Mary Johnson Ph. D.mary at labome dot comSynatom Research, Princeton, New Jersey, United States
Antibody storage ‘shelf life’ may range from weeks to years and depends on the intrinsic nature of the antibody and storage conditions. Optimal conditions for storage are unique to each antibody; nevertheless, some general guidelines can be applied. Antibodies must be stored at an appropriate temperature and pH range, and frequently, in the presence of concentrated (~1 M) substances like glycerol or sucrose, in order to retain activity and prevent aggregation. Table 1 summarizes the common storage conditions and other characteristics.
|aqueous, 4°C||25-50% glycerol or ethylene glycol, -20°C||frozen at -20 – -80°C or in liquid nitrogen||lyophilized|
|typical shelf life||1 month||1 year||years||years|
|antibody concentration||1-5 mg/ml||1-5 mg/ml||1-5 mg/ml||1-5 mg/ml|
|carrier proteins for dilution||BSA||BSA||BSA||no|
|sterile or antibacterial requirement||yes||usually||no||no|
|antioxidants||usually; 2-ME, DTT*||usually; 2-ME, DTT*||no||no|
|fluorescence conjugation||protect from light||protect from light||protect from light||protect from light|
|multiple uses of single aliquot||yes||yes||no; freeze-thaw cycles degrade antibodies||non-applicable|
Table 1. Antibody storage condition. * methionine can also be considered.
When storing antibodies below zero, it is important NOT to use frost-free refrigerators. Common household refrigerators are frost-free, and they go through free-thaw cycles repeatedly to prevent frost formation.
Conjugated antibodies should be stored in dark-color vials or in vials covered with aluminum foil.
Chemical modifications such as oxidation and proteolytic degradation of proteins occur at moderate temperature; however, the extent of these reactions is much more pronounced at high temperature. Antibodies are generally stored at ≤ 4℃ in clean and sterile glassware or polypropylene tubes. Storage at room temperature often leads to antibody degradation and/or inactivity, usually resulting from microbial growth. For short term storage (1 day to a few weeks), antibody stock solutions may be stored at 4°C without significant loss in activity.
Formation of ice crystals can destroy protein structure, and thus render antibodies ineffective. Cryoprotectants such as glycerol and ethylene glycol prevent the formation of hydrogen bond between water molecules, and thus decrease the freezing points when added to aqueous solution. The actual freezing point is dependent on the composition/concentration of the cryoprotectants, see Table 2.
Table 2. Glycerol and ethylene glycol freezing points (in Celsius) at different concentrations (in percentage %) in water.
Note: ethylene glycol is toxic, and must be handled with care.
When the storage temperature is below the freezing point for an antibody solution with cryoprotectants, the solution will solidify. However, instead of ice crystal formation, vitrification may occur, as in the cryopreservation of cells/embryos with DMSO. During vitrification, the structural integrity of antibodies is maintained.
For increased stability, glycerol or ethylene glycol is added to a final concentration of 50% and the antibody can be stored at -20°C. The antibody solution should be stored in small working aliquots to avoid repeated freeze-thaw cycles.
It is important to use sterile glycerol preparations, since glycerol can be contaminated with microbes.
Antibody preparations should always be sterilized through filtration using a 0.45 micron filter and must be handled aseptically to prevent microbial contamination.
Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.02-0.05% (w/v) or thimerosal at a final concentration of 0.01 % (w/v) inhibit microbial growth.
Sodium azide (NaN3) is toxic to most cells and organisms, and to humans (see figure 1) . However, gram-positive bacteria (streptococci, pneumococci, lactobacilli) are resistant towards sodium azide (see figure 2)  . Sodium azide inhibits cytochrome oxidase in the mitochondrial electron transport chain and induces, for example, apoptosis in RGC-5 cells . Antibodies in sodium azide solution should NOT be directly used in living cells or in in vivo studies. In addition, sodium azide interferes with most conjugation reactions. Sodium azide can be removed through gel filtration or dialysis.
A side note: sodium azide was used in the older generation of car airbags.
Protease inhibitors prevent proteolytic cleavage of proteins. Antibody proteolysis can be an important issue for storing ascite fluid and serum preparations since both ascite and serum preparations contain protease. Cold storage and/or protease inhibitors can be used to remediate this degradation.
Dilute antibody solutions (< 1 mg/ml) are more prone to inactivation and physical losses as a result of low-level binding to the storage vessel surfaces. Thus, it is advisable to keep antibody concentration as high as possible (e.g. >1 mg/mL) during storage.
Carrier or filler protein, such as purified bovine serum albumin (BSA) or gelatin, can be added to dilute antibody solutions to a final protein concentration of 1-5 mg/ml (0.1-0.5%).
A recent research indicates that the dry ice during protein storage and transport can cause acidification of the storage solution, and will cause proteins to aggregate (with or with precipitation), especially with acidic proteins (those proteins with pI less than 7) . Polyclonal IgG tends to be acidic, with a pI range of 4.7-7.5 . Gas-sealed vials and/or gas-sealed plastic bags (for bagging the vials) should be used to minimize the damage in addition to re-equilibrate any samples at -80°C freezer for a couple of days. Dri-Shield Moisture Barrier Bags from 3M, Thermo Scientific™ Nunc™ Cryoflex products, and IMPAK bags like 0203PM56OZETN and 05MP081OZE can be useful.
The effect of oxidation on antibody molecules has been extensively studied, especially for therapeutic antibodies. Methionine residues in the antibody chains tend to be the primary site of oxidation when antibodies are exposed to intense light and/or elevated temperature . Antibodies contain both intra-chain and inter-chain disulfide bonds. The commonly used anti-oxidants such as 2-ME and DTT may not prevent the disulfide rearrangement , whereas another antioxidant — methionine does not foster disulfide rearrangement, and it can be a better antioxidant during antibody storage [Mark Fitchmun, Somatek, San Diego, CA, US].
Very little loss of activity may occur when serum is directly stored for a decade at -20°C. However, once antibody is purified, some loss of activity has been seen over years, albeit very slowly. It also seems that glycerol may not be necessary for storage at -20°C for years or even decades if the anti bodes do not go through repeated freeze/thaw cycles. The stored antibodies should be in high concentration.
Monoclonal antibodies can be stored at -20°C in 50% glycerol.
It is also reported that monoclonal antibodies can be stored under saturated ammonia sulfate as pellets at 4°C or -20°C for many years without loss of activity, bacterial outgrowth or oxidation.
Lyophilization (freeze-drying, or drying from the frozen state) provides an alternative method of stabilizing antibodies that are not freeze-labile. In most situations, freeze-dried proteins can be stored at -20°C. Lyophilization requires costly equipment and is labor-intensive.
Conjugated antibodies in general should be stored in dark containers or covered in aluminum foil.
Alkaline phosphatase and other enzyme conjugates are particularly sensitive to freezing, and should in general be stored at 4°C after conjugation for short term.
Antibody conjugates are best stored for long term at -20°C with glycerol or ethylene glycol at a final concentration of 50%. Although some enzyme conjugates may be stored at -20°C without cryoprotectants, frozen stocks must be single use aliquots to prevent repeated freeze-thaw cycles.
Fluorescence can photobleach when exposed to light. As with any reagents conjugated with fluorescent tags, (not just antibodies), the antibody conjugates must be protected from light. Fluorophore-conjugated antibodies should be stored at 4°C and should never be frozen.