Choice Of Peptide Sequence For Antibody Production

If there are no constraints on antigen choice for a cytosolic or soluble polypeptide

or the rationale for choosing an epitope is ambiguous, a pragmatic approach to choosing a peptide

sequence for immunization is to synthesize the N-terminal and C-terminal sequences of the protein. These sequences are often found to be solvent exposed and mobile in crystallographic structures of proteins. There may be a higher likelihood that an antibody prepared against these sequences will work well in immunoblot analysis and in analyses of native proteins by immunoprecipitation. When using an N-terminal peptide, conjugation to the carrier should be achieved through the carboxyl terminus, so that the peptide will mimic its position in the protein. Similarly, a C-terminal peptide is best conjugated at its amino terminus.

Computer algorithms are frequently used to select epitopes from predicted protein sequences. The most frequently used are those that predict predominantly hydrophilic and hydrophobic portions of polypeptides, helping identify solvent-exposed regions on the surface of the protein. Programs such as those provided by the Wisconsin Genetics Computer Group, commercial programs, and others available on the World Wide Web12 are helpful. However, protein folding is not entirely understood. Unless a prediction of surface residues is based on modeling of a known protein three-dimensional structure, these hydrophilicity plots are not experimental data but only useful guides. If possible, more than one of these sites should be chosen to produce antibodies specific to a desired protein. More information about which peptide sequences bind to class I and class II major histocompatibility complex molecules is also being discovered. This knowledge base may help future peptideantigen design.

It is possible that a desirable peptide antigen may share by chance a region of homology or identity with a sequence in a totally unrelated protein. This can lead to experimental findings that are misleading or that require much analytic work to understand. Searching several databases using Web-based protocols such as BLAST can help prevent the occurrence of this type of problem. Modifications to the standard search protocols are usually necessary when short amino acid sequences are used in the query and have been well described elsewhere.

A major design concern about preparing sequence-specific antibodies is the choice of determinants common to a family of proteins or unique to one member of that family. This selection depends on the experiments a laboratory needs to perform. Specific and general reagents may be required. Programs located on the World Wide Web facilitate alignment of the amino acid sequences of multiple members of a protein family. Sequences or functional domains common to several related polypeptides can be highlighted. This alignment makes identification of sequences common to the same protein from multiple species straightforward, ensuring the general utility of the antibody reagent developed and enhancing its potential usefulness for discovering other related proteins. These same sequence alignments permit ready visualization of sequences that are found in only one member of a protein family or in one species of that family, aiding development of highly specific reagents.

by: Bio-Synthesis

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