Peptide Conjugation

Conjugating a peptide to a carrier protein is critical in generating anti-peptide antibodies. Finding the best coupling strategy to cross-link peptides to an appropriate carrier molecule is often overlooked during the peptide design stage. Selecting the most suitable carrier protein and choosing the correct conjugation chemistry will significantly improve the chance of producing high-titer antibodies for your antigen of interest. Peptides used for antiserum production require conjugation to a carrier since the peptide alone is usually insufficient in generating an immune response. Selection of a coupling strategy becomes extremely important since the conjugated immunogen should present an epitope similar to that observed in the native protein, with the correct orientation and flexibility. An alternative approach is using the MAP strategy for synthesizing the peptide to generate antibodies.

There are 3 items to consider when determining peptide conjugation

1. Carrier Protein: attachment of the peptide segment to a carrier protein is a key factor in eliciting an immune response.
   • KLH: Keyhole limpet hemocyanin is derived from marine mollusk
   • BSA: Bovine serum albumin is derived from cows
   • RSA: Rabbit serum albumin is derived from rabbits
   • OVA: Ovalbumin is derived from egg whites
   • Others
2. Location of peptide segment within the native protein: necessary in determining the appropriate conjugation chemistry of the peptide to the carrier protein for presenting the peptide segment to the immune system.
   • N-terminal region of the protein (within the first 100 amino acids)
   • Internal region (between the N-terminal and C-terminal regions)
   • C-terminal region of the protein (within the last 100 amino acids)
3. Conjugation Chemistry: the attachment site of the peptide to the carrier protein
   • EDC: peptide attachment to carrier protein via the carboxyl groups within the peptide sequence (D, E, and C-terminal carboxyl group)
   • Activated EDC: peptide attachment to carrier protein via the amino groups within the peptide sequence (K and N-terminal amino group)
   • MBS: peptide attachment to carrier protein via the thiol group of a cysteine residue within the peptide sequence.

Steps for choosing conjugation chemistry:

1. Look for "D" or "E" residues. You should not use EDC if there are "D" or "E" residues.
2. Look for "K" residues. You should not use Activated EDC if there are "K" residues.
3. If the peptide has "D" or "E" residues but no "K", use Activated EDC to conjugate at the N-terminus.
4. If the peptide has "K" residues but no "D" or "E", use EDC to conjugate at the C-terminus.
5. If the peptide has "D", "E", and "K" residues, add a cysteine (C) to one end and conjugate using MBS.
6. If a peptide has "D" or "E", "K", and C residues, there is no way to get a single site of conjugation at one end. You may consider changing the sequence. You are likely to get an immune response, even if there are multiple conjugation sites.
7. If you want the peptide conjugated at a specific end (N or C terminus), add a cysteine (C) on that side and conjugate using MBS.

Location of peptide segment within the native protein:

• Peptides from the C-terminus of the protein should be linked to the carrier through the N-terminus. This will present the peptide segment in similar representation as the native protein.
• Peptides from the N-terminus fo the protein should be linked to the carrier by their C-terminus.
• If the peptide segment is located internally of the native protein, conjugation can be done at the less hydrophilic end.

Multiple Antigen Peptide (MAP):

MAP is simply an alternative to protein conjugation.  It is recommended only for shorter peptide located either internally.