Identification of B-cell Epitope ofLeishmania donovaniand its application in diagnosis of visceral leishmaniasis

Abstract

Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P) and RRVAVLVLLDRL (P) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (PP) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (PP) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-PP antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-PP antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

Retracted: Evaluating the immunomodulatory responses of LdODC‐derived MHC Class‐II restricted peptides against VL

“…Humoral peptide efficacy was measured with active VL sera by ELISA, as described in methodology. 31 Initially, three concentrations (0.5, 1 and 2 μg per well) of each epitope were used to measure the effective dose of a peptide. Briefly, 2 μg/mL of each peptide was coated in 96-well plates with bicarbonate buffer (pH 9.6) and incubated overnight at 4°C.…”

Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis

“…Earlier, presence of anti-actin and anti-tubulin antibody was also reported [ 50 , 73 , 74 ]. Hypothetical protein (XP_003861271.1, spot C) successfully shown the efficiency of targeted MS derived peptide through Immuno chromatographic test (ICT) [ 9 ]. Above protein is under process of translational research for application.…”

“…To validate the MS derived peptide sequence, representing B cell epitopes of L . donovani proteins were synthesized commercially [ 9 ]. Spot ELISA were performed to validate the presence of antibody against these peptides in VL patients.…”

“…0.5 μg of SLA and synthetic peptide in PBS were loaded as a dot on ELISA plate coated with Nitro cellulose paper (NCP) for overnight, following protocol of Jamal et al . (2016) [ 9 ]. As positive and negative control, SLA and PBS was used.…”

“…Moreover, in our recent study B-cell epitopes RFFVQGDGIGQHSLQEALERR (P 1 ) and RRVAVLVLLDRL (P 2 ) from a hypothetical protein [Acc No: XP_003861458.1] of L . donovani were used as potential antigen based diagnostic as well as prognostic candidate in VL detection [ 9 ].…”