title: Identification of B-cell Epitope of Leishmania donovani and 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 (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) 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 (P1P2) 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-P1P2antibody 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-P1P2antibody 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.
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Fauzia
authors.initials: F
authors.lastname: Jamal
authors.affiliation: b Department of Bioinformatics , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Manas Ranjan
authors.initials: MR
authors.lastname: Dikhit
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Manish K
authors.initials: MK
authors.lastname: Singh
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Pushkar
authors.initials: P
authors.lastname: Shivam
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Sarita
authors.initials: S
authors.lastname: Kumari
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Sinha
authors.initials: S
authors.lastname: Pushpanjali
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.|c Department of Biotechnology , National Institute of Pharmaceutical Education and Research , Hajipur 844102 , India.
authors.forename: Amit K
authors.initials: AK
authors.lastname: Dubey
authors.affiliation: c Department of Biotechnology , National Institute of Pharmaceutical Education and Research , Hajipur 844102 , India.
authors.forename: Prakash
authors.initials: P
authors.lastname: Kumar
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Shyam
authors.initials: S
authors.lastname: Narayan
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Gupta
authors.affiliation: d Department of Clinical Medicine , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Krishna
authors.initials: K
authors.lastname: Pandey
authors.affiliation: d Department of Clinical Medicine , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: V N R
authors.initials: VNR
authors.lastname: Das
authors.affiliation: e Department of Immunology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Sanjiva
authors.initials: S
authors.lastname: Bimal
authors.affiliation: f Department of Molecular Biology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
authors.affiliation: a Department of Microbiology , Rajendra Memorial Research Institute of Medical Sciences , Patna 800007 , India.
authors.forename: Shubhankar K
authors.initials: SK
authors.lastname: Singh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000097:blood; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology / Q000469:parasitology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007893:Leishmania donovani / Q000737:chemistry / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D011817:Rabbits
chemical_list: D000913:Antibodies, Protozoan; D000936:Antigen-Antibody Complex; D000953:Antigens, Protozoan; D018985:Epitopes, B-Lymphocyte; D007106:Immune Sera
keywords: B-cell epitope; ICT; Leishmania; VL diagnosis; immunoinformatics