Protein Conjugates /Immunogenic Peptides
Peptides alone are often too small to generate an antigenic immune response. Therefore, the generation of anti-peptide antibodies requires conjugation to large carrier proteins. When administered in vivo, the immune system reacts to these peptide-protein conjugates resulting in antibody generation. However, the antibodies often target not only the peptide of interest but also the carrier protein and the linker region. Ideally, the undesired antibodies need to be removed by purification from the desired peptide-specific antibody.
Examples of frequently used carrier proteins for peptide conjugates are listed here:
- KLH (keyhole limpet hemocyanin, MW 4.5x105 - 1.3x107 Da) is the most frequently used carrier protein because of its high immunogenicity compared to other proteins. Note that the solubility of KLH in water is limited. This makes solutions sometimes appear cloudy, though this does not affect immunogenicity. KLH is routinely used for vertebrate research because there are no homologous vertebrate proteins resulting in little if any non-specific antibody activity. For invertebrate studies, it is best to use a carrier such as BSA or ovalbumin.
- BSA (bovine serum albumin, MW 67x103 Da) is a popular carrier protein for weakly antigenic compounds because of the high number of accessible Lys residues for conjugation.
- HSA (human serum albumin, MW 67 x103 Da).
- OVA (ovalbumin, MW 45x103) is a protein isolated from chicken eggs. It is often used as a control carrier protein to verify if antibodies produced with KLH/BSA are specific for the target peptide vs. the carrier protein.
The most common conjugation method for peptides to proteins is based on thiol maleimide chemistry – a highly specific and robust reaction. Generally the peptide is synthesized containing an additional cysteine residue that can be added C‑terminally or N‑terminally. For conjugation, the thiol provided by the cysteine is chemoselectively coupled to a maleimide modified carrier protein.
Conjugates are shipped as chilled solutions since it has been shown that either frozen solutions or reconstituted freeze dried materials show sub-optimal immunization results.
Multiple antigenic peptides (MAPs) are a popular alternative to the conjugation of peptides to carriers. They are composed of multiple peptide branches that are covalently bound to a central backbone composed of lysine residues. Because of the multiplicity of the peptides, MAPs are able to induce immune responses. Therefore, MAPs can be used for antibody production without the need for conjugation to a carrier protein. In general, multiple antigenic peptides are available with 4, 8, 12 or more branches, though those most frequently used contain 4 and 8 branches.