Serum samples used in this study were collected from the School of Tropical Medicine (STM), Kolkata. Twenty three VL patients were enrolled for the longitudinal study having single dose liposomal amphotericin B therapy (10mg/kg). Blood samples were collected before the treatment, active VL cases (AVL), one month after the treatment, cured VL cases (CVL), and about six months post treatment follow-ups (FU). Sera also collected from 23 other symptomatically similar diseases comprised of four samples each from malaria, tuberculosis, pneumonia, typhoid, and viral fever and one sample from liver abscess, systemic lupus erythematosus, and pancreatitis. Sample collection continued with 23 healthy people too as controls from the Indian Institute of Chemical Biology (IICB), Kolkata.
Parasite culture and purification of leishmanial antigens
L. donovani strain AG83 (ATCC© PRA-413™) of the parasite was regularly maintained in hamsters. Amastigotes were isolated from sacrificed hamsters and allowed to transform into promastigotes in culture medium (M199) with necessary supplements at 220C. Promastigotes were subcultured through fresh medium passages and 3rd to 5th passage cultures were harvested and centrifuged to get cell pellets. Cell pellets were then washed in PBS and stored at -200C until use.
Leishmania promastigote antigens, LAg, were purified from the cell pellet. In a typical experiment, cells were suspended in 5mM Tris-HCl having pH 7.4 and vortexed for 12 minutes (2 min for 6 times) to get the parasite membrane leaky. Parasites were then centrifuged to collect the ghost membrane pellet (2310 g, 10 min, 40C) which was then resuspended in the same buffer and subjected to ultrasonication (30 sec for 6 times). The suspension was centrifuged again to obtain the antigens in the supernatant (5190 g, 30 min, 40C). The concentration of LAg was estimated by Lowry’s methods and stored at -800C for further use. Soluble leishmanial antigen, SLA, was also purified from Leishmania promastigote culture similar to LAg with some modifications. Cell pellet was suspended in 1mM EDTA, 5 µg leupeptin, 1mM iodoacetamide and 1mM phenylmethylsulfonyl fluoride in 5mM Tris-HCl buffer, pH 7.4. Suspension was vortexed, centrifugation and sonicated as mentioned for LAg followed by solubilisation in (1% w/v) octyl-β-D-glucopyranoside at 40C for overnight. Next day solubilised suspension was centrifuged at 100,000g for 1h. The supernatant collected contains SLA which was dialyzed and finally stored at -800C after concentration estimation by Lowry’s method.
SDS-PAGE and electroelution
Proteins were first denatured by the reducing agent, βME, and resolved in 10% SDS-PAGE. Different molecular weight proteins were separated from LAg (10 μg/lane) and SLA (5 μg/lane). The pattern of LAg and SLA proteins were visualized by Coomassie Blue. Rf values for the molecular weights of the respective proteins were determined by the automated Image Lab software in comparison to the standards. For electroelution, 31, 34, 36, 45, 51, 63, 72, 91, and 97 kDa protein bands were excised from the LAg gel and subjected to electroelution as per the manufacture’s protocol (BioRad, Model-422). Subsequently, each protein was dialyzed against PBS. Electroeluted proteins after quantification were resolved on SDS-PAGE separately for reconfirmation of their molecular weights.
Indirect ELISA for antibody detection
Indirect ELISA to capture antibodies was performed on 96-well flat bottom plates (Nunc Maxisorp, Denmark). In brief, wells were coated with 1 μg/well concentration of purified and electroeluted proteins with phosphate buffer (100 μl/well) and incubated overnight in 40C. The next day, antigen-coated wells were blocked with 1% BSA in PBS (200 μl/well) for 2h at 370C. Subsequently, serum samples (1:2000) followed by peroxide conjugated antihuman IgG (1:4000) were applied to the wells in PBS buffer (100 μl/well) and incubated for 1h at 370C. Plates were washed in each step with PBS and Tween 20 to remove any non specific binding. Finally, wells were incubated in the substrate, o-phenylenediamine dihydrochloride (OPD), and H2O2 in the phosphate-citrate buffer (50 μl/well). The biological reaction was stopped with sulfuric acid and optical density values were acquired by using ELISA plate reader (RS232C, Thermo Scientific, USA) at 492 nm wavelength.
Stimulation of PBMCs and cytokines analysis
Peripheral blood mononuclear cells (PBMCs) from heparinized blood samples of one month cured VL patients and healthy individuals were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, USA) and finally resuspended in Medium RPMI 1640 with serum supplements and antibiotics. PBMCs obtained from each individual were cultured in triplicate (1x106 cells/well) with and without antigen stimulation. Stimulation of cured VL and healthy PBMCs with LAg (12.5 µg/ml) and cured VL PBMCs with electroeluted antigens (1.5 µg/ml) 31, 34, 51, 63, 72, and 91 kDa were performed at 370C in CO2 incubator. Supernatant from the cultures were collected after 96 h and stored at -200C for cytokine analysis. Level of cytokines, IFN-g, IL-12, IL-10 and TGF-β along with IFN-g/ IL-10 and IFN-g/ TGF-β were measured through ELISA according to the manufacturers’ instructions (BD OptEIA ELISA kit, BD Biosciences). Briefly, capture antibodies specific to the cytokines were coated in the wells in carbonate buffer overnight at 40C. Subsequently, wells were blocked and incubated with culture supernatants for 1h. Cytokine specific detection antibodies were used in the wells followed by TMB substrate. Optical density values were obtained in the ELISA reader at 450 nm.
Protein bands of molecular masses, 34 and 45 kDa were stained with Coomassie Blue and excised from 10% SDS-PAGE of LAg. The proteins imbibed in gel bands were digested through in-gel tryptic digestion kit according to the protocol provided by the manufacturers (ThermoFisher Scientific). Subsequently, co-crystallization of the digested peptides with matrix was done and subjected to MALDI-TOF/TOF for MS/MS spectra (Applied Biosystems). Data obtained from mass spectrometry were identified through the MASCOT search engine.
Statistical studies were conducted with Graph Pad Prism software version V. Two-tailed student t-test were performed for paired and unpaired samples in indirect ELISA. P values less than 0.05 was considered significant with 95% confidence intervals. Cut off values were determined by the Receiver-Operator Curve (ROC) where 100% sensitivity was obtained. Difference in the cytokine production was determined by the two-tailed unpaired student’s t-test.