Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens

J Immunol Methods. 2001 Jun 1;252(1-2):105-19. doi: 10.1016/s0022-1759(01)00341-6.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Protozoan / blood*
  • Antibodies, Protozoan / immunology
  • Antigens, Protozoan / immunology*
  • Buffers
  • Electrophoresis, Polyacrylamide Gel / methods
  • Enzyme-Linked Immunosorbent Assay / methods
  • Enzyme-Linked Immunosorbent Assay / standards*
  • Humans
  • Leishmania donovani / immunology
  • Leishmania donovani / isolation & purification*
  • Leishmaniasis, Visceral / blood
  • Leishmaniasis, Visceral / diagnosis*
  • Leishmaniasis, Visceral / immunology
  • Serologic Tests / methods
  • Serologic Tests / standards
  • Sodium Dodecyl Sulfate
  • Solutions

Substances

  • Antibodies, Protozoan
  • Antigens, Protozoan
  • Buffers
  • Solutions
  • Sodium Dodecyl Sulfate