SDS-PAGE showing the banding pattern of electrophoresed CICs antigens in VL-BT subjects compared to healthy, treated VL, relapse, reinfection and other diseases subjects.

Briefly, 50μg of proteins was electrophoresed (10% SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 or immunoblotted. Fig 3.a.i. SDS-PAGE comparing VL-BT subjects to healthy and treated VL cases. Molecular weight marker in lane 1; CICs antigen of VL-AT samples in lane-2 & 6,CICs antigen of VL-BT samples in lane-3 and healthy endemic and non-endemic in lane 4 and 5. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑). VL-AT (lane 2 & 6) is also showing comparatively down-regulated expression of 55kDa and 23kDaband. Fig 3.a.ii. SDS-PAGE showing the banding pattern of electrophoresed CICs antigens in VL-BT (relapse/reinfection) subjects compared to healthy and treated VL cases and SLA. Molecular weight marker in lane 1; CICs antigen of VL-BT samples in lane-2,3 (Reinfection/relapse) & 5; healthy endemic and non-endemic in lane 4 and 6; SLA in lane 7 and VL-AT in lane 8. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑). VL-AT (lane 8) and healthy endemic (lane 4) is also showing comparatively downregulated expression of 55 and 23kDaband.Fig 3.b. Immunoblotting data of SDS-PAGE (the gel of Fig 3.a.) showing immunoreactive bands in different study groups. Electrophoresed gel was transferred to NCP membrane and exposed to anti-leishmanial antibody HRP conjugated followed by substrate (DAB) exposure. Molecular weight marker in lane 1;CICs antigen of VL-BT samples in lane-2,3 and 8–10;VL-AT in lane 5; SLA in lane 7 and healthy in lane 4 and 6. 55 and 23 kDa can be recognized in all VL-BT samples (indicated by ↑). VL-AT (lane 5) is also showing comparatively downregulated expression of 55 kDa band. 23 kDa band is not recognizable in this sample. Fig 3.c. SDS-PAGE showing the banding pattern of electrophoresed CICs antigens of VL-BT subjects in comparison to healthy and treated VL cases and other diseases. Molecular weight marker in lane 1;CICs antigen of VL-AT samples in lane-2;VL-BT in lane 3; healthy endemic and non-endemic in lane 4 and 5; SLA in lane 6; Filaria in lane 7 and TB in lane 8. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑) in comparison to others. Fig 3.d. Immunoblotting data of SDS-PAGE (the gel of Fig 3.c.) showing immunoreactive bands in different study groups. Molecular weight marker in lane 1;CICs antigen of VL-AT samples in lane-2;VL-BT in lane 3; healthy in lane 4 and 5; SLA in lane 6; Filaria in lane 7, TB in lane 8, Asthma in lane 9 and viral in lane 10. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑) in comparison to others except low intensity band at 55kDa band appeared in VL-AT. Fig 3.e. Histogram showing comparative band intensity of 55 and 23 kDa SDS-PAGE gel and after immunoblotting. Relative band intensity was evaluated using Quantity one software. Band intensity of SLA in stained gel and after blotting was revealed as 10.5, 7.9 and 5.4, 11 respectively, confirmed the presence of the protein of similar molecular weight in L. donovani.