Overview:
To maximize the probability that antibodies against a synthesized peptide will recognize the native protein in the target assay, it’s critical to choose a peptide sequence that is predicted to correspond to a region of the native protein that is exposed in the target assay. Pacific Immunology® offers its clients antigen design assistance at no additional charge, and uses a unique and proprietary set of sequence analysis and protein folding prediction algorithms to maximize the potential to recognize the native protein in multiple assays. Here is an overview of antigen design principals that we consider.
Antigen Prediction Algorithms:
Pacific Immunology® has developed a unique and proprietary set of algorithms that allow us to analyze a protein and predict with above average certainty which regions of the protein will be exposed when the protein is folded into its native conformation. Many of these factors are based on our experience in developing tens of thousands of peptide antibodies.
Homology Considerations:
With regards to homology, there are two basic strategies to consider. The first is to choose a homologous peptide sequence that would allow a single antibody to recognize multiple similar 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:
Protein folding prediction algorithms continue to improve, and while they still can’t predict protein structures with 100% certainty, they can provide valuable insights with certain proteins. In some cases, 3D crystal structures for the target protein already exist and can help confirm accessible or exposed domains of the protein. It is important to note that even if the structure is known, exposed regions could still be inaccessible in certain assays.
Epitope Selection Strategy:
A typical peptide sequence of 15 amino acids in length will contain several epitope possibilities (5-7aa) against which individual antibodies will be generated. Ideally, this peptide sequence will correspond to a region of the protein that is exposed and accessible when the protein is folded into its native conformation. In general, these exposed regions often correspond to hydrophilic regions of the protein. In addition, there is typically some flexibility in the structure of the protein in these locations so that epitope accessibility is maximized.
Hydrophobicity:
Hydrophobicity of the amino acids is one factor that we take into account when selecting a peptide sequence to target. Hydrophilic regions have a higher probability of corresponding to exposed regions of the protein, while hydrophobic regions have a higher probability of corresponding to interior regions of the native protein. To counter the risk of selecting an epitope that is buried within the protein, Pacific Immunology® utilizes a proprietary combination of software algorithms to help predict peptide sequences that are likely to be exposed. One factor that is considered is the hydrophobicity of amino acids, with hydrophilic regions having a higher probability of being immunogenic and exposed on the native protein.
Targeting the N-terminal or C-terminal of the Protein:
While ideal immunogenic targets can be found throughout the length of the protein sequence, targeting the N or C terminal of the protein has traditionally been a popular approach in the industry since there seems to be a higher probability that these regions will be exposed. Peptide sequences that are located near either terminal and that score well according to our prediction algorithms may be somewhat less risky choices. However, we generally don’t recommend targeting the N-terminal of most proteins since this typically serves as s signaling peptide and is therefore often cleaved. Also, the C-terminal of membrane proteins has a higher probability of being too hydrophobic to be an ideal antigenic target.
Sequence Length:
For most projects, peptide sequences between 10 and 20 amino acids in length are ideal. Within this range, shorter sequence can offer greater specificity, but provide less epitope variability. Longer sequences, by contrast, offer a slight reduction in specificity, but offer increased epitope variability and therefore a higher probability of recognizing the native protein. In general, we recommend using a peptide of approximately 15 amino acids in length.
Carrier Protein Coupling Considerations:
To enhance 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 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. Antibodies against discontinuous epitopes (epitopes that exist when two discontinuous regions of a protein come together during folding) do exist, but it’s difficult to target these with peptide sequences or predict the structure of the epitopes.
Guarantees and Risks:
While Pacific Immunology® guarantees antibody titers against peptide sequences that it synthesizes and recommends, it unfortunately is not possible to guarantee how antibodies against that peptide sequence will work when assaying the native protein in a particular assay. The predictive algorithms that we use maximize the probability of recognizing the native protein in at least one assay such as a western blot. But, it’s important to understand that there is an element of risk associated with using any peptide sequence and that it isn’t uncommon to target several peptide sequences from a particular protein in order to find one that will work best in the target assay.