title: Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens.
abstract: An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 microg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref-ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 microg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n=22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.
authors.affiliation: Cellabs Pty Ltd, P.O. Box 421, Brookvale, NSW 2100, Australia. admin@cellabs.com.au
authors.forename: G H
authors.initials: GH
authors.lastname: Rajasekariah
authors.forename: J R
authors.initials: JR
authors.lastname: Ryan
authors.forename: S R
authors.initials: SR
authors.lastname: Hillier
authors.forename: L P
authors.initials: LP
authors.lastname: Yi
authors.forename: J M
authors.initials: JM
authors.lastname: Stiteler
authors.forename: L
authors.initials: L
authors.lastname: Cui
authors.forename: A M
authors.initials: AM
authors.lastname: Smithyman
authors.forename: S K
authors.initials: SK
authors.lastname: Martin
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood* / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology*; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards*; D006801:Humans; D007893:Leishmania donovani / Q000276:immunology / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D012698:Serologic Tests / Q000379:methods / Q000592:standards; D012967:Sodium Dodecyl Sulfate; D012996:Solutions
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002021:Buffers; D012996:Solutions; D012967:Sodium Dodecyl Sulfate