Single Domain Antibody (sdAb) Selection Under Application Conditions
The ability to refold to native conformation under extreme conditions and the extreme stability of phage provide a theoretical basis for stable sdAb variants identification. Here, Creative Biolabs describes the novel single domain antibody (sdAb) selection under application conditions to obtain novel binders.
Theoretical Basis
In modern biological research, the antibody fragments, such as scFv, Fab, and sdAbs, have been widely used in affinity purification, imaging, diagnostic assays, and therapy. However, these successful applications are not only determined by the antibody fragments' specificity and affinity but also the ability to bind the targets in the environment in which the antibody fragment is intended to functionally perform. In general, protein stability is determined by its capacity to maintain the native conformation and remain functions in extreme conditions, including high salt concentration, elevated temperatures, or extreme pH. Compared with conventional antibody, sdAb consists of a single domain unit and presents great stability. Besides, the refolding of sdAbs is more efficient compared to the unfolding of conventional antibody derived fragments.
The phage presents extreme stability to resist denaturation under extreme temperatures, high detergent concentration, extreme pH, and even chemical denaturants. In this case, display on phage aids in the identification of stable sdAbs.
sdAb Selection Under Application Conditions
To obtain the sdAbs with required stability, the variants can be selected from an immune library or engineered library via phage display. And the conditions under which the sdAbs displayed on phage bind to the target ideally should mimic the environment in which the sdAbs should be effective. The studies have shown that when preincubated for 10 min at 80°C and subsequently cooled down for 10 min at 4°C, more thermostable sdAbs can be obtained.
Fig.1 Schematic representation of the sdAb selection under application conditions.
Different Application Conditions
- The sdAbs targeting rotavirus should work in the intestine so that it should be inherently stable against the acidic conditions and proteases.
- The sdAbs targeting the fungus Malassezia furfur should recognize the target proteins in the condition with a high concentration of detergents.
- The sdAbs targeting human pancreatic lipase (HPL) and human gastric lipase (HGL) should be stable at low pH to survive the proteases in the stomach.
It is worth mentioning that the screening procedure should demonstrate the desired function of the sdAb under application conditions. The selected binders have to be produced, purified, and tested in vivo and in vitro for efficacy establish. In conclusion, sdAbs with appropriate characterizations can be selected under a variety of application conditions.
Case Study
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