Antigenic Peptide Identification

Antigenic Peptide Identification

While proteins are usually good antigens when used with appropriate host, most are not available in sufficient amounts or purities to generate antibodies. 
An alternative approach is to use a (poly) peptide stretch derived from the target protein sequence. 
Polypeptides (>100-150 aa) may be produced via the chemical synthesis of the corresponding coding DNA and further heterologous expression. As cost to generate synthetic ORFs is decreasing, the gene synthesis route is becoming more attractive. 
A more commonly used - and still more economical - approach is to design a shorter peptide sequence (10 to 30 aa) for total chemical synthesis followed by conjugation to a carrier protein thus yielding an excellent immunogen for anti-peptide antibodies production. This method offers many advantageous features including the ability to generate antibodies that may discriminate among homologous proteins, the possibility to purify by affinity chromatography the resulting antibodies using peptide-columns.

Antigenic determinants 

Successful anti-peptides antibodies will recognize peptide epitopes with high specificity on proteins in their native environment. Consequently, peptide epitopes must be accessible to the bulky antibodies macromolecules. 
The peptide must be selected from a region of the protein that naturally lies exposed to the outside solvation environment and even more so if the resulting antibody is to be of use in immunohistochemistry, confocal microscopy or flow-cytometry experiments where native 3D structures are sought for detection. The most likely accessible areas on protein molecules are those that are hydrophilic because they are most likely to be in contact with the aqueous environment. 
The peptide sequence should be unique, and not conserved in any known protein that may interfere with the specific experimental system used. Finally, the peptide must be immunogenic for the host animal. 
With the aid of sequence analysis software, one may be able to identify several peptide sequences that present the optimum features with regards to hydrophilicity, accessibility and flexibility.

Sequence selection 

Feasibility of chemical synthesis, purification and conjugation of the selected sequence(s) should be considered as well when designing the target peptide(s). Generally speaking, try and keep the ratio of hydrophilic to hydrophobic residues to 1:1. Avoid as much as possible reactive amino acids (C, M, W). Contact us with your sequence we will evaluate it for you.

Sequence length 

Experimental data seems to indicate that the smallest peptide antigen is 5-10 aa long. Although we have been successful with as small as 4 aa long peptides, it is recommended to use a minimum of 10 aa as peptide epitope. A good compromise between specificity, chemical synthesis difficulty and cost lies around 15aa long peptides. Using longer peptides (e.g. 18-25 aa) is possible and in many instances it may improve specificity.

Sequence termini 

For a sequence that lies internally within the target protein, one may consider neutralizing the terminal charges (NH3+ or COO-) by using C-amide or N-acetyl/formyl ends to more closely mimics the electronic environment of the epitope. 
For sequences derived from the C-terminal part of the protein, conjugation should be performed on the N-end of the peptide and vice-et-versa for N-end epitopes.

Multi-peptide co-immunization approach 

In some instances, and in order to improve the odds of producing an antibodies pool that will detect the native protein, one may choose several peptide epitopes within the same protein sequence and use them as antigens in a co-immunization protocol. Some increased background may be experienced.


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