthesized peptide will recognize the native protein in the target assay, antigen design is a key consideration when choosing a peptide sequence. Pacific Immunology is happy to offer its clients antigen design assistance at no additional charge, and offers the following overview of antigen design principals.
Homology Considerations:
With regards to homology, there are two basic strategies that can be followed. The first is to choose a homologous peptide sequence that would allow a single antibody to recognize multiple proteins. The second is to choose a unique sequence that would help ensure specificity to the target protein. Either approach can be taken into consideration when analyzing the protein sequence(s).
Protein Structure:
If the 3D structure of the protein is known, this can of course greatly assist in choosing exposed epitopes. But in most cases, this information is unknown and so sequences must be chosen based on predictive algorithms.
Epitope Selection Strategy:
In general, most ideal antigenic epitopes are hydrophilic, surface orientated and flexible1. This is recommended because in most natural environments, hydrophilic regions tend to reside on the surface of proteins, while hydrophobic regions tend to be located in the interior of the protein. Similarly, antibodies can only bind to epitopes found on the surface of proteins and tend to bind with higher affinity when those epitopes are flexible enough to move into accessible positions.
Hydrophobicity:
To counter the risk of selecting an epitope that is buried within the protein, Pacific Immunology utilizes a proprietary combination of software algorithms that help determine hydrophilic regions that are most likely to be immunogenic and exposed on the native protein.
Targeting the N-terminus or C-terminus:
While ideal immunogenic targets can be found throughout the length of the protein sequence, there is somewhat less risk in choosing high-scoring sequences that are also located within the N-terminus or C-terminus of the protein, since these regions have a higher tendency to be exposed in the protein’s native conformation. However, it is important to note that the C-terminus of membrane proteins is often too hydrophobic to be an ideal antigenic target.
Sequence Length:
For most projects, peptide sequences between 10 and 20 amino acids in length are recommended. Within this range, shorter sequence can offer greater specificity, but at the risk of being less likely to be exposed on the native protein. Longer sequences, by contrast, might be slightly less specific, but offer a higher probability of recognizing the native protein. In general, we recommend 15aa as an average peptide length, with slightly longer sequences being preferred to slightly shorter sequences.
Carrier Protein Coupling Considerations:
To permit enhanced presentation of the peptide sequence to the animal’s immune system, Pacific Immunology recommends choosing a peptide sequence that doesn’t contain internal cysteines, and then adding a cysteine to either the N-terminal or C-terminal of the peptide sequence. The peptide can then be coupled via the terminal cysteine, permitting maximum exposure of the peptide sequence in comparison to more general conjugation chemistries such as glutaraldahyde.
Continuous versus Discontinuous Epitopes:
Most antibodies target continuous epitopes - epitopes that represent a continuous sequence of amino acids. Antibodies will bind to these regions with high affinity provided that the sequence is not located within the protein's interior. In some cases, antibodies against discontinuous epitopes (epitopes that represent a fold in a peptide sequence or even a joining of two separate peptide chains) can be generated, although this requires that the peptide used for immunization has a secondary structure similar to the epitope and that the sequence length falls within the guidelines listed below.
Guarantees and Risks:
While Pacific Immunology guarantees antibody titers against peptide sequences that it synthesizes, it unfortunately isn’t possible to guarantee how antibodies against that peptide will work in any particular assay. Success rates using sequences that we recommend are generally quite high, but it is important to keep in mind that there is always an element of risk involved with any custom antibody project.
References
1) Van Regenmortel, M.H.V., 1986, Trends in Biochemistry, 11:36-39.