Peptides for Antisera Generation
Generation of anti-peptide antisera requires immunization of the host with peptide conjugated to a carrier protein in order to have a good chance of eliciting a strong immune response. Commonly used carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA). KLH is often favoured since there is no cross-reactivity with reagents used in subsequent ELISA or Western blot experiments.
The carrier protein may be conjugated using a variety of chemistries (MBS, EDC and activated EDC), the choice depending on the sequence of the peptide and the position of the peptide in the original protein sequence. It is recommended to conjugate the carrier protein at the N- or C-terminus of the peptide, rather than to an internal residue so that as much of the peptide sequence as possible is presented to the host immune system.
If the peptide originates from the N-terminal region of the protein sequence, coupling to the carrier protein should be at the C-terminus of the peptide. Conversely, if the peptide comes from the C-terminal region of the protein sequence, couple the carrier protein to the N-terminus of the peptide. If the peptide comes from an internal region of the protein the carrier protein may be added to either end of the peptide.
The MBS method couples the peptide and carrier protein via the thiol group of a Cys (C) residue. This method is not recommended if there is a Cys residue internally in the peptide sequence. The design process can incorporate a Cys at the N- or C-terminus of the peptide in order to use this method.
The EDC method couples the peptide and carrier protein via the carboxyl group of Asp (D), Glu (E) or the free carboxyl group of the C-terminal amino acid. This method is not recommended if there are Asp or Glu residues internally in the sequence.
The activated EDC method conjugates the carrier protein to the peptide via the amine group of Lys (K) or the free amine of the N-terminal amino acid. This method is not recommended if there is a Lys internally in the peptide sequence.
If the peptide sequence contains Cys, Asp, Glu and Lys internally it is not possible to conjugate the carrier protein specifically to the N- or C-terminus. It is recommended that the peptide sequence be redesigned to meet one of the above criteria for conjugation, in order to have the best chance of a strong immune response.
Epitope Specific Considerations
Prediction of the antigenicity of a peptide for antisera production can be attempted with a combination of predictive algorithms. Where possible the peptide should be conjugated to the carrier at the end that is furthest away from the peak of predicted antigenicity. If you have questions about predicting the antigenicity of peptides, please contact us.
It is also important to screen the peptide for homology with with other protein sequences, both for the species of the target tissue and the host immunized. An antibody generated against a peptide that has high homology with other proteins in the target species may be cross-reactive in that species, leading to false positive results. Where antibodies are specific for continuous peptide sequences they usually recognize epitopes between 5 and 15 amino acids in length. As a general rule, sequences that are identical for 6 consecutive residues have a high risk for antibody cross-reactivity, especially if both the sequence of the target peptide and those of the potentially cross-reactive peptides are located on the protein surface. For short, predicted epitope sequences, 5 to7 amino acids in length, it may be worth considering extending the sequence used for conjugation in order to provide sufficient spacing between the epitope and the carrier molecule, and also to reduce the potential for cross-reactivity with other proteins.
Peptides that have high homology with proteins of the host species to be immunized may not be very antigenic in that species. For proteins that are conserved in many mammalian species one should consider immunizing a phylogenetically more distant host species, such as chickens.
thinkpeptides is a brand of ProImmune