2 x 1 mg TDB Vaccine grade adjuvant.
10 ml sterile endotoxin-free physiological water (NaCl 0.9%)
Storage and Stability
TDB is provided as a sterile powder and shipped at room temperature. Store at -20°C. Lyophilized product is stable for 1 year at -20°C.
Trehalose-6,6-dibehenate (TDB) is a non-toxic synthetic analogue of the mycobacterial cell wall component trehalose 6,6’ dimycolate (TDM, also known as cord factor). TDB was recently shown to rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger a Th1/Th17 innate immunity. Incorporation of TDB combined with the synthetic amphiphilic cationic lipid compound dimethyldioctadecylammonium (DDA) into liposomes, has been shown to strongly enhance cellular and humoral responses against a protein antigen. Adjuvanticity of the cationic DDA:TDB liposomes and sustained protection against disease challenge has been demonstrated in particular with a tuberculosis vaccine candidate, and has good potential for application in a range of other diseases.
Preparation of Stock Suspension (1 mg/ml)
Add 100 µl DMSO to 1 mg TDB, heat at 60°C (approx. 15 - 30 seconds) and vortex.
- Once resuspended, immediately add 900 µl sterile physiological water (provided) or phosphate buffered saline, heat for 10 - 15 minutes at 60°C and homogenize by vortexing for 30 seconds.
Note: Following the addition of PBS, the suspension may appear slightly cloudy containing floating fine particles.
- Store at 4°C or prepare dilutions using a buffered solution for immediate use. Resupended product can be stored at 4°C for 6 months. Prior to each use, bring suspension to room temperature and homogenize by vortexing for 30 seconds.
Preparation of DDA: TDB Complex
The protocol described below is based on the method described in Henriksen-Lacey M. et al., 2010. Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen. J Control Release. 142(2):180-6.
- Prepare a 5 mg/ml solution of dimethyldioctadecylammonium bromide (DDA; not provided) in chloroform/methanol (ratio 9:1 v/v).
- Prepare a 5 mg/ml solution of TDB (dissolve 1 mg TDB using 0.2 ml chloroform/methanol (ratio 9:1 v/v)).
- Add 1 ml DDA solution (5 mg/ml) to 0.2 ml TDB solution (5 mg/ml) providing a ratio of 5:1 w/w (DDA:TBD).
- Use a roto-evaporator to remove the chloroform/methanol followed by flushing with N2, to produce a thin lipidic film containing the DDA:TDB complex.
- Store DDA:TDB complex at -20°C for 3 months or use immediately.
- Hydrate the lipidic film with 2 ml TRIS base buffer (10 mM; pH 7.4). Heat the suspension for 20 minutes at 60°C. Allow to cool before use. This suspension should not be stored, use within 1 day.
CAS number: 66758-35-8
Molecular weight: 987.43
Working concentration: 1 - 100 μg/mouse
Structure of TDB: