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Peptide Design for Immunization using Multiple Antigen Peptides

How to design peptides useful for immunization using multiple antigen peptides (MAPs)

The design of peptides to generate antibodies against protein epitopes is still considered an art. However, experienced researchers can use an educated approach for the design of immune response inducing peptide sequences for example by determining the amino acid sequence regions exposed to the surface of a protein. One approach is to review the folding of proteins and to search for β-turn loops where the amino acid residues at the turns are pointed to the surface of the proteins. This approach works well for proteins for which the 3D structures are available in a public data base such as the Pubmed Structure database [http://www.ncbi.nlm.nih.gov/structure/] or the RCSB PDB database [http://www.rcsb.org/pdb/home/home.do]. Another approach is to screen the protein sequences for sequence regions that have a high probability to induce immunization using bioinformatic methods. The following web link can help to achieve this:

http://www.proteinlounge.com/biosyn/pepfinder_home_in.asp

The multiple antigen peptides (MAP) system represents another approach to anti-peptide antibody elicitation. It has been reported that MAPs can be used for the production of high-titer anti-peptide antibodies as well as synthetic peptide vaccines. The system was first described by Dr. James Tam. A MAP contains a small immunologically inert core molecule of radially branched lysines dendrites onto which a number of peptide antigens are anchored. The result is a large macromolecule which has a high molar ratio of peptide antigen to core molecules and does not require further conjugation to a carrier protein. The α- and ε-amino functional groups of lysine residues are used to form a dendrimeric type core to which multiple peptide chains are attached. Depending on the number of lysines used to synthesize the core structure different numbers of peptide branches can be synthetically attached.

  1. What is the optimal length of the peptide? I recently read a paper that indicated 30 mer peptides perform better than 20 mer peptides in an ELISA based assay.
    • We have used many 20mer peptides for ELISA assays and have found no problem in their use. All have worked very well. However, in general we agree that longer peptides will bind stronger to their targets than shorter peptides if using ELISA plates.
    • The optimal length of peptides used for antibody production can vary from 15 to 22 amino acids. Class I peptides that are held within the MHC groove are usually 8 to 9 amino acid residues long. However, class II peptides are between 8 and 30 amino acid residues long.
  2. Do the peptides need to be conjugated to a carrier protein and if so what carrier is best? My concern here is steric hindrance if peptides themselves are conjugated to the beads. Therefore mentioned paper suggested that BSA was better than KLH, as KLH caused higher non-specific reactivity.
    • It is not necessary to conjugate peptide to carrier protein for assay development. If one has to be used, BSA or OVA are good carrier proteins that can be used for assay development. If a researcher wants to use carboxylated polystyrene Luminex beads for conjugation, one important point is that the conjugation condition for the carrier protein-peptide conjugate has to be optimized. The reason for this is that after the conjugation reaction many amine groups on the carrier proteins are occupied by peptides. This fact will make it difficult to allow the conjugation to the beads as well. The solution to this problem could be the use of different beads that have different functional groups or to control the conjugation degree of the peptide to the carrier protein. Another method to avoid carrier protein conjugation is to consider adding a spacer (such as a PEG spacer) to the N-terminus of peptides that contains a terminal amine group. This can be done by using a peptide synthesis approach to conjugate peptides to the beads. This method works very well and the peptides can be further conjugated to the carboxylated polystyrene Luminex beads.
  3. If MAPs are used are the primary amines on lysines still available for conjugation to microplex beads?
    • In the case of MAPs, the lysines located in the MAP core will not be available for conjugation. The reason for this is that the peptide chains are branched from the alpha and epsilon amine groups of the lysines. The only available groups will be the –COOH group of the MAP and the N-terminus of the 4 or 8 branches of the peptide. In this case, instead of carboxylated polystyrene Luminex beads, different beads with different functional groups will need to be used. The figure below illustrates the structure of a MAP: COOH group
      Structure of a MAP.
  4. What purity of the peptides should be used?
    • Both, the map core and the selected peptide are usually synthesized on a solid support. When cleaved off, the released MAPs usually exits as heterogeneous molecule with a purity of >85% which can be used for antibody production without further purification.