Antibodies present in serum
This is a very stable format for antibody storage. In -20°C or -70°C serum can be usually stored for years. In some specific cases this time can be shorter for anti-peptide antibodies. For very short time periods serum can be stored at + 4°C. In some cases more careful freezing with a first step at -20°C followed by -70°C can be beneficial.
Total IgG fraction (IgG antibodies purified on Protein G matrix)
Generally antibodies in this format 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 other 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 ug/ml).
Avoid freezing and thawing of IgY or 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 ug/ml). Egg yolk should NEVER be frozen as this will make difficulties for purification of antibodies. After 6 months of storage it might be more difficult to purify antibodies present in egg yolk.
Affinity purified antibodies
These 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:
Suggested storage conditions - to be tested:
* -20°C or -80°C
* + 4°C with preservatives like azide (0.02%) or merthiolate
* -20°C with glycerol; Final concentration of glycerol 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 precipiate following affinity purification. This can happed directly afterwards or overnight during cold storage. Some antigens will stimulate the production of a class of IgG which is called cryoglobulins, that means that they will precipitate in a cold. Warming up to room temperature can solve this problem. Antibody solutions can also be centrifuged to remove precipitates.
Chicken antibodies can also precipitate when stored in the cold overnight or after several weeks. Warming 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 in risk to be contaminated by bacterial growth, which is often the most common reason for protein inactivation.
* For larger volumes of affinity purified antibodies, filter-sterilise antibody sample and aliquot to avoid multiple 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 primary antibody at dilution at least 1:2000.
Alternative agents for preventing bactrial growth in antibody solution:
* Thimerosal at 0,01%
* Gentamicin sulfate at 50 ug/ml