Production and purification of polyclonal antibody against attenuated and wild type Leishmania infantum in dogs

Abstract

Antibodies are still widely used in several programs including early research, imaging, Targeting drug delivery system, Affinity chromatography, flowcytometry technic, diagnosis and treatment. Purification of antibody is a standard approach for detection of infection agent in different species. The reservoir hosts for Leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme–Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum. In this study we produced and purified polyclonal antibody against attenuated and wild type leishmania infantum in dogs. Anti-leishmania in dog serums precipitated with ammonium sulphate. The IgG recovered from ammonium sulphate precipitation was subject to ion exchange chromatography (IEC) and the purity of IgG was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. The purity of proteins were above 95% and then purified IgG was conjugated with FITC. We determined optimum titer of dog IgG by observation parasites under fluorescent microscope. The optimum dilution of prepared FITC conjugated dog IgG was 1: 400. This polyclonal antibody can be used for other applications in research, diagnosis and clinic.