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In order to obtain the best effect of producing antibodies, it is necessary to carefully design the antigen polypeptide, and the design should meet a basic condition: in the process of immunization, the antigen will not produce an excessive immune response, and at the same time it can produce generate antibodies that bind to the protein of interest.
It is generally recommended that the sequence length of the antigenic polypeptide be between 8-20 amino acid residues. If it is too short, there is a risk that the specificity of the polypeptide will not be strong and the binding ability between the generated antibody and the native protein will not be strong enough; however, if the sequence length exceeds 20, it will be possible to introduce secondary structure, and the resulting antibody may lose its specificity, and the longer the peptide chain, the more difficult it is to synthesize, and it is difficult to obtain high-purity products. With years of rich experience in the synthesis of difficult peptides, KS-V Peptide can efficiently and quickly provide high-purity finished antigen peptides to assist your research and development.
Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.
First of all, peptide antigens offer a smart solution for antibody generation in cases where the target protein’s amino acid sequence is known, but the protein itself is not available, or structurally too complex to be obtained as a structurally stable and intact protein (e.g. certain transmembrane receptors, ion channels, etc.). Moreover, the peptide immunogen approach offers flexibility in selecting and designing antigens for domains of the target protein as an additional advantage. Peptide antigens are also highly useful for generating antibodies against post-translationally modified binding sites on target proteins.
Other advantages are:
Although many peptide sequences can be immunogenic, not all are equally effective in generating antibodies with reactivity against the target protein. Successful antibody generation with peptide antigens depends on several factors that need to be considered in the design, including:
the accuracy of the target protein’s amino acid sequence
the predicted 2ary / 3airy structure of the intact protein
the selection of domains/regions of the protein to be mimicked by the peptide antigen
the need of conjugating peptide antigens to a larger carrier protein
the ease of synthesis of certain peptide sequences
Therefore, the following general recommendations apply to the design of peptide antigens:
Ensure that the correct species and protein sequence have been identified.
Select the peptide antigen from a solvent-accessible region of the native protein. Often, these are regions exposed at the protein surface that are in contact with the aqueous (hydrophilic) environment of the solvent. Certain areas of the protein may be inaccessible to antibodies, such as transmembrane regions or the centre of a globular protein.
An important advantage is that peptide antigens can be designed to cover a specific epitope of interest. Peptide antigen candidates are screened against a specific protein database. Certain domains may be also present in other proteins: these sequences should be avoided in order to minimize the likelihood of unwanted cross-reactivity and thus optimize your overall antibody-specificity. Sequences with minimal sequence homology are best selected in order to reduce unwanted off-target protein binding.
Depending on the 3D-structure of the targeted domain in the intact protein, constraining techniques may be required to optimally mimic the tertiary structure of the antigen. As one of the specialist in protein surface mimicry, KS-V has available various (partly proprietary) technologies for the design and synthesis of conformationally-constrained peptide antigens
gens are often too small to generate a significant immune response without prior conjugation to a carrier protein such as KLH, BSA or OVA. Usually the use of peptide – carrier protein conjugates is therefore recommended.
For the design of the peptide antigens the ease of synthesis of specific peptide sequences should also be considered. Both hydrophobic and hydrophilic residues should be included, and it is favorable for the peptide to incorporate amino acids that are immunogenicity-promoting (such as basic and aromatic amino acids). Hydrophobic amino acid content should best be kept below 50%, and long chains of hydrophobic residues should preferably also be avoided. For peptide solubility, at least one charged residue (arginine, glutamine, aspartic acid, or lysine) should be incorporated within every five amino acids. Peptide solubility can also be improved via conservative replacements or addition of polar residues to the N- or C-terminus. In designing the antigens preferably avoid complex regions, such as beta-sheets or alpha-helices, but instead aim for flexible regions.
KS-V applies its state-of-the-art capabilities and expertise for the synthesis of peptide antigens, including (if required) conformational constraints, PTM’s, conjugation or biotinylation. For most antibody generation projects a purity at least 85% is recommended for any peptide antigen. However, if desired we can provide antigens with higher purities also. We generally will synthesize approximately 10 mgs of peptide, which is usually sufficient for protein-conjugation, ELISA-screening and constructing an affinity matrix chromatography setup if desired.
