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FAQ: Some Options to Consider in the Development of Peptide Immunogens
 
The following information is offered by CRP to our clients requesting background on the development of effective peptide immunogens. These comments are a brief summary of our collective experience in this area. As always, the staff at CRP are available to help you deal with questions you may have about your ongoing or planned programs at CRP.

The design of an effective peptide immunogen is a complex subject, and the following comments are meant to provide a quick introduction to the key issues most commonly encountered in the design of a peptide immunogen.

General points that should be considered:

Peptide Length: As a general rule, the longer the better. Around eight to ten residues seems to be the smallest peptide most people try to use. Peptides of 15 to 20 residues are the most commonly used in developing antisera for western blot, histology and immuno-affinity or precipitation.

Conjugation to a Carrier Protein: The immune response to a peptide conjugated to a carrier protein is, with few exceptions, going to be stronger than will be seen for the peptide alone.

Carrier Protein: The most commonly used carrier today is KLH (Keyhole Limpet Hemocyanin) but albumins, generally Bovine, IgG's, PSG (Pumpkin Seed Globulin), and a number of other proteins can and have been used.

Conjugation Chemistry: Where possible, particularly when there is no cystine in the peptide sequence, consider the use of a sulfhydryl cross-linker through a cystine residue added at one end of the peptide. The decision about which end to add the cystine should be based on the ease of production. There are two exceptions to this: very short peptides and peptides from the C- or N-terminal end of the protein sequence. For short peptides, the use of a mixture of both C- and N-terminal linked peptides, or better, two separate programs on using an N-terminal coupling and the other a C-terminal coupling. Additional information on handling peptides from the C- or N-terminal end of a protein sequence is given below. Conjugations can also be performed using general reagents such as glutaraldehyde, or more specifically, using N-or C-terminal specific cross-linkers. Any of these may also cross-link through any side chains with the appropriate reactivity. For example, N-terminal reagents will react with the primary amine on the lysine side chain. Peptides linked to muliple copies of the carrier through their side chains are generally thought to be less effective as immunogens. Finally, choosing a peptide sequence to improve the likelyhood of a clean conjugation to a carrier is usually counter productive.

Other issues that are relevant to specific situatations are:

Do you have the complete or nearly complete sequence of the protein you want to pick peptides from? CRP offers, at no charge to its clients, a service designed to help identify those parts of a sequence most likely to illicit an immune response. This service may also be used to pick from a number of peptides, the one or two most likely to illicit an immune response. It will always remain the investigator's responsibility to select the peptide to be used.

Do you know or suspect the sequence to be highly conserved? Where data is available, use peptides whose sequences show the largest number of changes between the antigen source species and the planned antibody source species. Where the sequence data is not available, consider the use of chickens as the antibody source species.

Do you have only the C and/or N terminal sequences? Be certain that such sequences are conjugated to the carrier protein by their internal end. In these cases, consideration should be given to the addition of a cystine residue at the internal end of the peptide and the use of sulfhydryl cross-linking to the carrier protein.
 
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