Peptide Modifications: KLH, BSA, OVA Conjugates

Peptide-protein conjugates are used for antibody production against peptides. Peptides alone are mostly too small to elicit a sufficient immune response, so carrier proteins containing many epitopes help to stimulate T-helper cells, which help induce the B-cell response.

It is important to remember that the immune system reacts to the peptide-protein conjugate as a whole – so there will always be a portion of antibodies to the peptide, the linker and the carrier protein. Among the most common carrier proteins one can find:

• KLH (Keyhole Limpet Hemocyanin), a copper containing, non-heme protein found in arthropods and mollusca. It is isolated from Megathura crenulata and has a MW of 4.5 x 105 ~ 1.3 x 107 Da. KLH is the most commonly selected carrier due to its higher immunogenicity compared to BSA.


• BSA (Bovine Serum Albumin), a plasma protein in cattle, belonging to the most stable and soluble albumins. It has a MW of 67 x 103 Da – containing 59 lysines. About 30-35 of these primary amines are accessible for linker conjugation, which makes BSA a popular carrier protein for weak antigenic compounds.
  
  A disadvantage of BSA is, that it is used in many experiments as a blocking buffer reagent. If antisera against peptide-BSA conjugates are used in such assays, false positives can occur, because these sera also contain antibodies to BSA.


• OVA (Ovalbumin), a protein isolated from hen egg whites, with a MW of 45 x 103 Da. It is a good choice as second carrier protein to verify if antibodies are specific for the peptide alone and not the carrier protein (e.g. BSA).

Thiol group modifications (via Cys side chain) are used for KLH, BSA or OVA conjugation.

All modifications carrying thiol-reactive functional groups can be used. Among the most commonly used ones, you can find:

• Iodoacetamides
• Maleimides
• Alkyl halides



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