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Antibody types

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Technical information > Antibody production guide

Over the last 30 years, Agrisera has produced thousands of monoclonal and polyclonal antibodies for academic research, industry, diagnostics, and pharma, worldwide. Agrisera provides off-the-shelf antibodies, with prompt delivery, and reactivity to thousands of species. Our products are validated in at least two independent laboratories, and cited in thousands of publications since the year 2000.

Using the skill and knowhow we have gained over the years, we have created this guide, to take you through the different steps of antibody production. If you have any further questions, please contact [email protected].

You can find our educational poster "Tips and tricks for antibody production and validation process - how to obtain good results" here.

Three crucial components for successful antibody production


Antigen	Animals	Analysis

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 know 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.

In general, a suitable peptide is flexible, hydrophilic, surface oriented, continuous and easy to synthesize. Some additional factors that may increase the probability of generating a successful working peptide antibody are:

Access to a 3D structure of the protein to determine surface oriented as well as discontinuous and continuous epitopes
Knowledge on possible regions that should be targeted or avoided
Knowledge on possible post-translational modification sites (PTMs), e.g. phosphorylation, glycosylation, ubiquitination, methylation, acetylation, lipidation, proteolysis
Access to protein sequences or protein accession numbers, for targets which the resulting antibody should or should not cross-react with
Knowledge on whether the antibody should recognize an intra- or extracellular epitope on the protein
Knowledge on whether the target is a membrane or transmembrane protein
Knowledge on whether the N and C-terminus are exposed on your protein (often the case), and could thus be suitable regions for the antibody epitope

Proteins

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

Approaches to consider

Using a native protein isolated from a tissue (not feasible in case the 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

What will give better results, immunization with a peptide or a protein?

Each of these approaches have advantages and disadvantages. Antibodies bind to specific epitopes on a target protein, and the epitopes are five to six 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, while peptide antibodies have a higher specificity, 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. On the other hand, anti-peptide antibodies can increase the risk for possible cross-reaction, if the chosen sequence has high homology with other arbitrary proteins.

Finally, preparation of a fine emulsion of antigen (peptide or protein) and adjuvant is another important factor in order to secure efficient antibody production. With more than 30 years of experience, Agrisera has developed a good method to achieve this.

Animals

Keeping animals in natural and comfortable conditions will make them healthy and allow stimulation of their immune response. At Agrisera, the animals reside in spacious indoor premises with straw flooring. All animals are allowed to socialize and are monitored several times per day by trained animal care professionals. As diet is a big part of keeping the animals in good mental and physical condition, their food consists of high-quality animal feed and hay. (For more information about animal keeping, the Swedish animal protection law can be found here).

Prior to immunization each animal produces millions of antibody combinations. If clones producing antibodies to the desired antigen are not present already, or present only 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, while the other is better suited for immunoprecipitation.

Choice of species
There are no preferences here. The immune system of a bird, such as chicken, functions in a slightly different way compared to that of a rabbit, but both can produce functional antibodies (Chicken IgY, Rabbit IgG). In some cases, the antibodies 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 (50-70 ml serum or 200-400 mg total IgY). The final yield of a specific antibody can vary from 0.025 mg to 0.25 mg per ml serum produced and the amount required for a Western blot analysis can range from 0.5 µg to 5 µg 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 any possibilities that the serum or yolk already contains antibodies which bind to proteins in the proximity of your target protein (about ± 20 kDa from the target weight).

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 after which the specific antibodies bind to the peptide/protein within the column. The non-binding antibodies pass through and the antibodies bound to the column are washed with PBS and eluted with Glycine at a later stage. Read more »

Analysis

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:

Optimizing protein extraction and detection techniques (this is absolutely crucial in case your protein is of low abundance!)
Positive and negative controls (extracts from null mutants or overexpressors)
Proper antibody storage. Antibody stability and storage information can be found here
Reliable molecular weight markers, preferably with an IgG binding site that allows direct marker visualization on the blot

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.