Peptide antigens for antibody generation

AltaBioscience specialises in synthesising custom peptide antigens for use in antibody production and custom antibody generation. Synthetic peptides as immunogens have proven advantages over protein immunogens. By themselves, peptides are too small to elicit an immune response, so they must be presented as larger entities in order for the body to detect them and produce an antigenic response.

Our peptide antigen service offers two methods of peptide antigen production:

• multiple antigenic peptides (MAPS), both 8-branched or 4-branched
• conjugation to proteins such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or Ovalbumin (OVA).

Suitable for both monoclonal antibody production and polyclonal antibody production, our peptide synthesis experts can assist with peptide antigen design using our epitope design software, thereby suggesting antigenic peptide regions likely to provide the best immune response.

For purification, we can supply our ready-to-use reusable affinity columns, designed by AltaBioscience. These columns have your peptide antigen immobilised on a solid support and are ready for polyclonal antibody purification.

Whether you select MAPS synthesis or peptide-protein carrier conjugations for your immunogen program, we can also provide unconjugated linear peptides for use in blocking assays.

Peptide antigen options

Multi antigenic peptides (MAPs)

Multi-antigenic peptides (MAPs) are an effective method of antibody production using synthetic peptides. Eight identical peptide chains are synthesised and covalently joined to a common polylysine core. The molecular weight of these peptides (10kDa-15kDa) is ideal for them to be highly immunogenic without the need to couple to a carrier protein.

This technique gives very high titres and works for any epitope. For the extreme C-terminus of a protein, we recommend our alternative method of peptide antigen via conjugation to a protein.

Image shows 8 branched maps (8 linear peptides on poly-lysine core)

Peptide-carrier protein conjugation

We can also provide peptides covalently bound to carrier proteins such as KLH, BSA, or OVA. These can be useful when the epitope is at the peptide’s C-terminus.

In this process the peptide is synthesised in the conventional way with a Cys group at either the C-term or the N-term, then conjugated to the carrier protein in a separate process.

All our peptide-protein antigens are purified by dialysis and ready for use in antibody production.

The image shows peptide-protein conjugate

Peptide antigen design

With expertise spanning more than 45 years, our peptide chemists can assist with the design of peptide antigens. Using our software designed in-house, we look at both the antigenic potential of epitopes along the protein and also take into consideration the synthesis implications required for each specific peptide sequence identified. For selection, we aim to include flexible regions including both hydrophilic and hydrophobic amino acids, ensuring that a significant percentage of charged residues such as Lys, Arg, Glu and Asp can be incorporated, avoiding, where possible, any highly hydrophobic regions. Prolines, whilst favourable in the synthesis process, are not as helpful in peptide antigens as they produce a bend in the peptide backbone which alters shape. Cys-containing regions should also be avoided as these residues would generally be points of di-sulphide bridging in the protein.

Immunisation using two different peptides from one protein may increase the chance of successful antibody generation.

Antibody serum purification

Our laboratory has developed the technology to make peptides covalently bound to controlled pore glass (Peptide – CPG) for use as affinity columns. This material has many advantages over other types of support: it has good flow rates and is mechanically and chemically very robust with almost an indefinite lifetime.

Whilst we provide purification as part of our polyclonal antibody production service, we can also provide protocols for using our affinity purification columns should you wish to purify your own serum.

These columns can be used to purify antibodies raised by MAPs or peptide-protein conjugation methods.

• The columns are reusable and have a peptide loading of about 25umole/g.
• The pore size of the controlled pore glass is large enough for any antibody to enter freely.
• They can be made in the same synthesis operation as the peptide, ensuring a very cost effective synthesis option.

Our affinity columns can be used repeated times and offer a robust, ready-to-use product for antibody purification.

Peptide antigen applications and advantages

Peptide antigens are ideal for use in generating antibodies in both polyclonal and monoclonal antibody production services.

Peptides as antigens have several advantages over proteins when used as immunogens in antibody production. Native proteins may not be readily available or stable enough for use giving peptide antigen synthesis many advantages over protein antigens.

• Options for selecting targeted regions of a native protein provided the sequence is known.
• Peptide epitopes are well-defined and produced by a synthesis process that is well-established.
• Post-translational modifications can be incorporated with ease.
• Very cost-effective in comparison to protein antigens.
• It can be synthesised quickly for a rapid turnaround for use in antibody generation programs.

Peptide antigen structure

We offer free technical advice for selecting peptide antigens. Our team will not only consider the likely antigenicity of the epitope but also the implications regarding the peptide synthesis. Additionally, there are online antigen predictors such as Epitope Prediction and Analysis Tools – IEDB Analysis Resource

The following are general tips for epitope selection:

• Peptide length: Ideally, peptides should be 10 or more amino acids in length.
• Disulphide bridges : Avoid any Cysteine residues where possible. Cys residues in the natural protein would normally be cyclised. Cysteine is also used as a linker when conjugating peptide antigens to proteins such as KLH, OVA or BSA so is undesirable in a sequence.
• MAPs or peptide-protein conjugate : Our experience using either method has been highly successful. We would suggest that if the extreme C-terminal region of a protein is of interest, peptide-protein conjugation may be more suitable than MAPs in this instance.