BACKGROUND: For the diagnosis of visceral leishmaniasis (VL), rK39 antigen-based rapid test is widely used. Unfortunately, up to 32% healthy individuals from endemic region test positive with this antigen. There is an urgent need to search for a more specific antigen with sensitivity similar to rK39. METHODS: We identified a Leishmania donovani-specific 12.6-kDa (BHUP3) soluble promastigote antigen through sensitive western blot technique. The identified protein was partially purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the antigenic response of eluted protein was determined by western blot with different groups of individual sera. The diagnostic potential was further validated by enzyme-linked immunosorbent assay using serum of 100 VL patients, 93 nonendemic healthy control individuals, 110 endemic healthy control individuals, and 110 disease control individuals. Further, it was characterized by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time-of-flight analysis. RESULTS: On blotting, antibody against this protein was recognized by all (9/9) VL patient's sera, but it was absent in every control group (nonendemic healthy control and endemic healthy control). Sensitivity of the enzyme-linked immunosorbent assay was 88% (89/101), whereas the specificity for endemic healthy, nonendemic healthy, and different disease groups were 96% (106/110), 100% (93/93), and 97% (107/110), respectively. The two-dimensional gel electrophoresis showed a single spot, and matrix-assisted laser desorption/ionization-time-of-flight analysis revealed that it is a 113-amino-acid-long putative uncharacterized protein of 12.6-kDa anamorsin homolog matched completely with Leishmania major (GenBank accession number: Q4QIS1). CONCLUSION: Despite marginally lower sensitivity of BHUP3, excellent specificity warrants its further development as a tool for diagnosis of VL.
BACKGROUND: For the diagnosis of visceral leishmaniasis (VL), rK39 antigen-based rapid test is widely used. Unfortunately, up to 32% healthy individuals from endemic region test positive with this antigen. There is an urgent need to search for a more specific antigen with sensitivity similar to rK39. METHODS: We identified a Leishmania donovani-specific 12.6-kDa (BHUP3) soluble promastigote antigen through sensitive western blot technique. The identified protein was partially purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the antigenic response of eluted protein was determined by western blot with different groups of individual sera. The diagnostic potential was further validated by enzyme-linked immunosorbent assay using serum of 100 VL patients, 93 nonendemic healthy control individuals, 110 endemic healthy control individuals, and 110 disease control individuals. Further, it was characterized by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time-of-flight analysis. RESULTS: On blotting, antibody against this protein was recognized by all (9/9) VL patient's sera, but it was absent in every control group (nonendemic healthy control and endemic healthy control). Sensitivity of the enzyme-linked immunosorbent assay was 88% (89/101), whereas the specificity for endemic healthy, nonendemic healthy, and different disease groups were 96% (106/110), 100% (93/93), and 97% (107/110), respectively. The two-dimensional gel electrophoresis showed a single spot, and matrix-assisted laser desorption/ionization-time-of-flight analysis revealed that it is a 113-amino-acid-long putative uncharacterized protein of 12.6-kDa anamorsin homolog matched completely with Leishmania major (GenBank accession number: Q4QIS1). CONCLUSION: Despite marginally lower sensitivity of BHUP3, excellent specificity warrants its further development as a tool for diagnosis of VL.
Authors: P K Smith; R I Krohn; G T Hermanson; A K Mallia; F H Gartner; M D Provenzano; E K Fujimoto; N M Goeke; B J Olson; D C Klenk Journal: Anal Biochem Date: 1985-10 Impact factor: 3.365