Antibody Production FAQ

Usually the C or N-terminal of the protein is used, as those parts of the protein are exposed. Also, to mimic protein behavior, the synthesized peptide should have similar structure and charge as the protein it has been "cut out off". Therefore:

• Peptides derived from the C terminal should have N terminal modified by acetylation
• Peptides derived from the N terminal should have C terminal modified by amidation
• Peptides derived from an internal sequence should have both ends modified

The following points should also be considered:

• Are there any other proteins from the family of interest, where cross-reactivity should be avoided?
• Is the crystal structure of the protein (or homologous protein) known? This would be helpful for the peptide chemist in searching for the best peptide for antibody production.
• What is the final application of the produced antibodies? Native or denatured techniques?

Immunization can be done using native proteins, recombinant proteins, peptides, carbohydrates or other compounds of microbial, fungal or viral origin. Minimum molecular weight needed to induce sufficient immune response is 5-10 kDa. Biohazardous materials for immunizations are not accepted.

Important notes:

• If the antibodies are going to be used on the denatured target protein (example: Western blot, immunohistochemistry on fixed tissues), denatured forms of antigens are preferred (protein in inclusion bodies).
• If the antibodies are going to be used on native target proteins (example: immunoprecipitation), non-denatured forms of antigen are preferred (protein in solution free of denaturing agents).
• Not all peptide antibodies will recognize native protein, therefore a careful choice of peptide sequence is of crucial importance.
• Antibodies made against recombinant proteins expressed in bacteria can in some cases fail to recognize native protein. The reason for this might be incorrect folding of the protein antigen when expressed in bacterial cells.

No guarantees can be given in advance for a success of any immunization program.

Recommended references on the subject: "Monoclonal antibodies: principles and practice" by James W. Goding, 1996, ISBN 0-12-287023-9; Publisher: Academic Press. Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, 1999, ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press. Hjelm et al. (2012). Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes. PLOS ONE.

Advantages of using both IgG (rabbit, goat) and IgY (hen) antibodies developed against the same antigen:

• Independent confirmation that the expected target protein is detected.
• Antibody pools with distinct properties, complementing each other in different techniques (Western Blot, immunoprecipitation etc.), making double staining possible.
• A ground rule for immunization is to choose an animal that is genetically distant from the antigen source (e.g. hens are very suitable for production of antibodies against conserved mammalian proteins). For more information about IgY click here.

Antibodies present in serum
Serum is a very stable format for antibody storage. In -20°C or -70°C, serum can usually be stored for years. In some specific cases, the shelf-life can be shorter for anti-peptide antibodies. For very short periods of time, serum may be stored at + 4°C. In some cases, more careful freezing with a first step at -20°C, followed by -70°C is beneficial.

Total IgG fraction (IgG antibodies purified on Protein G matrix)
Generally, protein G purified antibodies are stable. They can be stored in -20°C or -70°C for years. For short-term storage, add some azide to a final concentration of 0.02 % (or another preservative).

Total IgY fraction (IgY antibodies purified by precipitation from egg yolk)
Purified IgY fractions are very stable, even at room temperature (although we do not recommend it as storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Avoid freezing and thawing of IgY, and storing it on dry ice. IgY antibodies can be stored at -20°C.

"The IgY preparations were stable over time. No loss of antigen recognition was observed after storage for 3 years at + 4°C". F. De Ceunick et al. Journal of Immunological Methods 252 (2001) 153-161.

Egg yolk
Antibodies in egg yolk should be stored at 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Egg yolk should NEVER be frozen as this will complicate purification of the antibodies. After 6 months of storage, purifying antibodies present in egg yolk might be somewhat difficult.

Affinity purified antibodies
Affinity purified antibodies are the most fragile. Caution should be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some will precipitate directly after the purification, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives to be tested:

• -20°C or -80°C
• +4°C with preservatives like azide (0.02%) or merthiolate
• -20°C with glycerol at a final concentration of 10 or 50%
• -20°C with BSA at final concentration of 0.05-0.5%

IgG
Mammalian, polyclonal antibodies can precipitate following affinity purification. This can occur directly after purification, or overnight during cold storage. Some antigens will stimulate the production of a class of IgG, called cryoglobulins, which precipitate at low temperatures. Heating the cryoglobulins up to room temperature can solve this problem. The antibody solutions can also be centrifuged to remove precipitates.

IgY
Chicken antibodies can also precipitate when stored in the cold, wither directly overnight, or after several weeks. Heating the IgY up to room temperature often helps to dissolve those precipitates. Otherwise, IgY solution can be centrifuged to remove precipitation prior to use. Antibody solutions stored without preservatives are at the risk of being contaminated by bacterial growth, which is one of the most common reasons for protein inactivation.

General recommendations:

• For larger volumes of affinity purified antibodies, filter-sterilize the antibody sample and aliquot the solution to avoid multiple freezing and thawing cycles.
• Ideal storage occurs at protein concentrations around 0.5-1 mg/ml.
• In cases of IgM antibody production, check protein stability in different storage conditions.

Important note: Sodium azide will inhibit horseradish peroxidase, as well as interfere with some coupling methods and biological assays. However, the amount present in IgY preparation (0.02 %) can be washed away in ELISA or Western Blot when IgY is used as primary antibody at dilutions of at least 1:2000.

Alternative agents for preventing bacterial growth in antibody solution:

• Thimerosal at 0.01%
• Gentamicin sulfate at 50 µg/ml