Isolation and characterisation of Leishmania donovani protein antigens from urine of visceral leishmaniasis patients

Abstract

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L . donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti- L . donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L . donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.

Urine-Based Antigen (Protein) Detection Test for the Diagnosis of Visceral Leishmaniasis

“…Here, we review in detail the strategy used to discovery these leishmanial biomarkers, the development of a sensitive and specific multiplexed capture ELISA, and the preliminary results of a clinical validation of this new and powerful monoclonal antibody-based antigen detection test for the diagnosis of VL. A recent publication describes six other new proteins of L. donovani identified in urine of VL patients using mass spectrometry coupled with immunoprecipitation of parasite antigens [66]. These proteins are not related to the markers described and reviewed herein and their utility as diagnostic markers have not yet been reported.…”