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 μg/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 μg/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.