Phage-nanobody as molecular marker for the detection of Leishmania tropica

Abstract

Cutaneous leishmaniasis (CL), caused by unicellular leishmania parasites, is one of the most important public health problems aggravated by the Syrian crises. Antibodies have long been implicated in the identification and isolation of leishmania antigens as well as in the detection and treatment of the pathogens. Phage display is one of the most effective ways for generating recombinant antibodies (like nanobodies), in addition displaying nanobody on phage could add great possibilities like antigen-detection and molecular imaging. This work aimed at the production of anti-L. tropica phage-nanobodies and the investigation of their potentials. Therefore, a standard camel immunization procedure was carried out using L. tropica cells followed by assessing the solicited immune response and the active participation of HCAbs in this response by ELISA. A relatively large nanobody “immune” library of 5 × 108 individual transformants, with 96.5% positivity, was prepared from the blood of the immunized camel. Phage display biopanning on this nanobody library showed that the phage-nanobody from third round and elution recognized several leishmania proteins similar to poly clonal anti leishmania by immune blotting. Five anti L. tropica phage-nanobodies (M13-NbLtc05, 06, 14, 24, 36) isolation from third elution were more specific to L. tropica than to L. major. Finally, M13-NbLtc36 labelled with FITC was capable to bind L. tropica cell higher than M13-NbhGH. In conclusion, phage display is an effective method to generate anti L.tropica nanobody. Moreover, Phage-nanobody was capable of detecting L.tropica cell by flow cytometric.