Antibodies present in serum
Serum is a very stable format for antibody storage. In -20°C or -80°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. Snap freezing in liquid nitrogen is not required.
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 -80°C for years. For short-term storage, add a preservative like 0.2 % ProClin300 (Sigma), which is less toxic alternative compare to sodium azide.
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), 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.
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. Therefore, fresh material should be processed as soon as possible.
Affinity purified antibodies
Affinity purified antibodies are the most sensitive. 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%
Problem: affinity purified antibodies precipitated during storage in 4°C
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.
Chicken antibodies can also precipitate when stored in the cold, wither directly overnight, or after several weeks. Warming them 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.
• For larger volumes of affinity purified antibodies, filter-sterilize and aliquot the antibody sample to avoid repeated freezing and thawing.
• Storage at protein concentration around 0.5-1 mg/ml.
• In case of IgM especially 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 the primary antibody, at a dilution of at least 1:2000.
Alternative agents for preventing bacterial growth in antibody solution:
• Thimerosal at 0.01%
• Gentamicin sulfate at 50 µg/ml
• Kathon™ CG