Identification of major antigens of Leishmania donovani using kala azar sera

The study was conducted with the prime objective of isolating an antigen from the crude preparation of whole promastigotes (Leishmania donovani) with a view to future exploitation in serodiagnosis and production of monoclonal antibodies. Soluble antigen, prepared from promastigotes isolated from actively growing cultures, was fractionated by gel filtration chromatography over a column of Sephadex G200. Three peaks of proteins could be recovered. The antigenic reactivity of different fractions was checked against sera of kala azar patients by immunoelectrophoresis (IEP), rocket immunoelectrophoresis (RIEP) and enzyme-linked immunosorbent assay (ELISA). The first peak was found to be highly reactive in comparison with other peaks. SDS-PAGE and western blot analysis of this antigen revealed a major antigen of promastigotes in the vicinity of 65 to 66 kDa, while a weakly reactive triplet could also be detected in the low molecular weight region.Abbreviations IEP Immunoelectrophoresis - RIEP Rocket Immunoelectrophoresis - ELISA Enzyme-Linked Immunosorbent Assay - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - BHI Brain Heart Infusion - HBSS Hanks' Balanced Salt Solution - NCP Nitrocellulose Paper - DAB Diamino Benzidine Tetrahydrochloride - kDa Kilo Daltons