Soluble Leishmania antigen (SLA) was prepared as previously described (Gidwani et al., 2011 (link)). Briefly, L. donovani amastigotes from clinical isolates (Kala-azar Medical Research Center, Muzaffapur, Bihar, India) were grown in Medium 199, Hanks’ Balanced Salts (M199; Thermo Fisher) until transformed into promastigotes, then cultured. 2 × 109 stationary-phase promastigotes were harvested from culture and centrifuged at 3900 g for 20 minutes to obtain parasite pellet, which was washed three times with cold 1x PBS and resuspended in solution (10 mM Trizma® hydrochloride solution (TRIS-HCl; Sigma-Aldrich), 1 mM pH 8.0 ethylenediaminetetraacetic acid (EDTA; Amresco), 1.6 mM phenylmethanesulphonyl fluoride (PMSF; HiMedia, Mumbai, India), and 50 μg/ml N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin; Amresco)) at a concentration of 2 × 109 parasites/ml. The parasite suspension was sonicated 4-5 times for 15 s at 10 Hz and centrifuged at 27,000 g for 30 minutes at 4°C. The lipid layer was removed from the surface of supernatant and the remaining solution was ultracentrifuged at 100,000 g for 4 hours at 4°C. The supernatant was removed, dialysed against the PBS overnight and stored at −80°C until use. Protein was measured using a Micro BCA Protein Assay Kit (Thermo Fisher) as per manufacturer’s instructions.
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