Choosing the best antigen

The best antigen will mimic as closely as possible the molecule which is the target of the antibody in the final application.

The target is a protein

If the target is a protein, the best antigen is certainly the protein itself. Any difference between the antigen and the natural target will result in an antibody perfectly matching the antigen, but not recognizing the target in its natural context.

Some important factor have to be taken into account:

What is the complexity of my antigen?

The antigen complexity is important, as it is directly proportional to the complexity of the antibody which will be generated. The most complex antigens will contain numerous epitopes, all being a potential target for the host’s immune system, which will generate as many different antibodies.

How can the host influence immunization?

Because each animal is different, they will all produce a different antibody mix, some epitopes being recognized as more dominant by the immune system of each host, hence more targeted. Collectively, the polyclonals obtained from individual immunizations should recognize the complex antigen very efficiently.

What is the impact on my application?

But if the final assay targets one specific epitope only, it is likely that the polyclonal obtained from one immunization will perform better compared to another one. In this case, using a more specific antigen better matching the antibody application may be useful.

Protein antigen production

Advantages of using a peptide as antigen

Specific antigens like peptides contain a few different epitopes.


Accordingly, a polyclonal generated from a peptide immunization will be more specific than a polyclonal obtained using the whole protein as the antigen.

Protein detection

An anti-peptide antibody may perfectly detect the corresponding protein, provided that there is not sequential, conformational, or post-translation modification difference between the peptide and the protein region from which it originates.

Peptide antigen production

Using a protein as the antigen


When a protein is to be used as an antigen, it is most often produced recombinantly. Care must be taken that the recombinant and the native protein forms can differ. For example, the protein in its native environment may be associated to other protein partners, which may hide dominant epitopes not accessible to the antibody in a cellular context. Also, the protein may be post-translationally modified, while the recombinant protein may not be.

The antigen can be:

When producing proteins in E. coli, inclusion bodies are obtained in many cases. Within inclusion bodies, the protein retains some secondary structures, but the tertiary structure is not folded. Using inclusion bodies as the antigen is practically possible, but the antibody fraction which targets non-natural epitopes within the inclusion bodies will for sure not recognize the protein in its 3D from.

Depending on the protein, and the application, the antibody fraction which targets epitopes common to the inclusion bodies and the folded protein may be good enough to allow detecting the protein in its natural environment.

Obtaining a correctly folded protein from inclusion bodies requires a denaturation/renaturation process which is strictly protein-dependent, and always challenging. Urea is often used as the denaturing agent. But incubating a protein with urea for prolonged periods will lead to carbamylation at the N-termini of proteins/peptides and at the side chain amino groups of Lys and Arg residues.

Therefore, using a protein with urea as the antigen will generate antibodies against these non-natural epitopes. Because Lys and Arg residues are often exposed on the protein surface, part of the polyclonal antibodies generated with this strategy will fail binding to the native protein form. This limitation must be carefully considered before using urea solutions to generate antibodies.

GuHCl is a powerful protein denaturing agent, which does not lead to carbamylation as urea. But GuHCl is toxic and cannot be injected in animals. If a protein denatured with GuHCl is to be used as an antigen, dialysis will be a pre-requisite.

If one wants to use a denatured protein as the antigen, injecting an SDS-PAGE gel fragment may be an option. The limit here is that this cannot apply to small rodents and is not compatible with a Speedy 28-days program.

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