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Peptides created through GenScript's OptimumAntigen™ Design Program are optimized using the industry's most advanced antigen design algorithm. Each peptide is measured against several protein databases to confirm the desired epitope specificity. Benefits of using the OptimumAntigen™ Design Tool include avoidance of unexposed epitopes, ability to specify desired cross-reactivity, strong antigenicity of chosen peptide, identification of the best conjugation and presentation options for your desired assay(s), use of built in peptide tutorial for synthesis and solubility, and guaranteed immune response.
The following criteria are considered to optimize the likelihood of surface exposure and antigenicity:
1. Sequence length: The typical length of the peptide antigen for generating anti-peptide antibodies is in the range of 10-20 residues.
Why?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. Peptide sequences with length of 10-20 residues minimize synthesis problems since they are reasonably soluble in aqueous solution and may have some degree of secondary structure.
2. Hydrophilic, surface-oriented, and flexible: Peptide antigens are produced against epitope sequences which are most likely to be hydrophilic, surface-oriented, and flexible.
Why? Most naturally occurring proteins in aqueous solutions have their hydrophilic residues on the protein surface and hydrophobic residues buried in the interior. The strategy of optimizing antigen design to choose epitopes with hydrophilic, surface-exposed sequences is based on the fact that antibodies tend to bind to epitopes on the protein surface. Additionally, it has been shown that epitopes have a high degree of mobility, thus flexibility is also taken into consideration.
3. Targeting the N-terminus or C-terminus: Either the C-termini or N-termini are frequently chosen for generating anti-peptide antibodies.
Why?The C-termini and N-termini of proteins are often exposed and have a high degree of flexibility making either a good epitope candidate for antibody production. Though the C-termini is usually a good choice, if the target protein is an integral membrane protein and the C-terminus is part of the transmembrane segment, this sequence will be too hydrophobic to be a good candidate. In this type of circumstance, the N-terminus is a better choice. Also, the leader sequence should not be included in the sequence selected for antibody generation.
4. Continuous versus Discontinuous Epitopes: The OptimumAntigen™ Antigen Design tool will preferentially choose continuous epitopes (linear epitopes) instead of discontinuous epitopes.
What does this mean? Epitope sequences in proteins generally consist of 6-12 amino acids and can be classified as continuous or discontinuous. Continuous epitopes consists of a contiguous sequence of amino acids in a protein. Anti-peptide antibodies will bind to continuous epitopes in native protein when the epitope sequence is exposed on the surface of the protein. Discontinuous epitopes consist of sequences of amino acids that are not contiguous but are brought together when the peptide chain folds or by the juxtaposition of two separate polypeptide chains. Anti-peptide antibodies may or may not recognize a discontinuous epitope depending on whether the peptide used has a secondary structure similar to the epitope and/or if the protein epitope has enough continuous sequence for the antibody to bind with a lower affinity.
5. Algorithms: Our peptide antigen design algorithm takes into account hydrophilicity/hydrophobicity, secondary structure, common sequence motifs, and integrates commercially available algorithms to optimize the peptide design.
Hydrophilicity/Hydrophobicity Our antigen design tool integrates several well-known strategies for predicting hydrophilicity/hydrophobicity. Hydrophilicity plots as described by Hopp and Woods assign an average hydrophilicity value for each residue in the sequence and the highest point of average hydrophilicity for a series of contiguous residues is usually at or near an antigenic determinant. A slightly different algorithm described by Kyte and Doolittle evaluates the hydrophilic and hydrophobic tendencies of the sequence. This profile is useful for predicting exterior versus interior regions of the native protein.
Secondary Structure Secondary structure can be identified by the algorithms developed by Chou and Fasman or Lim. Knowledge of structure regions including alpha-helix, beta-sheet and beta-turn aids the selection process of a potentially exposed, immunogenic internal sequence for antibody generation. Surface region or regions of high accessibility often border helical or extended secondary structure regions. In addition, sequence regions with beta-turn or amphipthic helix character have been found to be antigenic.
Sequence Motifs We avoid common sequence motifs such as the RGD motif, the helix-loop-helix sequence, GTP binding sites, and SH2 domains which may cause cross-reactivity. We also avoid sequences associated with certain biological activities, such as autolytic cleavage hormonal activity, and undesired post- translational modifications.
Integration of multiple algorithms We incorporate many commercial software packages such as MacVector™, DNAStar™, and PC-Gene™ since the use of only one of these cannot offer the optimization gained from the combination of all. Combined use of several predictive methods may result in a success rate as high as 86% in predicting antigenicity.
6. Epitope Analysis and Coupling Strategy:Conjugation chemistries that best mimic epitope presentation are carefully selected.
What is the purpose? Peptides alone are usually too small to elicit an immune response sufficient to generate antibodies. This is why peptides of interest are conjugated to carrier proteins containing many epitopes that stimulate T-helper cells. These T-cells induce the B-cell response which generates the antibodies. Some of these antibodies will target the linker region and the carrier protein itself in addition to the peptide of interest. These non-specific antibodies can be removed during the purification or screening process. Specificallly, our coupling strategy maximizes epitope exposure within structural motifs and optimizes orientation to the termini.
Based on our records, at least 99.6% of the orders (pie chart on the right) for which peptide antigens are designed, synthesized and conjugated by GenScript received a higher ELISA titer (≥1:64,000) than guaranteed.
Designing the optimal synthetic peptide antigen is a crucial first step towards producing high quality custom antibodies. If you're struggling with choosing the best antigen for generating a custom antibody, our proven Peptide Antigen Database can help! Find the optimal peptide antigen for your protein of interest today.
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