Strategic Decision Making:

Peptide vs. Recombinant Antigen

Compare the general features of both approaches:

Peptide Antibody Projects	Antibodies against recombinant proteins
General: No need for a purified protein to be used as an antigen.	General: Most promising antigen
Epitope: Highly precise epitope information, which is helpful in designing antibodies that discriminate between isoforms, splicing variants, mouse vs. human or other species, …	Epitope: More robust antibodies due to reactivity with (in most cases) more than a single epitope. May lead to stronger labelling of the antigen (more binding sites).
Preferred method, recommended and straightforward to raise antibodies against posttranslational modifications (phosphorylations, cleavage products, …) to reproduce a previous successful project if there are published data available from the scientific literature.	Preferred method whenever a recombinant protein is available. Usually very immunogenic when applied as inclusion bodies or as soluble antigen. Only in exceptionally rare cases (e.g. when proteins are extremely well conserved between different species) coupling to a carrier protein may be required. If in doubt, please let us check the relevant amino acid sequence.
Affinity purification highly recommended. The peptide of interest is only a minor fraction of the whole immunogenic mixture used for injecting an animal. Other components include the carrier protein and mycobacteria with a myriad of proteins (used as adjuvant). Accordingly, it is not unusual that crude peptide antisera (as well as total antibody fractions derived thereof) are quite "dirty" and it is difficult to judge whether a possibly faint band in a strong background is specific or not. Therefore, it is mandatory to purify peptide antisera on a peptide column. This is also valid for testing purposes.	Affinity purification mostly dispensable. As a rule, purification tags (GST, MBP, His6, …) are very immunogenic. Actually, about half of the antibodies reacting with the fusion protein may be directed against the tag. Experience shows that tag specific antibodies rarely interfere with conventional assays and usually do not cause recognizable background. However, if required, these antibodies can be pre-absorbed / depleted by fractional affinity purification on a second affinity column with the tag sequence only (e.g. a poly-His matrix is available upon request).
Risk assessment 1. The chance to obtain an antibody reactive with the synthetic peptide used for immunization is close to 100%. Mostly, there are even high titers and one can expect to get an average minimum yield of 2.5-5 mg of peptide specific antibody from 50 ml of serum. 2. Despite careful epitope selection and use of highly purified peptides there remains a considerable risk that peptide specific antibodies may not recognize the natural (denatured or native) protein. This problem is well known and is addressed as a topic at antibody conferences [Greenbaum et al., 2007]. 3. Peptide antibodies recognize a small epitope (typically 5-8 aa), which by chance, also occurs in identical or similar form in different unrelated proteins. Therefore peptide antibodies, might specifically (!) cross-react with these proteins. Often identities of consecutive 5 amino acids (and sometimes even 3-4 aa) are sufficient for such a cross-reactivity. On the other hand, statistically, identical 5 aa motifs are essentially inevitable, even with very careful peptide selection.	Risk assessment With a recombinant protein as antigen, essentially all projects will yield antibodies that can be affinity purified (if required) and will react with the respective antigen. ------------------------------- Reference:Greenbaum J.A. et al. (2007). Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools. J. Mol. Recognition 20 (2):75-82.

Peptide Antibody Projects

Antibodies against recombinant proteins

General:

No need for a purified protein to be used as an antigen.

General:

Most promising antigen

Epitope:

Highly precise epitope information, which is helpful in designing antibodies that discriminate between isoforms, splicing variants, mouse vs. human or other species, …

Epitope:

More robust antibodies due to reactivity with (in most cases) more than a single epitope. May lead to stronger labelling of the antigen (more binding sites).

Preferred method, recommended and straightforward

to raise antibodies against posttranslational
modifications (phosphorylations, cleavage products, …)
to reproduce a previous successful project
if there are published data available from the scientific literature.

Preferred method whenever a recombinant protein is available.

Usually very immunogenic when applied as inclusion bodies or as soluble antigen.

Only in exceptionally rare cases (e.g. when proteins are extremely well conserved between different species) coupling to a carrier protein may be required. If in doubt, please let us check the relevant amino acid sequence.

Affinity purification highly recommended.

The peptide of interest is only a minor fraction of the whole immunogenic mixture used for injecting an animal. Other components include the carrier protein and mycobacteria with a myriad of proteins (used as adjuvant). Accordingly, it is not unusual that crude peptide antisera (as well as total antibody fractions derived thereof) are quite "dirty" and it is difficult to judge whether a possibly faint band in a strong background is specific or not. Therefore, it is mandatory to purify peptide antisera on a peptide column. This is also valid for testing purposes.

Affinity purification mostly dispensable.

As a rule, purification tags (GST, MBP, His6, …) are very immunogenic. Actually, about half of the antibodies reacting with the fusion protein may be directed against the tag. Experience shows that tag specific antibodies rarely interfere with conventional assays and usually do not cause recognizable background. However, if required, these antibodies can be pre-absorbed / depleted by fractional affinity purification on a second affinity column with the tag sequence only (e.g. a poly-His matrix is available upon request).

Risk assessment

1. The chance to obtain an antibody reactive with the synthetic peptide used for immunization is close to 100%. Mostly, there are even high titers and one can expect to get an average minimum yield of 2.5-5 mg of peptide specific antibody from 50 ml of serum.

2. Despite careful epitope selection and use of highly purified peptides there remains a considerable risk that peptide specific antibodies may not recognize the natural (denatured or native) protein. This problem is well known and is addressed as a topic at antibody conferences [Greenbaum et al., 2007].

3. Peptide antibodies recognize a small epitope (typically 5-8 aa), which by chance, also occurs in identical or similar form in different unrelated proteins. Therefore peptide antibodies, might specifically (!) cross-react with these proteins. Often identities of consecutive 5 amino acids (and sometimes even 3-4 aa) are sufficient for such a cross-reactivity. On the other hand, statistically, identical 5 aa motifs are essentially inevitable, even with very careful peptide selection.

Risk assessment

With a recombinant protein as antigen, essentially all projects will yield antibodies that can be affinity purified (if required) and will react with the respective antigen.

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Reference:Greenbaum J.A. et al. (2007). Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools. J. Mol. Recognition 20 (2):75-82.

Conclusions

From these considerations it is evident that except for special cases (see above) recombinant proteins or protein fragments are often more promising and also more economical antigens than peptides. Even inclusion bodies are superb antigens. If interested, please ask for our inclusion bodies purification protocol (poor yields of recombinant proteins often are a hint that the protein may end up in inclusion bodies).

Helpful Information

Strategic Decision Making:

Peptide vs. Recombinant Antigen