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Immunization with just peptide antigens is very often not successful, as peptide are often too small to generate significant immune responses on their own. In order to solve this problem, the peptides should be conjugated to large carrier proteins, such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or Ovalbumin (OVA). To ensure the effective and successful generation of anti-peptide antibodies, the use of peptide – protein conjugates is always recommended.
KLH (MW 4.5×105 – 1.3×107 Da) is the most commonly used carrier protein, most likely because it has a significantly higher immunogenicity as compared to other proteins. KLH-conjugated peptides often have limited solubility in water, which results in a cloudy appearance. The turbidity does not affect its immunogenicity and the resulting solution can be used for successful immunizations.
BSA (bovine serum albumin, MW 67×103 Da) is one of the most stable and soluble albumins available. It contains 59 lysines, of which approx 30-35 are accessible for conjugation. Therefore BSA is a popular carrier protein for weakly antigenic compounds.
OVA (ovalbumin, MW 45×103) is a protein isolated from chicken eggs. It is often used as a control carrier protein to verify that antibodies are specific for the target peptide rather than the carrier protein (e.g. KLH/BSA).
The most frequently applied conjugation method for peptides to proteins is based on ‘thiol–maleimide’ chemistry. For this conjugation, the peptide is synthesized with an additional cysteine residue added to the N- or C-terminus, so that it may be linked to the carrier protein. The thiol provided by the cysteine is subsequently chemoselectively coupled to a maleimide modified carrier protein.
The conjugates are supplied as chilled solutions since it has been shown that either reconstituted freeze dried materials or frozen solutions deliver sub-optimal immunization results