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.

Fig 1. Sample processing and analysis workflow used in this study.

Asterisks indicate immunocapture methods. Anti-1S2D, -DD8 and -LV9 are rabbit antibodies against these respective strains of L. donovani. VLu, concentrated proteins from VL urine; EHCu, concentrated proteins from urine of Endemic Healthy Controls; uAg, material captured from VL urine by anti-L. donovani antibodies; LC-MS/MS, liquid chromatography tandem mass spectrometry.

Fig 2. Sudanese VL patients’ urine (VLu) and immunocaptured urine antigen (uAg) detected by rabbit anti-L. donovani DD8 and -LV9 by western blot.

Regions submitted to mass spectrometry are broadly indicated by boxes. A) western blot, B) corresponding gel stained for proteins, C) the same gel as B, stained for carbohydrates. Molecular mass marker (M) sizes in kiloDaltons. Lanes are: lysate antigen of L. donovani strain DD8; uAg, VL urine material eluted from an anti-L. donovani DD8 immunochromatography column; VLu, concentrated VL urine; EHCu, concentrated urine from endemic healthy controls. Vertical lines indicate where the photographs were spliced to remove non-relevant lanes from the original images, and realigned based on the molecular weight marker.