Ms mc2155 strain  (from the collection of the National Reference Laboratory of Tuberculosis, Pedro Kouri Institute, Cuba) was used. Cultures were grown in 1% (w/v) yeast extract (Merk, Germany), 0.5% (v/v) glycerol (Riedel de Haen, Germany), 0.4% (v/v) Tween 80 (Fluka, Germany), in 8% nutrient broth (Biocen, Cuba) for 48h with agitation (200 rpm) at 37ºC. The purity of the culture was evaluated by Ziehl-Neelsen staining .
Total lipid extraction from Ms was accomplished using the technique described by Bligh and Dyer  and the liposomes containing these lipids were obtained by the dehydration-rehydration method . The size of the vesicles was determined by transmission electron microscopy (TEM) and dynamic light scattering using a Brookhaven ZetaPlus (Brookhaven Instruments Worcs, UK).
Fifty female BALB/c mice (6-8 weeks) were used in the experiments. The procedure was carried out according to the international regulations for laboratory animal experimentation . Five groups of animals (n=10) were inoculated subcutaneously with either PBS; BCG, (Moreau strain, EPB ”Carlos J. Finlay”, Cuba) (106 CFU); LMs composed of 1mg of total lipids from Ms; LMs-A (LMs 1mg + alum Alhydrogel, Sigma, 1 mg) or LMs-M (LMs 1mg + Montanide, Seppicc, France). Two doses at 0 and 21 days were administered. The group immunized with BCG only received one dose on day 0. Blood samples were collected 42 days after the first immunization. The blood was centrifuged and the sera stored at -20ºC until use.
Sera from TB patients, PPD+ and PPD- healthy subjects were collected from the Universiti Sains Malaysia Hospital (HUSM). The protocols of the study were conducted according to the ethical guidelines as approved by the Human Ethics Committee of USM and written informed consent from participants was obtained.
An indirect ELISA was performed to measure the anti-LMs IgG response. Briefly, the plates were coated with 10µg/mL of the LMs formulation for 16h at 4°C and sera from mice were diluted at 1:50 and incubated for 1h at 37°C. A HRP conjugated rabbit anti mouse IgG (Sigma) at a dilution of 1:3000 was used and incubated for 1h at 37°C. The reaction was developed with a solution (100 µl /well) of o-phenylendiamine (OPD; Sigma) (5 mg in 12.5 ml 0.1 M sodium citrate buffer, pH 5 + 5 µl 30% (v/v) H2O2). After 20 to 30 min the reaction was stopped with 2N H2SO4 (50µl/well) and the absorbance was read at 492nm. To evaluate the cross-reactivity against the cell wall antigens (CWA) of MTb, a cocktail of these antigens was used to coat the ELISA plate at a concentration of 10µg/mL for 16h at 4°C.
A similar procedure was also followed to measure the anti-LMs IgG response in sera from TB patients, PPD+ and PPD- healthy subjects except that the secondary antibody used was a HRP conjugated rabbit anti human IgG (Sigma) at 1:6000 dilution.