Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: Implication for diagnosis of Kala-azar

Objective(s): Leishmaniasis is endemic in ۸۸ countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c mice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of ۱۰ to ۱ in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.Results: Approximately ۲۵ positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as ۸D۲ FVI۶, ۸D۲ FVI۳, ۶G۲ FV۴ and ۶G۲ FV۳. Results from isotype determination showed the IgG۲b sub-class in ۶G۲FV۲ and ۸D۲FVI۶ monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

Keywords:

Axenic amastigotes , Hybridoma cells , Leishmaniases , Leishmania infantum , Monoclonal antibodies