Antigen selection for antibody generation

Isca Biochemicals have long experience in antigen peptide selection and design.  Antigen peptides must be carefully selected to offer the best chance of raising antibodies which will recognise the target protein whilst avoiding cross-reactivity with proteins containing potentially homologous protein sequences.

We always recommend starting with a protein selected from Uniprot or the NCBI, as these are reputable sources of protein sequence with reliable gene-translation protocols. They also provide secondary and tertiary structure predictive algorithms which further enhance selection.  Antigenic peptides are conventionally 10-20 amino acids long, offering a compromise between antigenicity and synthetic ease and the objective then is to identify a peptide sequence which contains mixed charge and is not membrane-bound or internally locked. 

Other important considerations are solubility, poly-glycine and poly- alanine chains, the N terminal amino acids and the use of aspartic acid.

• Insoluble sequences are a problem to synthesise, purify and to manipulate. Native insoluble sequences are likely to be associated with membranes, which makes them poor choices for antibody work.

• Poly-glycine or alanine sequences are unlikely to be successful in antigen development because of their lack of charge and small side-chains.

• N-terminal sequences of proteins also must be treated with caution as they are likely to be signal sequences. They may be post-translationally cleaved from the target sequence in vivo, creating a false antigen target. N-terminal glutamic acid should be avoided as it will form an N-terminal pyroglutamate. This risk can be eliminated by adding an N-terminal acetyl group.

• Aspartic acid should be treated with caution as it is prone to form aspartimide, especially with an adjacent glycine.

As synthetic peptides alone are generally too small to trigger an immune response, peptide antigens are usually conjugated to a carrier protein before they are used in immunisation and attention to conjugation chemistry is required to present the peptide appropriately. Where a cysteine is available internally, this can be used as a conjugation point, but this is not essential as a non-native cysteine can be incorporated at a terminal. The carrier proteins KLH, OVA and BSA are regularly employed to conjugate synthetic peptides via a range of chemistries and we can advise on the most suitable.

Isca Biochemicals produces many high quality, fully characterised peptides for antibody production.