Antibody production guide

Three crucial components for successful antibody production

Antigen for immunization Host animal for antibody production Testing picture




1. Antigen

Antibodies can be produced against a wide range of different antigens. Most commonly used antigens are proteins and synthetic peptides.

- Peptides

If you do not have access to a target protein but knows its full length sequence, a synthetic peptide derived from the protein sequence can be used for antibody production. By carefully designing a suitable peptide, antibodies raised against the peptide can be used to detect the protein in a target assay.

Some general key considerations which will increase the probability of generating a succesful working peptide antibody:

  • access to a 3D structure of the protein make is possible to determine surface oriented, discontinous and continous epitopes
  • are there regions that should be targeted or avoided?
  • are there any known post-translational modification sites? Example of PTMs: phosporlytation, glycosylation, ubiquitination, methylation, acetylation, lipidation, proteolysis
  • access to protein sequences or protein accession numbers, for which the resulting antibody should or should not cross react with
  • should the antibody recognize an intra- or extracellular epitope on the protein?
  • N and C-terminus are often exposed parts of the proteins and thus commonly used. Could this be adapted for your protein?
  • in general, a suitable peptide is flexible, hydrophilic, surface oriented, continous and easy to synthesize

Agrisera is happy to help with design of a suitable antigen. The design is based on your criteria together with estimated suitability for synthesis, conjugation and immunization. If you are choosing a peptide yourself you are welcome to send us the sequence for a statement regarding immunogenicity and solubility. Read more »

- Proteins

Proteins (native or denatured) isolated from a tissue or recombinantly produced, are frequently used for antibody production. How the proteins are produced vary from case to case depending on access of suitable tissue and final use of the antibody. Basically, antibodies produced against native proteins tend to react best with native proteins (Immunoprecipitation) and antibodies produced against denatured proteins to proteins subjected to denaturing conditions (Western Blot, IHC). However, well functioning antibodies can be generated without necessarily follow these rules. Given that adjuvants can have a degenerative effect on the protein, the guidelines are also not entirely obvious. The purity of a protein is also a factor to consider. The higher purity, the greater the proportion of specific antibodies will be. If the protein is impure, it can be separated on SDS gel and the gel piece can be used for immunization. If the protein is expressed with a fusion partner, antibodies will be generated against the fusion partner as well and it might be necessary to find a strategy to remove those non-specific antibodies. This can be done by cleavage of the tag prior to immunization or by affinity purification.

Are you are interested in producing antibodies against a protein? Agrisera offers a full service for production of high quality recombinant proteins. Read more »

What will give better results, immunization with a peptide or a protein? Each of these approaches has advantages and disadvantages. Antibodies bind to specific epitopes on a target protein and the epitopes are two to three amino acids in a row (linear epitope) or distant amino acids brought together in a 3D structure. Antibodies against proteins or protein fragments will recognize several epitopes compared to peptide antibodies that are considered to be mono-specific as they target a more limited epitope. Anti-peptide antibodies are thus preferred if cross-reaction with other protein family members needs to be avoided. Anti-peptide antibodies can on the other hand increase the risk for possible cross-reaction, if the chosen sequence has high homology with other arbitrary proteins.

Approaches to consider:

  • using a native protein isolated from a tissue (not feasible in case target protein is of very low abundance or very similar to other protein family members)
  • using a recombinant protein. A fragment to be overexpressed needs to be carefully chosen, if cross-reactivity with other members of a protein family needs to be avoided

Finally, preparation of fine emulsion of antigen and adjuvant is another important factor to secure efficient antibody production. With 30 years of experience, Agrisera has developed a good method to achieve this.

2. Animals

Keeping animals in natural conditions will make them healthy and allow stimulation of their immune response. Each animal produces, prior to immunization, millions of antibody combinations that can fit potential proteins to be encountered in the future. If clones producing antibodies to the desired antigen are not present already or present in very low amounts it might be difficult to obtain a good response. Thus, it can be beneficial to use more than one animal per antigen. When a number of animals are immunized with the same antigen, resulting antibodies will bind to similar, but not identical epitopes. Therefore antibodies produced in two animals can in some cases be used with different success in various techniques, e.g. one works better in a western blot, another in immunoprecipitation.

Choice of species?
There are no preferences here. The immune system of a bird, such as chicken, act in a slightly different way compared to rabbit, but both can produce functional antibodies (chicken IgY, rabbit IgG). In some cases, they will act differently in different techniques and thus complement each other. For projects requiring large amounts of antibodies, chicken or a goat can be preferable. In general, a standard production program is sufficient to meet the need for a research laboratory (50-70ml serum or 200-400mg total IgY). The final yield of a specific antibody can vary from 0.025mg to 0.25mg per ml serum produced and the amount required for a Western blot analysis can range from 0.5µg to 5µg of purified serum in 10 ml incubation buffer.

Pre-immune screening
Animals eat a plant-based diet and therefore a check of serum or yolk before immunization is started is recommended, especially for antigens derived from plants, algae and bacteria. This will help to exclude a possibility that a serum or yolk already contains antibodies which bind to proteins in the proximity of your target protein (about ± 20 kDa from the target weight). After immunization is performed, specific antibodies can be used as crude serum or as affinity purified.

Affinity purification
Specific antibodies are obtained by coupling a protein or peptide to a specific matrix-column. The immune serum is applied to the column where upon the specific antibodies bind to the peptide/protein. The non-binding antibodies pass through and the antibodies bound to the column are washed with PBS and eluted with Glycine. Read more »

3. Testing

It is important to evaluate the antibody in your own assay to see how it works. During the time the antibody is produced, all necessary preparations can be done. Proteins can be blotted in advance and stored dry in RT for up to six months. When you evaluate the antibody we recommend using:

  • fresh samples
  • optimizing protein extraction and detection technique (this is absolutely crucial in case your protein is of low abundance!)
  • including positive and negative controls (extracts from null mutants or overexpressors)
  • antibodies stored properly. Antibody stability and storage information can be found here
  • reliable molecular weight markers, preferably with IgG binding site that allows direct marker visualization on the blot (for example Magic Marks, Invitrogen)

Re-using of a primary antibody solution during initial testing is not recommended, especially not if you work with quantitative Western Blot.

If suitable controls are difficult to obtain, saturation of antibody with peptide/protein used for immunization can be an alternative. In case of proteins with low tissue abundance, it is of crucial importance to use sensitive detection systems based on chemiluminescence, such as Agrisera ECL Bright or to work with a specific tissue fragment or fraction.

If you need any help with immunological techniques, you are welcome to visit our western blot trouble-shooting or contact us with specific questions.