Home / Antibody Production Guide

1. Antigen
Careful preparation and choice of antigen is crucial to secure success of your antibody production project.
If you are choosing a peptide yourself use immunogenicity prediction software. Antibody producing companies usually offer peptide selection services. If your antigen is a highly conserved mammalian protein - non-mammalian species are recommended to use for antibody production, as for example chickens (chicken immunonoglobulin of yolk from which antibodies are purified is called IgY).

One frequently asked question is: What will give better results, immunization with a peptide or a recombinant protein?

Possible approaches to consider: 
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).
B. Recombinant protein. If impure, separate it on SDS gel and use as a gel piece. A fragment to be overexpressed needs to be carefully chosen, if cross-reactivity with other members of a protein family is to ba avoided.
C. Peptide. N or C-terminus is usually recommended to begin with. Anti-peptide antibodies are preferred if cross-reaction with other protein family members needs to be avoided. Anti-peptide antibodies are considered to be mono-specific, as they target more limited epitope pool as compared to a whole native protein or a recombinant protein fragment.

Each of these approaches has advantages and disadvantages. Antibodies bind to specific epitopes on a target protein. The epitopes are two or three amino acids in a row (linear epitope) or distant amino acids brought together in the 3D structure. Antibodies to proteins or protein fragments will recognize several epitopes compared to peptide antibodies (which are usually 7-20 amino acids long). In some cases this can increase a risk for possible cross-reactions. However, any of the approaches listed above can result in production of useful antibodies. When few animals are immunized with the same antigen, resulting antibodies will bind to similar, but not identical epitopes (Hjelm et al. 2012). Therefore antibodies produced in two animals might be in some cases used with different success in various techniques, e.g. one works better in a western blot, another in immunoprecipitation. 

Preparation of fine emulsion of antigen and adjuvant is another important factor to secure efficient antibody production. 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 in very low amounts it might be difficult to obtain a good response. It can thus be beneficial to use more than one animal per antigen.
Animals eat a plant-based diet. Therefore, a check of serum or yolk before immunization is started (pre-immune screening) 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 a your target protein (about ± 20 kDa from a target weight), for which antibodies are already present before immunization. After immunization is performed specific antibodies can be used in a format of serum or might require so called affinity purification - protein or peptide is coupled to a specific matrix (usully min. 1 mg of protein and 5 mg of peptide are used and serum from immunized animal is passed through such column. From case to case, affinity purification of specific antibodies will or sometime will not remove contaminating bands. In case of very low amounts of protein are available for affinity purification of specific antibodies, it can be done using this approach.

 

3. Testing
We recommend using fresh samples, optimizing protein extraction (this is absolutely crucial in case your protein is of low abundance!) and detection technique as well as including positive and negative controls (extracts from null mutants or overexpressors) in antibody validation process. If these are difficult to obtain, saturation of antibody with peptide/protein used for immunization can be of help. In case of proteins with low tissue abundance, it is of crucial importance to use sensitive detection systems based on chemiluminescence, such as ECL (Lumigen) or West Pico (Pierce) or 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. Re-using of a primary antibody solution during initial testing is not recommened, especially not if you work with western blot quantitation approach. Antibody stability and storage information can be found here.

 

Antibody production outcome cannot be predicted in advance, but if all components of this process are carefully carried out, the possibility of success increases dramatically. 

1. Obtain antigen
- purify native protein
- overexpress your protein of interest. There is no specific preference for a fusion partner. However, a His tag is much smaller than, for example, GST or MBP. Besides antibodies to your target protein or peptide there will always be a pool of antibodies produced to the fusion partner
- design and order synthesis of a peptide. Agrisera offers a service of peptide design and synthesis. Useful information to provide in such a case is post-translation modifications, crystal structure of protein of interest or related protein (if known)

2. Choose animal species
There are no preferences here. The immunological system of a bird, such as a chicken, acts in a slightly different way than the one of a rabbit but both can produce useful antibodies (chicken IgY , rabbit IgG). In some cases they will each work in various techniques and complement each other. For projects with a need for large quantity of antibodies a hen that can substitute 20 rabbits or a goat can be of choice. Depending on the final dilution of antibody in specific assay (1: 1000 or 1: 10 000) and if there is a need to perform affinity purification, usually a standard production program (50-100 ml of serum) is enough to satisfy the need of a research laboratory. The final yield of a specific antibody can vary from 0.025 mg to 0.25 mg per ml of produced serum and the amount required for an assay like a Western blot can vary from 0.5 ug to 5 ug of purified serum in 10 ml incubation buffer.

3. Perform pre-immune serum screening
Use the same material and load the same amount on the gel as is expected in your future work with the produced antibody.  

 

Prepare material for testing during the time your antibody is produced, which with a standard protocol takes about 15 weeks.
Knowing when the first samples from antibody production are going to arrive, secure that there are membranes prepared for testing. Proteins can be blotted in advance and stored dry in RT for up to 6 months. When the antibody for testing arrives, it is important to evaluate if a band of right molecular weight is detected - therefore we recommend to include positive and negative controls, or if not feasible, perform peptide/protein neutralization assay and use reliable molecular weight markers, preferably with IgG binding site that allows direct marker visualization on the blot (as for example Magic Marks, Invitrogen). Secondary antibodies of high quality are certainly also of importance.