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In order to obtain the best effect of producing antibodies, it is necessary to carefully design the antigen polypeptide, and the design should meet a basic condition: in the process of immunization, the antigen will not produce an excessive immune response, and at the same time it can produce generate antibodies that bind to the protein of interest.
It is generally recommended that the sequence length of the antigenic polypeptide be between 8-20 amino acid residues. If it is too short, there is a risk that the specificity of the polypeptide will not be strong and the binding ability between the generated antibody and the native protein will not be strong enough; however, if the sequence length exceeds 20, it will be possible to introduce secondary structure, and the resulting antibody may lose its specificity, and the longer the peptide chain, the more difficult it is to synthesize, and it is difficult to obtain high-purity products. With years of rich experience in the synthesis of difficult peptides, KS-V Peptide can efficiently and quickly provide high-purity finished antigen peptides to assist your research and development.
Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.
First of all, peptide antigens offer a smart solution for antibody generation in cases where the target protein’s amino acid sequence is known, but the protein itself is not available, or structurally too complex to be obtained as a structurally stable and intact protein (e.g. certain transmembrane receptors, ion channels, etc.). Moreover, the peptide immunogen approach offers flexibility in selecting and designing antigens for domains of the target protein as an additional advantage. Peptide antigens are also highly useful for generating antibodies against post-translationally modified binding sites on target proteins.
Other advantages are:
Although many peptide sequences can be immunogenic, not all are equally effective in generating antibodies with reactivity against the target protein. Successful antibody generation with peptide antigens depends on several factors that need to be considered in the design, including:
the accuracy of the target protein’s amino acid sequence
the predicted 2ary / 3airy structure of the intact protein
the selection of domains/regions of the protein to be mimicked by the peptide antigen
the need of conjugating peptide antigens to a larger carrier protein
the ease of synthesis of certain peptide sequences
Therefore, the following general recommendations apply to the design of peptide antigens:
Ensure that the correct species and protein sequence have been identified.
Select the peptide antigen from a solvent-accessible region of the native protein. Often, these are regions exposed at the protein surface that are in contact with the aqueous (hydrophilic) environment of the solvent. Certain areas of the protein may be inaccessible to antibodies, such as transmembrane regions or the centre of a globular protein.
An important advantage is that peptide antigens can be designed to cover a specific epitope of interest. Peptide antigen candidates are screened against a specific protein database. Certain domains may be also present in other proteins: these sequences should be avoided in order to minimize the likelihood of unwanted cross-reactivity and thus optimize your overall antibody-specificity. Sequences with minimal sequence homology are best selected in order to reduce unwanted off-target protein binding.
Depending on the 3D-structure of the targeted domain in the intact protein, constraining techniques may be required to optimally mimic the tertiary structure of the antigen. As one of the specialist in protein surface mimicry, KS-V has available various (partly proprietary) technologies for the design and synthesis of conformationally-constrained peptide antigens
gens are often too small to generate a significant immune response without prior conjugation to a carrier protein such as KLH, BSA or OVA. Usually the use of peptide – carrier protein conjugates is therefore recommended.
For the design of the peptide antigens the ease of synthesis of specific peptide sequences should also be considered. Both hydrophobic and hydrophilic residues should be included, and it is favorable for the peptide to incorporate amino acids that are immunogenicity-promoting (such as basic and aromatic amino acids). Hydrophobic amino acid content should best be kept below 50%, and long chains of hydrophobic residues should preferably also be avoided. For peptide solubility, at least one charged residue (arginine, glutamine, aspartic acid, or lysine) should be incorporated within every five amino acids. Peptide solubility can also be improved via conservative replacements or addition of polar residues to the N- or C-terminus. In designing the antigens preferably avoid complex regions, such as beta-sheets or alpha-helices, but instead aim for flexible regions.
KS-V applies its state-of-the-art capabilities and expertise for the synthesis of peptide antigens, including (if required) conformational constraints, PTM’s, conjugation or biotinylation. For most antibody generation projects a purity at least 85% is recommended for any peptide antigen. However, if desired we can provide antigens with higher purities also. We generally will synthesize approximately 10 mgs of peptide, which is usually sufficient for protein-conjugation, ELISA-screening and constructing an affinity matrix chromatography setup if desired.