An Insight for the Future Development of Diagnostic Tool by Exploiting Novel Leishmania Donovani Recombinant Hypothetical Protein

Abstract

Introduction: Assenting diagnosis of Visceral Leishmaniasis (VL) relies on the detection of Leishmania donovani (L. donovani) in splenic and bone marrow specimens obtained by invasive techniques. Thus, the development of inexpensive, non-invasive serological test encompassing high specificity, sensitivity and diagnostic efficacy is urgently needed. Aim: To assess the significance of recombinant proteins possessing B-cell epitopes in VL diagnosis. Materials and Methods: Employing immunoinformatics approach, the B-cell epitope footprint of L. donovani hypothetical proteins (derived from earlier studies) were decrypted holding good antigenic character with numerous epitopes. L. donovani hypothetical proteins XP_003860226.1 and XP_003861271.1 were first time cloned as His-tagged fusion proteins and purified as novel recombinant protein antigens, designated rLdhyb and rLdhyc respectively. Sanger sequencing method was exploited to sequence gene insert (GeneBank accession number MH479406). Results: B-cell epitopes revealed 100% conservancy with L. infantum. Immunoinformatics data revealed no significant sequence similarity with homo sapien and the causative agent of other diseases like tuberculosis, typhoid, malaria etc., resembling in symptoms to VL. Sequencing chromatogram of cloned gene Ldhyb and Ldhyc revealed 98% and 94% identity with L. donovani. ELISA revealed the absolute specificity with sensitivity of 95.4% for rLdhyb and 91% for rLdhyc. Area under curve for rLdhyb, rLdhyc and SLA were 0.99, 0.99 and 0.961, with standard error 0.002, 0.007 and 0.019 respectively. The in silico data was coherently supported by in vitro result. Conclusion: Absolute specificity, high sensitivity and diagnostic efficacy (rLdhyb: 98%; rLdhyc: 97%) advocated their excellent biomarker property. The present findings provide some basic insights for the future development of novel hypothetical proteins based non-invasive diagnostic tool for VL detection.

Keywords

• b-cell epitopes
• diagnostic efficacy
• immunoinformatic
• sanger sequencing
• sensitivity